CN106232878A - The biomarker of MELK activity and the method that uses it - Google Patents
The biomarker of MELK activity and the method that uses it Download PDFInfo
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Abstract
The present processes relates to following beyond thought discovery, human eukaryote initiation factor 4B (eIF4B) the 406th (as, serine residue) phosphorylation level, or the corresponding phosphorylated amino acid of its ortholog thing, can serve as MELK enzyme (such as kinases) activity and/or the biomarker of carcinogenic activity.The present processes further relates to following beyond thought discovery, human histone H3 the 3rd (as, threonine residues) and/or the 10th (as, serine residue) and/or the 11st (as, threonine residues) or the phosphorylation level of corresponding phosphorylated amino acid of its ortholog thing, it is also possible to as MELK enzyme (such as kinases) activity and/or the biomarker of carcinogenic activity.
Description
This application claims the U.S. Provisional Application 61/954,046 submitted on March 17th, 2014 and carry November 12 in 2013
The priority of the U.S. Provisional Application 61/902,877 handed over.Entire contents is incorporated herein by reference.
Background technology
Protein kinase maternal embryo leucine zipper kinases (MELK), it is known that its participation regulation cell cycle progress,
Cell proliferation, apoptosis and mRNA montage (Badouel et al. (2006) Cell Cycle 5:883-889and
Badouel et al.(2010)Exp.Cell Res.316:2166-2173).By gene expression spectrum analysis, MELK is also by really
Recognize relevant to kinds cancer, including mammary gland in breast carcinoma, pulmonary carcinoma, bladder cancer, lymphoma and cervical cancer cell and animal model
Formation (Komatsu et al. (2013) Int.J.Oncol.42:478-506 of tumor;Pickard et al.(2009)
Breast Cancer Res.11:R60;Hebbard et al.(2010)Cancer Res.70:8863-8873;Lin et
al.(2007)Breast Cancer Res.9:R17;WO 2004/031413;WO 2007/7013665;and WO 2006/
085684).Despite this association, the most do not carry out the tumorigenic functional analysis of MELK mediation, swelling of MELK mediation
Mechanism and (through extending) that tumor occurs are used for determining that these tumors of regulation occur useful reagent algoscopy to be also unknown.This
The shortage understood prevents the qualification to the biomarker reliably reporting MELK enzyme and/or carcinogenic activity.Although known certain
The inhibitor of the kinase activity of a little targeting MELK (see, e.g., Chung et al. (2012) Oncotarget 3:1629-
1640), but the tumorigenic biomarker to confirming MELK mediation is still had to have clear and definite demand in field, to provide fast
The effective means of speed assess the anti-cancer therapies of targeting MELK.
Summary of the invention
The application based on, be at least partially based on the 406th (such as, the silk ammonia of finder eukaryotic initiation factor 4B (eIF4B)
Acid residue) phosphorylation level be maternal embryo's leucine zipper kinases (MELK) activity reliable biological label, be suitable for
With measurement MELK enzymatic activity clinically before clinical.Similarly, the application based on, be at least partially based on the of finder's histone H 3
3 (such as, threonine residues) and/or the 10th (such as, serine residue) and/or the 11st (such as, threonine residues)
Phosphorylation level be the reliable biological label of maternal embryo's leucine zipper kinases (MELK) activity, be suitable for before clinic and
Measure MELK enzymatic activity clinically.
On the one hand, it is provided that one is used for identifying that suppression people maternal embryo leucine zipper kinases (MELK) or its direct line are same
The kinases of source thing or the method for the reagent of carcinogenic activity, including: a) comprise i) people MELK or its direct line with the contact of described reagent same
Source thing and ii) human eukaryote initiation factor 4B (eIF4B) or the sample of its ortholog thing, and b) determine that described reagent suppresses people
The ability of corresponding phosphorylated amino acid in 406th serine phosphorylation of eIF4B or people's eIF4B ortholog thing, wherein
Phosphorylation minimizing identifies this reagent suppression people MELK or the kinases of its ortholog thing or carcinogenic activity.At an embodiment
In, by by ammonia corresponding in the amount of the in people eIF4B in sample the 406th serine phosphorylation or people's eIF4B ortholog thing
The amount of base acid phosphoric acid compares with compareing, and determines the described in people eIF4B the 406th serine phosphorylation or people
The suppression of corresponding phosphorylated amino acid in eIF4B ortholog thing.In another embodiment, comparison refers to, compared to
There is not the amount of the relatively early time point of reagent or sample contact reagent, the 406th serine phosphorylation of people eIF4B in sample
The amount of corresponding phosphorylated amino acid in amount or people's eIF4B ortholog thing.In another embodiment, by by sample
In the amount of the 406th serine phosphorylation of people eIF4B or people's eIF4B ortholog thing the amount of corresponding phosphorylated amino acid with
The ratio of people eIF4B 406 or its ortholog thing total amount compares with compareing, and determines the to people eIF4B the 406th silk
The suppression of corresponding phosphorylated amino acid in propylhomoserin phosphorylation or people's eIF4B ortholog thing.In another embodiment, right
According to referring to, compared to there is not reagent or this ratio of the relatively early time point at sample contact reagent, 406 serines in sample
The ratio of corresponding phosphorylated amino acid in people's eIF4B ratio of phosphorylation or people's eIF4B ortholog thing.Implement at another
In mode, described method also includes determining the amount of the protein translated from the RNA of the 5'UTR with highly structural, optionally,
Wherein said protein selects free bone marrow cell carcinoma oncogene (c-Myc), the chain inhibitor of apoptotic proteins X-(XIAP) and bird ammonia
The group of the composition of acid decarboxylase (ODC1).In another embodiment, described method also includes determining that reagent is the most directly tied
Close to described people eIF4B or its described ortholog thing, or described people MELK or the step of its described ortholog thing.At another
In individual embodiment, the group of sample choosing freely external, in vitro and internal sample composition.In another embodiment, sample packages
Containing cell (such as, cancerous cell, such as cancer select free MELK or eIF4B expand or any cancer of overexpression, have MELK or
The group that any cancer of eIF4B Activating mutations and MELK or eIF4B are formed by any cancer of other kinase activations).At another
In individual embodiment, cell is to obtain from experimenter.In another embodiment, sample choosing freely organize, whole blood, serum,
Blood plasma, cheek are scraped, saliva, cerebrospinal fluid, urine, feces and bone marrow composition group.In another embodiment, 406 serines
In the people eIF4B amount of phosphorylation or people's eIF4B ortholog thing, the amount of corresponding phosphorylated amino acid is by using specificity
The reagent of people eIF4B or the corresponding phosphorylated human eIF4B ortholog thing being attached to 406 serine phosphorylations carries out immunity
Measure (such as, radioimmunoassay, western blot analysis, proximity ligation assay, immunofluorescence assay, the enzyme determined
Immunoassay, immunoprecipitation assay, chemoluminescence method, Immunohistochemical assay, Dot blot measure or slit print
Mark detection method).In another embodiment, enzyme immunoassay is a kind of Sandwich ELISA, and its use is special
Property combine capture antibody or the fragment of people eIF4B or corresponding people's eIF4B ortholog thing and specifically combine 406 silks
The detection antibody of people's eIF4B ortholog thing of people eIF4B or the corresponding phosphorylation of propylhomoserin phosphorylation or fragment.At another
In embodiment, described people eIF4B or its ortholog thing, and/or described people MELK or its ortholog thing, including table 1 institute
The nucleotide sequence of row or aminoacid sequence.In another embodiment, reagent is little molecule, or antibody or its antigen binding fragment
Section.In another embodiment, reagent is by the amount of the people eIF4B of 406 serine phosphorylations or people's eIF4B ortholog thing
In the amount of corresponding phosphorylated amino acid reduce at least 50%.
On the other hand, it is provided that one is used for assessing reagent and suppresses people MELK or its ortholog thing to swash in subject
The method of the curative effect of enzymatic activity, including: a) first time point detection the 406th serine phosphorylation in experimenter's sample
The amount of people eIF4B or in the amount of the ortholog thing corresponding to people's eIF4B amino acid sites phosphorylation;B) after using reagent
One or more subsequent point in time repeat step a);C) comparison step a) and described b) in the 406th serine phosphorylation
The amount of people eIF4B or the amount of its ortholog thing, wherein compared with at least one time point follow-up, in the of first time point
The amount of the people eIF4B of 406 serine phosphorylations or at the ortholog thing corresponding to people's eIF4B amino acid sites phosphorylation
Measure higher, show reagent suppression people MELK or the kinase activity of its ortholog thing.In some embodiments, the 406th silk
In the amount of the people eIF4B of propylhomoserin phosphorylation or people's eIF4B ortholog thing, the amount of corresponding phosphorylated amino acid is surveyed by immunity
Fixed determine, the reagent that this immunoassay use can be specifically binding to the 406th serine phosphorylation people eIF4B or
(such as, radioimmunoassay, western blot analysis, ortho position connect skill to people's eIF4B ortholog thing of corresponding phosphorylation
Art, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemoluminescence method, Immunohistochemical assay,
Dot blot measures or slit engram detection method).In another embodiment, enzyme immunoassay is a kind of sandwich enzyme-linked exempts from
Epidemic disease algoscopy, its use specifically combine the capture antibody of people eIF4B or corresponding people's eIF4B ortholog thing or fragment and
Specifically combine the inspection of people's eIF4B ortholog thing of people eIF4B or the corresponding phosphorylation of the 406th serine phosphorylation
Survey antibody or fragment.In another embodiment, in the group of sample choosing freely in vitro and internal sample composition.Real at another
Executing in mode, containing cancerous cell, (such as, cancerous cell choosing freely contains MELK or eIF4B and expands or overexpression any sample packages
Cancer, it is made up of by any cancer of other kinase activations any cancer and MELK or eIF4B of MELK or eIF4B Activating mutations
Group).In another embodiment, sample choosing freely organize, whole blood, serum, blood plasma, cheek are scraped, saliva, cerebrospinal fluid, urine,
Feces and the group of bone marrow composition.In another embodiment, the sample in step a) and/or step b) is taken from experimenter's
A part for single sample.In another embodiment, the sample in step a) and/or step b) is taken from the conjunction of experimenter
And a part for sample.In another embodiment, during first time point and subsequent point in time, experimenter has accepted
Treatment of cancer, complete treatment of cancer and/or be in from cancer alleviation.In another embodiment, described people eIF4B or
Its ortholog thing, and/or described people MELK or its ortholog thing, including the nucleotide sequence listed by table 1 or aminoacid sequence.
In another embodiment, reagent is little molecule, or antibody or its Fab.In another embodiment, institute
State reagent by the amount of the people eIF4B of the 406th serine phosphorylation or in people's eIF4B ortholog thing corresponding phosphorylation
Amino acid whose amount reduces at least 50%.
On the other hand, it is provided that a kind for the treatment of suffers from the method for the patient of cancer, suppression people is used including to experimenter
406th serine phosphorylation of eIF4B or in its ortholog thing the reagent of the phosphorylation of corresponding phosphorylated amino acid,
Thus treatment suffers from the patient of cancer.In one embodiment, reagent is used with pharmaceutically acceptable dosage form.Separately
In one embodiment, reagent is little molecule, or antibody or its Fab.In another embodiment, reagent is straight
Connect and be attached to described people eIF4B or its ortholog thing, or described people MELK or its ortholog thing.At another embodiment
In, cancer is selected free MELK or eIF4B amplification or any cancer of overexpression, is had any of MELK or eIF4B Activating mutations
The group that cancer and MELK or eIF4B are formed by any cancer of other kinase activations.In another embodiment, described people
EIF4B or its ortholog thing, and/or described people MELK or its ortholog thing, including the nucleotide sequence listed by table 1 or amino
Acid sequence.In another embodiment, reagent is little molecule, or antibody or its Fab.Another embodiment party
In formula, described reagent by the amount of the people eIF4B of the 406th serine phosphorylation or in people's eIF4B ortholog thing corresponding
The amount of phosphorylated amino acid reduces at least 50%.In another embodiment, this method also includes using one or more
Other anticarcinogen.
On the other hand, it is provided that a kind of for determining people MELK or the function of its ortholog thing or the method for activity,
Including: a) detect the amount of the people eIF4B of the 406th serine phosphorylation in sample or corresponding to people's eIF4B amino acid sites
The amount of people's eIF4B ortholog of phosphorylation;B) after operation sample and/or after operation same sample or test sample,
In same sample or test sample, one or more subsequent point in time repeat step a);And c) comparison step a) and described b) middle inspection
Survey the 406th serine phosphorylation people eIF4B amount or the people corresponding to people's eIF4B amino acid sites phosphorylation
The amount of eIF4B ortholog, wherein with at least one subsequent point in time and/or to same sample or test sample at least one times
Subsequent operation is compared, first time point change the 406th serine phosphorylation people eIF4B amount or corresponding to people
The amount of people's eIF4B ortholog of eIF4B amino acid sites phosphorylation, indicates people MEL or the function of its ortholog thing
Or activity is changed.In some embodiments, the amount of the people eIF4B of the 406th serine phosphorylation or corresponding to
The amount of people's eIF4B ortholog of people's eIF4B amino acid sites phosphorylation is determined by immunoassay, and this immunoassay make
The people eIF4B of reagent people eIF4B or the corresponding phosphorylation that can be specifically binding to the 406th serine phosphorylation straight
It is congener (such as, radioimmunoassay, western blot analysis, proximity ligation assay, immunofluorescence assay, enzyme immunity
Algoscopy, immunoprecipitation assay, chemoluminescence method, Immunohistochemical assay, Dot blot measure or slit engram inspection
Survey method).In another embodiment, enzyme immunoassay is a kind of Sandwich ELISA, and it uses specifically
In conjunction with capture antibody or the fragment of people eIF4B or corresponding people's eIF4B ortholog thing with specifically combine the 406th silk ammonia
The detection antibody of people's eIF4B ortholog thing of people eIF4B or the corresponding phosphorylation of acid phosphoric acid or fragment.At another
In embodiment, the group of sample choosing freely external, in vitro and internal sample composition.In another embodiment, described sample
Algoscopy based on cell is used including cell or method.In another embodiment, described cell be select free MELK or
EIF4B amplification or any cancer of overexpression, any cancer having MELK or eIF4B Activating mutations and MELK or eIF4B quilt
Cancerous cell in the group of any cancer composition of other kinase activations.In another embodiment, sample choosing is freely organized, entirely
Blood, serum, blood plasma, cheek are scraped, saliva, cerebrospinal fluid, urine, feces and bone marrow composition group.In another embodiment, step
Rapid same sample a) and/or in step b) or test sample are taken from a part for the single sample of experimenter.At another
In embodiment, what same sample in step a) and/or step b) or test sample were taken from experimenter merges the one of sample
Part.In another embodiment, during first time point and subsequent point in time, experimenter accepted treatment of cancer,
Complete treatment of cancer and/or be in from cancer alleviation.In another embodiment, described people eIF4B or its direct line are same
Source thing, and/or described people MELK or its ortholog thing, including the nucleotide sequence listed by table 1 or aminoacid sequence.At another
In embodiment, described sample is freely contacted by sample operations choosing with test agent, by the upstream letter of sample with MELK signal path
Number contact, and sample is contacted with MELK inhibitor form group.In another embodiment, test agent is little point
Son, or antibody or its Fab.In another embodiment, test agent is by the 406th serine phosphorylation
The amount of people eIF4B or the amount reduction at least 50% of corresponding phosphorylated amino acid in people's eIF4B ortholog thing.
On the other hand, it is provided that a kind of for identifying suppression people MELK or its ortholog thing kinases or the examination of carcinogenic activity
The method of agent, including: a) comprise i) people MELK or its ortholog thing and ii with reagent contact) human histone H3 or its lineal with
Source thing;And b) determine the 3rd threonine phosphorylation of reagent suppression human histone H3 or in human histone H3 ortholog thing
Corresponding phosphorylated amino acid;And/or it is same at the 10th serine phosphorylation of human histone H3 or the direct line of human histone H3
Corresponding phosphorylated amino acid in the thing of source;And/or the 11st threonine phosphorylation of human histone H3 or human histone H3's is straight
Being the ability of corresponding phosphorylated amino acid in congener, the phosphorylation wherein reduced identifies suppression people MELK or it is lineal same
Source thing kinases or the reagent of carcinogenic activity.In one embodiment, to the described in human histone H3 the 3rd threonine phosphorylation
And/or it is corresponding in the 10th serine phosphorylation and/or the 11st threonine phosphorylation, or human histone H3 ortholog thing
The suppression of phosphorylated amino acid be by with the human histone H3 and/or the 10th of the 3rd threonine phosphorylation in matched group sample
The human histone H3 of position serine phosphorylation and/or the amount of the human histone H3 of the 11st threonine phosphorylation, or human histone
In H3 ortholog thing, the amount of corresponding phosphorylated amino acid is compared and is relatively determined.In another embodiment, comparison is
Refer to, relative to relatively time point morning, the 3rd threonine phosphorylation and/or the 10th in sample after unused reagent or sample contact reagent
In the amount of the human histone H3 of position serine phosphorylation and/or the 11st threonine phosphorylation or human histone H3 ortholog thing
The amount of corresponding phosphorylated amino acid.In another embodiment, to the described in human histone H3 the 3rd threonine phosphorylation
And/or it is corresponding in the 10th serine phosphorylation and/or the 11st threonine phosphorylation or human histone H3 ortholog thing
The suppression of phosphorylated amino acid, is by by the 3rd threonine phosphorylation and/or the 10th serine phosphorylation and/or the 11st
In the amount of human histone H3 of position threonine phosphorylation or histone H 3 ortholog thing the amount of corresponding phosphorylated amino acid with
In sample, the ratio of histone H 3 or its ortholog thing total amount is compared with a control and determines.At another embodiment
In, comparison refers to, compared to unused reagent or by relatively early time point, the 3rd threonine phosphorylation in sample of sample with reagent
And/or the 10th serine phosphorylation and/or the human histone H3 ratio of the 11st threonine phosphorylation or human histone H3 straight
It it is corresponding phosphorylated amino acid ratio in congener.In another embodiment, method also includes determining mitotic
The amount of specific proteins.In another embodiment, described method also includes determining whether reagent is bonded directly to described people
Histone H 3 or its described ortholog thing, or described people MELK or the step of its described ortholog thing.Another embodiment party
In formula, the group of sample choosing freely external, in vitro and internal sample composition.In another embodiment, sample packages contains cell, example
Such as cancerous cell, (such as, cancerous cell selects free MELK or histone H 3 amplification or any cancer of overexpression, has MELK or group egg
The group that any cancer of white H3 Activating mutations and MELK or histone H 3 are formed by any cancer of other kinase activations).Separately
In one embodiment, cell is to obtain from experimenter.In another embodiment, sample choosing freely organize, whole blood, blood
Clearly, blood plasma, cheek scrape, saliva, cerebrospinal fluid, urine, feces and the group of bone marrow composition.In another embodiment, the 3rd Soviet Union's ammonia
The amount of the human histone H3 of acid phosphoric acid and/or the 10th serine phosphorylation and/or the 11st threonine phosphorylation or people's group
In albumen H3 ortholog thing, the amount of corresponding phosphorylated amino acid is to be specifically bound to the 3rd threonine phosphoric acid by use
Change or the 10th serine phosphorylation or the human histone H3 of the 11st threonine phosphorylation or corresponding phosphorylated human histone
The reagent of H3 ortholog thing carries out what immunoassay determined.In another embodiment, immunoassay are radioimmunities
Mensuration, western blot analysis, proximity ligation assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay,
Chemoluminescence method, Immunohistochemical assay, Dot blot measure or slit engram detection method.At another embodiment
In, described enzyme immunoassay is Sandwich ELISA, and it uses and specifically combines human histone H3 or corresponding
Capture antibody or the fragment of human histone H3 ortholog thing and specifically combine the 3rd threonine phosphorylation or the 10th silk
Propylhomoserin phosphorylation or the human histone H3 ortholog of the human histone H3 of the 11st threonine phosphorylation or corresponding phosphorylation
The detection antibody of thing or fragment.In another embodiment, described human histone H3 or its ortholog thing, and/or described
People MELK or its ortholog thing, including the nucleotide sequence listed by table 1 or aminoacid sequence.In another embodiment, examination
Agent is little molecule, or antibody or its Fab.In another embodiment, described reagent is by the 3rd threonine phosphorus
Amount and/or the people of the human histone H3 of acidifying and/or the 10th serine phosphorylation and/or the 11st threonine phosphorylation organize egg
In white H3 ortholog thing, the amount of corresponding phosphorylated amino acid reduces at least 50%.
On the other hand, it is provided that one is used for assessing reagent and suppresses people MELK or its ortholog thing to swash in subject
The method of the curative effect of enzymatic activity, including: a) in first time point detects experimenter's sample the 3rd threonine phosphorylation and/
Or the amount of the human histone H3 of the 10th serine phosphorylation and/or the 11st threonine phosphorylation or corresponding aminoacid phosphoric acid
The amount of the human histone H3 ortholog thing changed;B) the one or more subsequent point in time after using reagent repeat step a);
And c) amount of the human histone H3 of phosphorylation or people's group of corresponding aminoacid phosphorylation in comparison step a) and described step b)
The amount of albumen ortholog thing, wherein the 3rd threonine phosphorylation and/or the 10th serine phosphorylation and/or the 11st Soviet Union
The amount of the human histone H3 ortholog thing of the amount of the human histone H3 of propylhomoserin phosphorylation or corresponding aminoacid phosphorylation is the
One time point is higher than at least one subsequent point in time, shows reagent suppression people MELK or the kinase activity of its ortholog thing.?
In some embodiment, the 3rd threonine phosphorylation and/or the 10th serine phosphorylation and/or the 11st threonine phosphoric acid
In the amount of the human histone H3 changed or human histone H3 ortholog thing, the amount of corresponding phosphorylated amino acid passes through immunoassay
Determining, this immunoassay use and are specifically binding to the human histone H3 of the 3rd threonine phosphorylation or the 10th silk
The human histone H3 of propylhomoserin phosphorylation or the human histone H3 of the 11st threonine phosphorylation, or the people of corresponding phosphorylation organizes egg
The reagent of white H3 ortholog thing.In another embodiment, immunoassay are that radioimmunoassay, western blot are divided
Analysis, proximity ligation assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemoluminescence method, immune group
Weave chemistry algoscopy, Dot blot measure or slit engram detection method.In another embodiment, enzyme immunoassay is one
Planting Sandwich ELISA, it uses and specifically combines human histone H3 or corresponding human histone H3 ortholog thing
Capture antibody or fragment and specifically combine the 3rd threonine phosphorylation or the 10th serine phosphorylation or the 11st Soviet Union
The detection antibody of the human histone H3 ortholog thing of human histone H3 or the corresponding phosphorylation of propylhomoserin phosphorylation or fragment.?
In another embodiment, the group of sample choosing freely in vitro and internal sample composition.In another embodiment, sample packages contains
(cancerous cell selects free MELK or histone H 3 amplification or any cancer of overexpression, has MELK or histone H 3 to live cancerous cell
Change any cancer and MELK suddenlyd change or the histone H 3 group by any cancer composition of other kinase activators).Real at another
Executing in mode, sample choosing is freely organized, whole blood, serum, blood plasma, cheek are scraped, saliva, cerebrospinal fluid, urine, feces and bone marrow form
Group.In another embodiment, the sample in step a) and/or step b) is taken from of single sample of experimenter
Point.In another embodiment, what the sample in step a) and/or step b) was taken from experimenter merges one of sample
Point.In another embodiment, during first time point and subsequent point in time, experimenter has accepted treatment of cancer,
Complete treatment of cancer and/or be in from cancer remission.In another embodiment, described human histone H3 or its direct line are same
Source thing, and/or described people MELK or its ortholog thing, including the nucleotide sequence listed by table 1 or aminoacid sequence.At another
In embodiment, reagent is little molecule, or antibody or its Fab.In another embodiment, described reagent will
The human histone H3's of the 3rd threonine phosphorylation and/or the 10th serine phosphorylation and/or the 11st threonine phosphorylation
In amount or human histone H3 ortholog thing, the amount of corresponding phosphorylated amino acid reduces at least 50%.
On the other hand, it is provided that a kind of method of patient suffering from cancer for treatment, suppression is used including to experimenter
3rd threonine phosphorylation of human histone H3 and/or the 10th serine phosphorylation and/or the 11st threonine phosphorylation or
The reagent of corresponding phosphorylated amino acid in its ortholog thing, thus treatment suffers from the patient of cancer.At some embodiment
In, reagent is used with pharmaceutically acceptable dosage form.In another embodiment, reagent is little molecule, or antibody or
Its Fab.In another embodiment, reagent is bonded directly to described human histone H3 or its ortholog thing,
Or described people MELK or its ortholog thing.In another embodiment, cancer select free MELK or histone H 3 to expand or
Any cancer of overexpression, there are MELK or any cancer of histone H 3 Activating mutations and MELK or histone H 3 by its separate excitation
The group of any cancer composition of enzyme activition.In another embodiment, described human histone H3 or its ortholog thing, and/
Or described people MELK or its ortholog thing, including the nucleotide sequence listed by table 1 or aminoacid sequence.At another embodiment
In, reagent is little molecule, or antibody or its Fab.In another embodiment, described reagent is by the 3rd Soviet Union's ammonia
The amount of the human histone H3 of acid phosphoric acid and/or the 10th serine phosphorylation and/or the 11st threonine phosphorylation or people's group
In albumen H3 ortholog thing, the amount of corresponding phosphorylated amino acid reduces at least 50%.In another embodiment, described
Method also includes using one or more other anticarcinogen.
On the other hand, it is provided that a kind of for determining people MELK or its ortholog thing function or the method for activity, including:
A) the 3rd threonine phosphorylation and/or the 10th serine phosphorylation and/or the 11st threonine phosphorylation in detection sample
The amount of human histone H3 or the amount of the human histone H3 ortholog thing of corresponding aminoacid phosphorylation;B) described sample is being operated
And/or after operating same sample or test sample, the one or more follow-up times in described same sample or test sample
Point repeats step a);And c) amount of the human histone H3 of phosphorylation or corresponding aminoacid in comparison step a) and described step b)
The amount of the human histone H3 ortholog thing of phosphorylation, wherein with at least one subsequent point in time and/or to same sample or survey
Sample subsequent operation the most at least one times is compared, at the 3rd threonine phosphorylation of first time point and/or the 10th serine phosphorylation
The amount of the human histone H3 of change and/or the 11st threonine phosphorylation or the human histone H3 of corresponding aminoacid phosphorylation are lineal
Congener amount change, show people MELK or its ortholog thing function or activity are regulated.Some embodiment party
In formula, the people of the 3rd threonine phosphorylation and/or the 10th serine phosphorylation and/or the 11st threonine phosphorylation organizes egg
In the amount of white H3 or human histone H3 ortholog thing, the amount of corresponding phosphorylated amino acid is determined by immunoassay, this
Immunoassay use and are specifically binding to the 3rd threonine phosphorylation or the 10th serine phosphorylation or the 11st threonine
The reagent of the human histone H3 ortholog thing of human histone H3 or the corresponding phosphorylation of phosphorylation.Another embodiment party
In formula, immunoassay are that radioimmunoassay, western blot analysis, proximity ligation assay, immunofluorescence assay, enzyme are exempted from
Epidemic disease algoscopy, immunoprecipitation assay, chemoluminescence method, Immunohistochemical assay, Dot blot measure or slit engram
Detection method.In another embodiment, enzyme immunoassay is a kind of Sandwich ELISA, and it uses specifically
In conjunction with capture antibody or the fragment of human histone H3 or corresponding human histone H3 ortholog thing with specifically combine the 3rd
Threonine phosphorylation or the 10th serine phosphorylation or the human histone H3 of the 11st threonine phosphorylation or corresponding phosphoric acid
The detection antibody of the human histone H3 ortholog thing changed or fragment.In another embodiment, sample choosing freely the most external, from
Body and the group of internal sample composition.In another embodiment, described sample includes that cell or described method use based on carefully
The algoscopy of born of the same parents.In another embodiment, cell is to select free MELK or histone H 3 to expand or overexpression any
Cancer, there are MELK or any cancer of histone H 3 Activating mutations and MELK or histone H 3 by any cancer of other kinase activators
Cancerous cell in the group of disease composition.In another embodiment, described sample choosing freely organize, whole blood, serum, blood plasma, cheek
Scrape, saliva, cerebrospinal fluid, urine, feces and bone marrow composition group.In another embodiment, step a) and/or step b)
In same sample or test sample be taken from the part of single sample of experimenter.In another embodiment, step
A) same sample and/or in step b) or test sample are taken from the part merging sample of experimenter.Real at another
Executing in mode, during first time point and subsequent point in time, experimenter has accepted treatment of cancer, has completed treatment of cancer
And/or it is in from cancer alleviation.In another embodiment, described human histone H3 or its ortholog thing, and/or institute
State people MELK or its ortholog thing, including the nucleotide sequence listed by table 1 or aminoacid sequence.In another embodiment,
Described sample is freely contacted by the operation choosing of sample with test agent, the stream signal of sample with MELK signal path is contacted, with
And sample is contacted with MELK inhibitor the group of composition.In another embodiment, test agent is little molecule, or antibody or its
Fab.In another embodiment, test agent is by the 3rd threonine phosphorylation and/or the 10th serine phosphorus
Corresponding phosphoric acid in the amount of the human histone H3 of acidifying and/or the 11st threonine phosphorylation or human histone H3 ortholog thing
Change amino acid whose amount and reduce at least 50%.
It is also understood that, according to the retrievable knowledge of those skilled in the art, the particular implementation of the application can be with this
More than one method described in literary composition is used along.
Accompanying drawing is sketched
Fig. 1 shows the interaction of MELK Yu eIF4B.Flag-MELK is expressed at MDA-MB-468 cell conditional.Will
Mitosis lysate carries out anti-Flag co-immunoprecipitation, carries out Tandem Mass Spectrometry Analysis subsequently.Left figure shows from immunoprecipitation
The peptide of middle recovery amount.Right figure shows the checking of the interphase interaction of mitosis period MELK and eIF4B.Need note
Meaning be Flag-MELK be to carry out doxycycline induction.
Fig. 2 shows the result of the peptide library selection of the optimal substrate motif for determining MELK.Upper drawing shows use weight
Group total length MELK carries out the phosphorylation in vitro result in steric peptide storehouse.Every kind of peptide comprises one and is fixed on relative to described
The residue of one of 9 positions of phosphate acceptors (such as, serine or threonine) that central authorities are fixing.By reactant point sample to film
On, and described point is exposed to fluorophor storage screen.Bottom panel show to use and quantify and the generation of standardized on-screen data
Sequence identifier.It should be noted that MELK is to having strong selectivity relative to the arginine of phosphoacceptor site-3.
Fig. 3 shows that MELK is in vitro by the 406th of eIF4B the serine phosphorylation.With the Flag-of immunoprecipitation
EIF4B (wild type) or Flag-eIF4B (S406A) carries out vitro kinase to the kinase domain of recombinant full-lenght MELK or MELK
Measure.The strong phosphorylation of the 406th serine of eIF4B can be observed in the presence of MELK.When with sudden change eIF4B
(S406A) when replacing wild type (wt) eIF4B, this phosphorylation disappears.
Fig. 4 shows that MELK can not be by the 422nd of eIF4B the phosphorylation in vitro.With the Flag-eIF4B of immunoprecipitation
(wild type) or Flag-eIF4B (S422A) carry out vitro kinase mensuration to the kinase domain of recombinant full-lenght MELK or MELK.
By immunoblotting, reaction is analyzed.
Fig. 5 shows that MELK regulates the 406th serine phosphorylation of eIF4B in vivo.Left figure shows and knocks out MELK
Compromise the phosphorylation of the 406th serine of eIF4B.When doxycycline presence or absence, by using at nocodazole
Reason collect that MDA-MB-468 cytotostatic expresses can the bobby pin MELK (shMELK) of tetracycline induction (tet-on), and carry out
Immunoblotting.Right figure shows the phosphorylation of the 406th serine of MELK inhibitor infringement eIF4B.Use solvent or 200nM
OTSSP167 (a kind of MELK inhibitor) (Chung et al. (2012) Oncotarget 3:1629-1640) process and have silk
The MDA-MB-468 cell of division 30 minutes.Lysate is used for immunoblotting.
Fig. 6 shows that use mTOR inhibitors (such as, rapamycin and Torin 1) processes mitotic cell 30 minutes
With the comparative result using MELK inhibitor (such as, OTSSP67) to process these cells.Result show MELK suppression rather than
MTOR suppresses, the phosphorylation of the 406th serine of suppression eIF4B.
Fig. 7 shows that knocking out MELK or eIF4B when mitosis reduces the protein abundance of XIAP, c-Myc and ODC1.With
Doxycycline or Vehicle controls process MDA-MB-468 and MDA-MB-231 stably expressing tet-on shMELK or sh-eIF4B
Cell.The retardance induced by nocodazole obtains the mitotic cell at prometaphase.It should be noted that XIAP, c-
The mRNA of Myc and ODC1 has been demonstrated containing structural 5'UTR, and their aggregate level keeps constant.
Fig. 8 shows that MELK knocks out the fluorescein driven during reducing mitosis by the 5 ' UTR of c-Myc or ODC1
The translation of enzyme.The bi-cistronic vectors being instructed to is transfected when doxycycline presence or absence and stably expresses tet-on
The MDA-MB-231 cell of shMELK.In transfection two days later, gather in the crops the mitotic cell of nocodazole retardance and carry out fluorescence
Element enzymatic determination.Ratio (RL/FL) with the value standardization Renilla luciferase of control vector Yu Fluc.Need note
Meaning, in every pair of columnar body shown in the figure showing relative RL/FL ratio, left side column corresponds to Dox
(-), right side column corresponding to Dox (+).
Fig. 9 shows that MELK is in vitro by the 3rd of human histone H3 the threonine, the 10th serine and the 11st Soviet Union's ammonia
Acid phosphoric acid.Recombined human histone H 3 exists at ATP in the case of restructuring MELK (kinase domain) presence or absence
In the case of 30 DEG C hatch 30 minutes.Reaction is terminated by adding SDS sample buffer.Then use instruction antibody that sample is carried out
Immunoblotting.
Figure 10 shows that MELK knocks out and reduces the 3rd threonine of histone H 3, the 10th serine and the 11st Soviet Union
Propylhomoserin rather than the mitotic phosphorylation of the 28th serine.Stably the MDA-MB-468 of transduction tet-on shMELK is thin
Born of the same parents, use or do not use doxycycline (200ng/ml) to process induced gene silence.Then by cell nocodazole
(200ng/ml) 20 hours are processed.Mitotic cell is obtained by the method for shaking to cause drop (shake-off), and cell lysate is by using
Instruction antibody carries out immunoblotting.
Figure 11 shows MELK to knock out not affects the phosphorylation of aurora kinase, it is known that aurora kinase is the of human histone H3
The kinases of 10 serines.Stably the MDA-MB-468 cell of transduction tet-on shMELK uses or does not use doxycycline
(200ng/ml) induced gene is processed reticent.Cell nocodazole (200ng/ml) processes 20 hours.Mitotic cell
Being obtained by the method for shaking to cause drop (shake-off), cell lysate carries out immunoblotting by using instruction antibody.
Figure 12 shows the phosphoric acid of the 10th serine of the histone H 3 that MELK inhibitor suppresses MELK to induce in vitro
Change.Restructuring histone H 3 restructuring MELK (kinase domain) do not exist or in the presence of, at OTSSP167 (200ng/
Ml final concentration) in the case of presence or absence 30 DEG C cultivate 30 minutes.Reaction is terminated by adding SDS sample buffer.Then
Sample uses instruction antibody to carry out immunoblotting.
Figure 13 show the inhibitory action to MELK inhibit the 3rd threonine of histone H 3, the 10th serine and
11st threonine rather than the mitotic phosphorylation of the 28th serine.By nocodazole, (200ng/ml, 20 is little
Time) cell cycle arrest induced obtains the mitotic cell at prometaphase.By the chemical small molecule inhibitor of MELK
OTSSP167 processes cell 30 minutes with the concentration of instruction.Then cell lysate is prepared for immunoblotting.
Detailed Description Of The Invention
The present processes relates to following beyond thought discovery, the 406th of human eukaryote initiation factor 4B (eIF4B)
The phosphorylation level of (e.g., serine residue), or the corresponding phosphorylated amino acid of its ortholog thing, can serve as MELK enzyme
(such as kinases) activity and/or the biomarker of carcinogenic activity.Specifically, the phosphorus of the 406th serine of such as eIF4B
Acidifying reduces (such as, the 406th serine phosphorylation by suppressing MELK to mediate directly or indirectly), with reduction MELK enzyme
Activity (such as, kinase activity) is corresponding with the carcinogenic effect that MELK mediates.Such biomarker is for before clinic and face
Bed application is particularly advantageous, because the phosphorylation state of eIF4B itself is directly related with MELK proto-oncogene.Similar
Ground, the present processes is directed to following beyond thought discovery, the 10th (e.g., the serine residue) of human histone H3 or its
The phosphorylation level of the corresponding phosphorylated amino acid of ortholog thing, and/or the 11st (e.g., the threonine of human histone H3
Residue) or the phosphorylation level of corresponding phosphorylated amino acid of its ortholog thing, can serve as MELK enzyme (such as kinases)
Activity and/or the biomarker of carcinogenic activity.Specifically, the 3rd threonine phosphorylation of such as human histone H3 reduces (example
As, the 3rd threonine phosphorylation by suppressing MELK to mediate directly or indirectly) and/or the 10th silk of human histone H3
Propylhomoserin phosphorylation reduces (such as, by the 10th serine phosphorylation suppressing MELK to mediate directly or indirectly) and/or people
11st threonine phosphorylation of histone H 3 reduces (such as, by 11 the Soviet Union's ammonia suppressing MELK to mediate directly or indirectly
Acid phosphoric acid), corresponding with the carcinogenic effect that MELK mediates with the MELK enzymatic activity (such as, kinase activity) reduced.Such
Biomarker is for before clinic and clinical practice is particularly advantageous, because the phosphorylation state of histone H 3 and the former cancer of MELK
Gene itself is directly related.In some embodiments, can consider to make the 406th of employment eIF4B according to the application
Serine, the 3rd threonine of human histone H3, the 10th serine and/or the 11st of human histone H3 the of human histone H3
Position threonine, and the corresponding phosphorylated amino acid of its ortholog thing any, including its combination in any.Enforcement at other
In mode, it is not subject to according to the application, the 28th serine of people eIF4B or the corresponding phosphorylated amino acid of its ortholog thing
MELK regulates, and does not the most use it.
A.MELK, eIF4B and histone H 3 molecule
MELK member in " MELK " used herein finger protein kinases superfamily, also referred to as " PEG3 kinases ", " albumen
Kinases Eg3 ", " protein kinase " and " serine/threonine-protein kinase MELK ".At least there are nine kinds of splicing variants codings
Nine kinds of different people's MELK hypotypes.The people MELK hypotype 1 (NP_ that people's MELK transcript variant 1 (NM_014791.3) coding is long
55606.1).People's MELK transcript variant 2 (NM_001256685.1) lacks one in 3 ' coding regions compared with transcript variant 1
Exon, but remain reading frame and resulted in the hypotype (NP_001243614.1) shorter than hypotype 1.People MELK transcribes
Variant 3 (NM_001256687.1) lacks an exon compared with transcript variant 1 in 5 ' coding regions, but remains and read
Frame has also resulted in the hypotype (NP_001243616.1) shorter than hypotype 1.People MELK transcript variant 4 (NM_
001256688.1) lack two continuous print exons compared with transcript variant 1 in 5 ' coding regions, but remain reading frame
And resulted in the hypotype (NP_001243617.1) shorter than hypotype 1.People's MELK transcript variant 5 (NM_001256689.1)
Start translation from another start codon, lack an exon compared with transcript variant 1 in 5 ' coding regions, thus cause
Create hypotype (NP_001243618.1) that is shorter than hypotype 1 and that have different N-ends.People MELK transcript variant 6 (NM_
001256690.1) start translation from another start codon, lack two companies compared with transcript variant 1 in 5 ' coding regions
Continuous exon, but remain reading frame thus result in hypotype (NP_ that is shorter than hypotype 1 and that have different N-ends
001243619.1).People's MELK transcript variant 7 (NM_001256691.1) from another start codon start translation, with turn
Record variant 1 is compared and is lacked two exons in 5 ' coding regions, but remains reading frame thus resulted in shorter than hypotype 1
And have different N-end hypotype (NP_001243620.1).People's MELK transcript variant 8 (NM_001256692.1) with transcribe change
Allosome 1 compare lack in 5 ' coding regions three exons and downstream the start codon in frame start translation, result in
The hypotype (NP_001243621.1) shorter than the N-end of hypotype 1.Finally, people MELK transcript variant 9 (NM_001256693.1)
Lack in 5 ' coding regions compared with transcript variant 1 two continuous print exons and downstream the start codon in frame start to turn over
Translate, resulted in the hypotype (NP_001243622.1) shorter than the N-end of hypotype 1.Regulate MELK autophosphorylation and at target egg
The protein structure domain of the activation of white upper kinase activity and architecture basics are known (to refer at least Cao et al. (2013) PLoS
One 8:e70031 and Canevariet al. (2013) Biochemistry 52:6380-6387).
Mice MELK nucleic acid (NM_010790.2) and aminoacid (NP_034920.2) sequence can be from American National biology skills
The GenBank data base that art information centre safeguards is obtained by open channel.Straight at other species MELK in addition to mice and people
Be nucleic acid and the peptide sequence of congener be also known, including, such as chimpanzee MELK (XM_001169038.3, XP_
001169038.1,XM_001168991.3,XP_001168991.1,XM_001168745.3,XP_001168745.1,XM_
001168775.3,XP_001168775.1,XM_003951427.1,XP_003951476.1,XM_520578.4,XP_
520578.3,XM_001168822.3,XP_001168822.2,XM_003312085.2,XP_003312133.1,XM_
003951428.1, XP_003951477.1, XM_003312086.2, and XP_003312134.1), monkey MELK (XM_
001115076.2 and XP_001115076.2), Canis familiaris L. MELK (XM_003431578.1, XP_003431626.1, XM_
538730.3, XP_538730.2, XM_003431579.1, and XP_003431627.1), cattle MELK (NM_001111260.1 and
NP_001104730.1), rat MELK (NM_001108662.1 and NP_001102132.1), chicken MELK (NM_
001031509.1 and NP_001026680.1) and Brachydanio rerio MELK (NM_206888.2 and NP_996771.2).
" eIF4B " used herein refers to eukaryotic translation initiation factor 4B member in eukaryotic translation initiation factor family, also
It is referred to as " EIF-4B " and " PRO1843 ".People's eIF4B nucleic acid (NM_001417.4) and aminoacid (NP_001408.2) sequence can
Obtained by open channel from the GenBank data base of American National Biotechnology Information central service.At other in addition to people
Nucleic acid and the peptide sequence of species eIF4B ortholog thing are also known, such as mice eIF4B (NM_145625.3 and NP_
663600.2), chimpanzee eIF4B (XM_003313676.1, XP_003313724.1, XM_001142097.3, and XP_
001142097.3), monkey eIF4B (NM_001195808.1 and NP_001182737.1), Canis familiaris L. eIF4B (XM_853888.2, XP_
858981.2, XM_853812.2 and XP_858905.2), cattle eIF4B (NM_001035028.2 and NP_001030200.1), big
Mus eIF4B (NM_001008324.1 and NP_001008325.1) and chicken eIF4B (XM_003643408.2 and XP_
003643456.2).It addition, " the 406th serine " of eIF4B refers to the amino acid number of people eIF4B.Therefore, art technology
Personnel will readily appreciate that the 406th serine of people's eIF4B polypeptide is conservative in numerous species, although these are the most residual
Base may be cited in this article, but the present processes is equally applicable to corresponding with the 406th of described people eIF4B the serine
The hypotype of other species, congener and ortholog thing in corresponding residue (such as, the aminoacid of phosphorylation).
" Histone H3 " used herein refers to the H3 member of histone family, and it includes for forming eukaryotic cell core
The albumen of minibody structure.There is the chromatin being centered around around protein with the form of nucleosome in eukaryotic cell, this is chromatin
Minimum subunit and include being wrapped in around eight aggressiveness of core histones about 146-147 DNA base to (two H2A,
H2B, H3 and H4).Mammalian cell has 3 known histone H 3 sequence variants, be expressed as H3.1, H3.2 and
H3.3, these three sequence variant high conservative, difference is only at several aminoacid sequences.Histone H 3 in many cell processes
The post translational modification of residue critically important, the 10th serine and/or the 28th serine phosphorylation are to cell division and propagation
Regulate and control critically important.The histone H 3 of the 10th serine phosphorylation is biomarker-specific thing for mitotic cell, with
Mitosis Idiotype biomarker known to other is similar to, the MPM-2 of such as phosphorylation, phosphorylation retinoblastoma
Albumen 1 (Rb), phosphorylation cdc2, BubR1, cell periodic protein B 1, cdc25c, cdk1, cdc27 and analog etc..Any silk
Propylhomoserin, threonine or tyrosine residue can be phosphorylated.In some embodiments, other positions that may be phosphorylated
Point includes the 3rd threonine, the 6th threonine, the 11st threonine, the 31st serine, the 41st tyrosine, the 57th silk
Propylhomoserin, the 80th threonine and the 107th threonine.
" Histone H3 " used herein both can refer to single H3.1, H3.2 or H3, it is also possible to refers to all of which.
These aminoacid sequences include a methionine as No. 1 residue, when protein is processed, it is by cut.Therefore,
Such as, the 10th serine (Ser) in the 11st serine correspondence the application in histone aminoacid sequence in table 1 below.
These three protein variants is to be encoded by least 15 kinds of different genes/transcripton.The sequence of encoding histone H3.1 variant
Row can be obtained by open approach, HIST1H3A (NM_003529.2;NP_003520.1)、HIST1H3B(NM_
003537.3;NP_003528.1)、HIST1H3C(NM_003531.2;NP_003522.1)、HIST1H3D(NM_003530.3;
NP_003521.2)、HIST1H3E(NM_003532.2;NP_003523.1)、HIST1H3F(NM_021018.2;NP_
066298.1)、HIST1H3G(NM_003534.2;NP_003525.1),HIST1H3H(NM_003536.2;NP_
003527.1)、HIST1H3I(NM_003533.2;And HIST1H3J (NM_003535.2 NP_003524.1);NP_
003526.1).The sequence of encoding histone H3.2 variant can be obtained by open approach, HIST2H3A (NM_
001005464.2;NP_001005464.1)、HIST2H3C(NM_021059.2;And HIST2H3D (NM_ NP_066403.2)
001123375.1;NP_001116847.1).The sequence of encoding histone H3.3 variant can be obtained by open approach,
H3F3A(NM_002107.3;And H3F3B (NM_005324.3 NP_002098.1);NP_005315.1).Specifying information refers to
U.S. patent publication No. 2012/0202843.Additionally, the peptide sequence of histone H 3 ortholog thing, and encode these polypeptide
Nucleotide sequence, be known in many species, including, such as, histone H 3 .1 ortholog thing (NM_ in mice
013550.4;NP_038578.2), chimpanzee (XM_527253.4;XP_527253.2), monkey (XM_001088298.2;XP_
001088298.1), Canis familiaris L. (XM_003434195.1;XP_003434243.1), cattle (XM_002697460.1;XP_
002697506.1), rat (XM_001055231.2;And Brachydanio rerio (NM_001100173.1 XP_001055231.1);NP_
001093643.1).Histone H 3 .2 ortholog thing (NM_178215.1 in mice;NP_835587.1), chimpanzee (XM_
524859.4;XP_524859.2), monkey (XM_001084245.2;XP_001084245.1), Canis familiaris L. (XM_003640147.1;XP_
003640195.1), cattle (XM_002685500.1;XP_002685546.1), rat (NM_001107698.1;NP_
001101168.1), chicken (XM_001233027.2;And Brachydanio rerio (XM_002662732.1 XP_001233028.1);XP_
002662778.1).Similarly, histone H 3 .3 ortholog thing (XM_892026.4 in mice;XP_897119.3), monkey
(XM_001085836.2;XP_001085836.1), cattle (NM_001099370.1;NP_001092840.1), rat (NM_
053985.2;NP_446437.1), chicken (NM_205296.1;And Brachydanio rerio (NM_200003.1 NP_990627.1);NP_
956297.1) it is known.Histone H 3 (such as, the 3rd threonine, the 10th serine, the 11st Soviet Union of detection phosphorylation
Other residues of propylhomoserin and histone H 3) antibody of phosphorylation and to prepare the method for these antibody be known in this field
's.It addition, such as, histone H 3 " Ser-10 " refers to the amino acid number of human histone H3.Therefore, those skilled in the art will hold
10th serine of readily understood human histone H3 polypeptide is conservative in numerous species, although these specific residues are at this
Literary composition may be cited, but the present processes is equally applicable to the Asia of the 10th serine corresponding to described human histone H3
The corresponding residue (such as, the aminoacid of phosphorylation) of the ortholog thing of type, congener and other species.3rd threonine
Equally applicable with the 11st threonine.
The invention provides representational MELK, eIF4B and Histone H3 ortholog thing (such as, at least at table 1
With in embodiment) as follows:
Table 1
People MELK (hypotype 1) cDNA sequence (NM_014791.3)
People MELK (hypotype 1) aminoacid sequence (NP_55606.1)
People MELK (hypotype 2) cDNA sequence (NM_001256685.1)
People MELK (hypotype 2) aminoacid sequence (NP_001243614.1)
People MELK (hypotype 3) cDNA sequence (NM_001256687.1)
People MELK (hypotype 3) aminoacid sequence (NP_001243616.1)
People MELK (hypotype 4) cDNA sequence (NM_001256688.1)
People MELK (hypotype 4) aminoacid sequence (NP_001243617.1)
People MELK (hypotype 5) cDNA sequence (NM_001256689.1)
People MELK (hypotype 5) aminoacid sequence (NP_001243618.1)
People MELK (hypotype 6) cDNA sequence (NM_001256690.1)
People MELK (hypotype 6) aminoacid sequence (NP_001243619.1)
People MELK (hypotype 7) cDNA sequence (NM_001256691.1)
People MELK (hypotype 7) aminoacid sequence (NP_001243620.1)
People MELK (hypotype 8) cDNA sequence (NM_001256692.1)
People MELK (hypotype 8) aminoacid sequence (NP_001243621.1)
People MELK (hypotype 9) cDNA sequence (NM_001256693.1)
People MELK (hypotype 9) aminoacid sequence (NP_001243622.1)
Mice MELK cDNA sequence (NM_010790.2)
Mice MELK aminoacid sequence (NP_034920.2)
People's eIF4B cDNA sequence (NM_001417.4)
People's eIF4B aminoacid sequence (NP_001408.2)
Mice eIF4B cDNA sequence (NM_145625.3)
Mice eIF4B aminoacid sequence (NP_663600.2)
Monkey eIF4B cDNA sequence (NM_001195808.1)
Monkey eIF4B aminoacid sequence (NP_001182737.1)
Cattle eIF4B cDNA sequence (NM_001035028.2)
Cattle eIF4B aminoacid sequence (NP_001030200.1)
Rat eIF4B cDNA sequence (NM_001008324.1)
Rat eIF4B aminoacid sequence (NP_001008325.1)
Human histone H3.1 aminoacid sequence (NP_003520.1)
1 martkqtark stggkaprkq latkaarksa patggvkkph ryrpgtvalr eirryqkste
61 llirklpfqr lvreiaqdfk tdlrfqssav malqeaceay lvglfedtnl caihakrvti
121 mpkdiqlarr irgera
Mice histone H 3 .1 aminoacid sequence (NP_038578.2):
1 martkqtark stggkaprkq latkaarksa patggvkkph ryrpgtvalr eirryqkste
61 llirklpfqr lvreiaqdfk tdlrfqssav malqeaceay lvglfedtnl caihakrvti
121 mpkdiqlarr irgera
Human histone H3.2 aminoacid sequence (NP_001005464.1):
1 martkqtark stggkaprkq latkaarksa patggvkkph ryrpgtvalr eirryqkste
61 llirklpfqr lvreiaqdfk tdlrfqssav malqeaseay lvglfedtnl caihakrvti
121 mpkdiqlarr irgera
Mice histone H 3 .2 aminoacid sequence (NP_835587.1):
1 martkqtark stggkaprkq latkaarksa patggvkkph ryrpgtvalr eirryqkste
61 llirklpfqr lvreiaqdfk tdlrfqssav malqeaseay lvglfedtnl caihakrvti
121 mpkdiqlarr irgera
Human histone H3.3 aminoacid sequence (NP_002098.1):
1 martkqtark stggkaprkq latkaarksa pstggvkkph ryrpgtvalr eirryqkste
61 llirklpfqr lvreiaqdfk tdlrfqsaai galqeaseay lvglfedtnl caihakrvti
121 mpkdiqlarr irgera
Mice histone H 3 .3 aminoacid sequence (NP_032237.1):
1 martkqtark stggkaprkq latkaarksa pstggvkkph ryrpgtvalr eirryqkste
61 llirklpfqr lvreiaqdfk tdlrfqsaai galqeaseay lvglfedtnl caihakrvti
121 mpkdiqlarr irgera
Due to the degeneracy of genetic code or owing to encoding or there is " inessential ", " guarding ", " stereoisomer " or " non-
Conventional " aminoacid and different nucleic acid and protein molecule (such as, MELK, eIF4B of different plant species and their direct line
Congener) will not substantially change enzyme (such as kinases) regulating power of MELK and/or the tune of the 406th serine to eIF4B
Energy-conservation power, within therefore these are also included within scope of the present application." conserved amino acid replacement " refers to that amino acid residue is replaced by
There is the amino acid residue of similar side chain.The stereoisomer (such as, D-aminoacid) of 20 kinds of conventional amino acid, non-natural ammonia
Base acid such as α, α-disubstituted aminoacid, N-alkyl amino acid, lactic acid and other unconventional aminoacid can also be herein
The suitable ingredients of described polypeptide.Exist between aminoacid sequence and the nucleotide sequence of coded protein of specific protein known and
Clear and definite corresponding relation, this correspondence is defined (as follows) by genetic code.Similarly, the nucleotide sequence of specific nucleic acid
There is known and clear and definite corresponding relation with by the aminoacid sequence of this nucleic acid coding, this correspondence is defined by genetic code.
Genetic code important and well-known feature be its degeneracy, be used for manufacturing egg accordingly, for great majority
The aminoacid of white matter, can use more than one triplet coding nucleotide (such as, as shown above).Therefore, many different
Nucleotide sequence can encode given aminoacid sequence.These nucleotide sequences are because produce identical in all organisms
Aminoacid sequence and be considered as function equivalence (although some organism relative to translate other nucleotide sequences can be more
Effectively translate a part of nucleotide sequence).Additionally, once in a while, it appeared that purine or pyrimidine in given nucleotide sequence
The variant that methylates.This coding not affected between triplet nucleotide codon and corresponding aminoacid that methylates closes
System.Additionally, skilled artisan will appreciate that the nucleotide of specific cryptosystem that how to suddenly change specifically to change based on relevant
The aminoacid of password sublist coding.Other required nucleic acid and/or amino acid modified site-directed mutation and PCR can be used to be situated between
The induced-mutation technique led carries out through engineering approaches.
" nucleic acid " can be to take multiple coding biomarker described herein form (such as, DNA, mRNA and cDNA)
Any one.Such as, these biomarker nucleic acid include DNA (such as, genomic DNA and cDNA), and it comprises required gene
Sequence all or in part, or the complementary series of these sequences or hybridized fragment.Biomarker nucleic acid molecules also includes RNA,
It comprises the sequence all or in part of required gene, or the complementary series of described sequence, the most all of thymine residue quilt
Uracil residues replaces." polynucleotide transcribed " are a kind of polynucleotide (such as, RNA, cDNA, or RNA's or cDNA is similar
Thing), its biomarker at least partially and in the application transcribes all or part of complementary or same of the mature rna of generation
Source, and transcribe normally post-treatment (such as, montage, if any) and the reverse transcription of transcripton.
As exchanged term " homology " or " homogeny " of use herein, refer to two polynucleotide sequences or more than two
Sequence similarity between peptide sequence, and homogeny is tightened up comparison.Phrase " homogeny or percent homology " and " phase
The same sex or homology % " refer to when two or more polynucleotide sequences or two or more peptide sequence compare, sequence
Similarity percentage.Two or more sequence similarity values are in optional position interval for 0-100%, or 0-100% is interval
Any integer value.Homogeny or similarity can be by comparing for comparative purposes and the position of each sequence arranged is come really
Fixed.When being occupied by identical nucleotide base or aminoacid by the position in comparative sequences, then described molecule in this position is
Identical.Similarity between polynucleotide sequence or the degree of homogeny be identical on the position that polynucleotide sequence is total or
The function of the amount of the nucleotide of coupling.The homogeny degree of peptide sequence is the same amino acid of the position being had by peptide sequence
The function of amount.Homology between peptide sequence or the degree of similarity are the amino acid whose of the total position of peptide sequence
The function of amount.Term " basic homology " refer at least 50% homology, at least 60% homology, at least 65% homology, at least 70% with
Source, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology or more (example
As, about 96% homology, 96.5% homology, 97% homology, 97.5% homology, 98% homology, 98.5% homology, 99% homology,
99.5% homology, 99.6% homology, 99.7% homology, 99.8% homology, 99.9% homology or more).Some embodiment party
In formula, biomarker nucleic acid molecule encoding comprises protein or its part of aminoacid sequence, this aminoacid sequence and dimension
Hold such as, the ability of phosphorylation eIF4B, phosphated lanolin and/or by the protein of the ability of MELK phosphorylation or its portion
The enough homologies of aminoacid sequence divided.
Mathematical algorithm can be used to carry out comparative sequences and measure the percent homology of two sequences.Can use
Clustal method is compared.Multiple alignment parameters includes GAP point penalty=10, Gap LENGTH PENALTY=10.DNA is contrasted, joins
Can be Htuple=2, Gap point penalty=5 to alignment parameters, Window=4 and Diagonal saved=4.For protein
Contrast, pairing alignment parameters can be Ktuple=1, Gap point penalty=3, Window=5 and Diagonals Saved=5.Phase
As, homogeny percentage ratio Needleman and Wunsch algorithm between two aminoacid sequences determine
(J.Mol.Biol. (48): 444-453 (1970)), it has been included in the GAP program (providing online) of GCG software kit, has used
Blossom 62 matrix or PAM250 matrix, Gap Weight is 16,14,12,10,8,6 or 4, Length Weight 1,2,3,4,5 or 6.
In another embodiment, the homogeny percentage ratio between two nucleotide sequences be with the GAP program of GCG software kit (
Line provides) determine, use NWSgapdna.CMP matrix and Gap Weight be 40,50,60,70 or 80 and Length Weight 1,2,
3,4,5 or 6.In another embodiment, the homogeny percentage ratio between two aminoacid or nucleotide sequence is to use
E.Meyers and W.Miller (CABIOS, 4:11-17 (1989)) algorithm determines, it has been merged in ALIGN program (2.0 editions)
(providing online), uses PAM120 weight residue table, and gap LENGTH PENALTY is 12, and gap point penalty is 4.
Prepare nucleic acid method (MELK that such as, is known in the art being translated from there is the nucleic acid in structural 5th ' district,
EIF4B and/or mRNA, including standard hybridization, PCR and/or nucleic acid technology.The nucleic acid of amplification can be cloned into suitably
Carrier, and characterized by gene sequencing.
" biomarker protein " is the biomarker coding by the application or corresponding protein.Term " egg
White matter " and " polypeptide " be used interchangeably herein.In one embodiment, described protein and MELK described herein and/
Or eIF4B and/or whole aminoacid sequence at least 50%, 60%, 70%, 80%, 90% and 95% of histone H 3 protein
Or more (such as, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%,
99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more) homology.Additionally, MELK described herein
And/or the biologically-active moiety of eIF4B and/or histone H 3 protein is all contained in wherein, with MELK as herein described and/or
EIF4B and/or histone H 3 protein fragments (such as domain or motif, this fragment energy phosphorylation eIF4B, phosphorylation group egg
White H3 and/or by MELK phosphorylation) have at least 50%, 60%, 70%, 80%, 90% and 95% or more (such as,
95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,
99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more than) homology.Typically, biologically-active moiety (peptide, example
As, have 5,10,15,20,30,35,36,37,38,39,40,50,100,150,200,250,300,350,400,450 or more
The peptide of amino acid length) comprise a domain or motif, such as MELK kinase domain, comprise by the amino of MELK phosphorylation
The eIF4B domain of acid residue, the phosphorylated substrate motif of such as MELK mediation, it is relative to serine/threonine (people
406th serine of eIF4B or the 422nd serine) there is an arginine at-3 or comprise by the ammonia of MELK phosphorylation
The histone H 3 domain of base acid residue, such as, comprise the human histone H3 district of the 10th serine.It addition, other biological is active
Part, other regions disappearance of wherein said protein, can be prepared by recombinant technique and by one specifically described herein
Or various active assesses.
The method preparing protein (such as MELK and/or eIF4B and/or histone H 3) is known in the art, including example
As, prepare from cDNA from the beginning marking protein or by being subsequently added aminoacid initial amino acid in suitable cell
(referring to Current Protocols, John Wiley&Sons, Inc., New York).Additionally, prepare the side of protein fragments
Method is known in the art, including with suitable protease cracking protein or produce coded protein fragment nucleic acid fragment
And in suitable cell this nucleic acid fragment of follow-up expression.The method preparing mutein, such as, by exchange or/and delete
Except one or more aminoacid, it is known in the art.
B. diagnostic method
The method providing identification reagent (such as little molecule and antibody), described reagent suppression people MELK or its ortholog thing
Carcinogenic activity and/or kinase activity, including: a) with reagent contact containing i) people MELK or its ortholog thing and ii) people's eucaryon
Initiation factor 4B (eIF4B) or the sample of its ortholog thing;B) measure the 406th of described reagent suppression people eIF4B
The ability of corresponding aminoacid phosphorylation in serine phosphorylation or its ortholog thing, wherein, phosphorylation reduction identifies suppression
People MELK or the kinases of its ortholog thing or the reagent of carcinogenic activity.Similarly, it is provided that identification reagent is (if little molecule is with anti-
Body) method, described reagent suppression people MELK or the carcinogenic activity of its ortholog thing and/or kinase activity, including: a) with examination
Agent contact is containing i) people MELK or its ortholog thing and ii) human histone H3 or the sample of its ortholog thing;B) survey
Determine reagent suppression the 3rd threonine of human histone H3 and/or the 10th serine and/or the 11st threonine phosphorylation or it is straight
Being the ability of corresponding aminoacid phosphorylation in congener, wherein, phosphorylation level reduction identifies suppression people MELK or it is lineal
The kinase activity of congener or the reagent of carcinogenic activity.These methods are also referred herein as drug screening assays, generally
Including screening candidate/test compound or reagent and MELK and/or eIF4B and/or histone H 3 protein interaction (example
As, in conjunction with) ability, this interaction regulates eIF4B phosphorylation, the regulation phosphorylated residue of eIF4B and MELK by MELK
The interaction of the target of the intracellular signal transduction of mediation, regulates phosphorylated histone H3, and/or regulation group egg by MELK
The interaction of the intracellular signal transduction target that the phosphorylated residue of white H3 mediates with MELK.There are one or more these energy
The test compound of power or reagent can serve as medicine, treat with distortion, abnormal and/or unwanted MELK and/or
The disease that the activity of eIF4B and/or histone H 3 expression of nucleic acid and/or protein is characterized, such as cancer.Candidate/testization
Compound includes, such as, and little organic and inorganic molecule (such as, combination and the molecule of acquisition in natural product storehouse).Similarly, anti-
Body reagent, such as, be attached to MELK, the phosphorylation driving mediated with common MELK in the way of MELK regulation residue phosphorylation
The mode of regulation eIF4B activity be attached to MELK and/or the driving regulation histone of phosphorylation mediated with common MELK
The mode of H3 activity is attached to MELK, and these reagent are all useful.Those skilled in the art also can easily prepare other and adjust
Joint reagent, such as aptamers, antisense RNA, siRNA, these reagent can be with MELK nucleic acid and/or protein interaction with impact
EIF4B or the phosphorylated histone H3 of MELK mediation (refer to, at least Chung et al. (2012) 3:1629-1640;WO
2013/109388;WO 2012/016082;WO 013/045539;It is the most all incorporated herein by).
Term " sample ", " tissue samples ", " experimenter's sample ", " subject cell or tissue samples " or " specimen ", often
Individual referring both to is organized or external (such as, the cultivation), in vitro or internal (such as, the primary cell of separation) of experimenter from experimenter
The set of the similar cellular of sample acquisition.The source of tissue samples can be from organ that is fresh, freezing and/or that preserve, group
Knit the solid tissue of sample, biopsy or aspirate;Blood or blood constituent;Body fluid, such as whole blood, serum, blood plasma, cheek
Scrape, saliva, cerebrospinal fluid, urine, feces and bone marrow, amniotic fluid, peritoneal fluid or interstitial fluid;Experimenter gestation or growth course in any
The cell of time.Tissue samples may comprise and the compound that mixes of natural fabric non-natural ground, such as preservative, anticoagulant
Agent, buffer agent, fixative, nutrient, antibiotic etc..Sample is also possible that cancerous cell, such as ovarian cancer, pulmonary carcinoma, breast carcinoma
With multiple myeloma cells or any cancer, wherein MELK and/or eIF4B and/or histone H 3 amplification or overexpression, have
One activation sudden change or by other kinase activations.
Term " experimenter " and " patient " are used interchangeably.In this article, term " experimenter " and " experimenters " refer to
Thing, such as, mammal include non-primate (such as, cattle, pig, horse, donkey, goat, camel, cat, Canis familiaris L., Cavia porcellus, rat,
Mice, sheep) and primate (such as, monkey, such as Macaca inus, gorilla, chimpanzee and people).
Term " suppresses " feeling the pulse with the finger-tip scale statistics of variables to significantly reduce, such as the 3rd threonine phosphorylation and/or the 10th
Serine phosphorylation and/or the histone H 3 of the 11st threonine phosphorylation, eIF4B, MELK of the 406th serine phosphorylation
The reduction of enzymatic activity (such as, kinase activity), tumour progression etc..This statistically significant reduce compared with matched group can be to
Few 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, such as, according to method described herein
The testing compound used and analyze can comprise the real inhibitor (such as, kinase activity) of MELK enzymatic activity, this suppression
By with do not use MELK part or beyond compared with after preset time, the amount of the eIF4B of the 406th serine phosphorylation, the 3rd
The amount of the histone H 3 of threonine phosphorylation, the amount of histone H 3 of the 10th serine phosphorylation, and/or the 11st threonine
The amount of the histone H 3 of phosphorylation reduces at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more
Many.In one embodiment, term " MELK inhibitor " is a kind of material, the least molecule, antibody, antisensenucleic acids, little dry
Disturb nucleic acid, the phosphorylation of the 406th serine of its interference people eIF4B or eIF4B406 ortholog thing corresponding phosphorylation position
Point, the phosphorylation of the 3rd threonine of human histone H3 or the human histone H3 corresponding phosphorylation site of ortholog thing, people organizes egg
The phosphorylation of the 10th serine of white H3 or the human histone H3 corresponding phosphorylation site of ortholog thing, and/or human histone
The phosphorylation of the 11st threonine of H3 or the human histone H3 corresponding phosphorylation site of ortholog thing.Exemplary MELK suppression
Agent is known in the art, and as OTSSP167, SIM-A, Bryamycin and anti-MELK antibody are disclosed, such as, exists
Chung et al.(2012)Oncotarget3:1629-1640;WO 2013/045539;WO 2013/109388;And WO
In 2012/016082;It is the most all incorporated herein by.
" knots modification " of term biomarker or " the change level " of biomarker refer to compared with check sample, this Shen
Being increased or decreased of the expression of biomarker, modification and/or activity at least one of sample please.
If biomarker amount respectively than normal level more or less than the mark of amount method therefor for assessment
Quasi-error, or at least twice of normal value, three times, four times, five times, ten times or more times, then the biology in subject
The amount of label is the high or low of " significantly " compared with normal amount.Or, if amount respectively than biomarker normal amount (example
As, the Average expression level of several check sample biomarkers in check sample or the application) high or low at least about 2 times,
At least about 3 times, at least about 4 times, at least about 5 times, then the biomarker in subject amount can compared with normal amount
It is considered as the high or low of " significantly ".
" possible " described herein refers to the probability increased, i.e. have at least 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%,
120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or higher (any model being included
Enclose) may project, object, event or people can occur.Therefore, in one embodiment, may suppression MELK mediation
The reagent of eIF4B phosphorylation have the 406th serine phosphorylation of suppression people eIF4B of increase or the direct line of people eIF4B with
The probability of the corresponding phosphorylated amino acid of source thing.In another embodiment, the histone H 3 phosphorus of MELK mediation may be suppressed
The reagent of acidifying has the 3rd threonine phosphorylation of suppression human histone H3 of increase, the 10th serine phosphorylation and/or the
11 threonine phosphorylation, or the probability of the human histone H3 corresponding phosphorylated amino acid of ortholog thing.
The testing compound (at least partly) of the application can be by the number of ways in combinatorial library method known in the art
In any one obtain, including biological libraries method;Space can Addressable Parallel solid phase or solution phase libraries method;Need deconvolution
Synthetic library method;" pearl one compound " library method;With the synthetic library method using affinity chromatograph to select.Biological libraries method is limited to
Peptide library, and other four kinds of methods are applicable to peptide, non-peptide oligomer or compound Small molecular libraries (Lam, K.S. (1997)
Anticancer Drug Des.12:145)。
Example for the method for molecular library synthesis can find in the art, such as, exist: DeWitt et al.
(1993)Proc.Natl.Acad.Sci.U.S.A.90:6909;Erbet al.(1994)
Proc.Natl.Acad.Sci.USA91:11422;Zuckermann et al.(1994)J.Med.Chem.37:2678;Cho
et al.(1993)Science261:1303;Carrellet al.(1994)Angew.Chem.Int.Ed.Engl.33:
2059;Carellet al.(1994)Angew.Chem.Int.Ed.Engl.33:2061;With at Gallop et al. (1994)
In J.Med.Chem.37:1233.
Library of compounds can be presented on solution (e.g., Houghten (1992) Biotechniques 13:412-421),
Or on magnetic bead (Lam (1991) Nature 354:82-84), chip (Fodor (1993) Nature 364:555-556), antibacterial
(Ladner USP 5,223,409), spore (Ladner USP'409), plasmid (Cull et al. (1992)
ProcNatlAcadSci USA 89:1865-1869) or phage (Scott and Smith (1990) Science249:
On 386-390);(Devlin(1990)Science 249:404-406);(Cwirlaet al.(1990)
Proc.Natl.Acad.Sci.87:6378-6382);(Felici(1991)J.Mol.Biol.222:301-310);(Ladner
Front).
In some embodiments, wherein to the 406th serine phosphorylation of people eIF4B or people's eIF4B ortholog thing
In the suppression of corresponding phosphorylated amino acid be by with the people eIF4B of the 406th serine phosphorylation in matched group sample
In amount or people's eIF4B ortholog thing, the comparison of the amount of corresponding phosphorylated amino acid determines.Comparison may refer to, and compares
In unused reagent or with by the amount described in the relatively early time point of described reagent and described sample contact, the 406th silk in sample
In the amount of the people eIF4B of propylhomoserin phosphorylation or people's eIF4B ortholog thing corresponding phosphorylated amino acid amount.EIF4B's
Phosphorylation level typically by measure phosphorylation eIF4B protein amount and the most unphosphorylated eIF4B
The amount standardization of the amount gross protein measured and be analysed to phosphorylating protein in sample determines.At testing compound
In the presence of the response phosphorylation level that calculates and substrate or background phosphorylation level (such as, do not use testing compound or executing
Relatively early time point with after testing compound) thus preset time point not by the shadow of absolute amount difference of indicator albumen
Ring.
Can be with selection differential time point or predetermined time to reach in sample after using testing compound to cell
406th serine phosphorylation level have correction compared to matched group after the significant difference with statistical significance.Predetermined
Time, this species diversity be probably maximum, but this is optional, depend on test other parameters.Although additionally,
That ratio calculation specifically described herein is conducive to provide useful comparison is digital, but calculates the phosphorylation using testing compound
Antipode between eIF4B level and matched group, and experimenter to be measured with compare antipode between experimenter, being also can
Use and be effective.
In some embodiments, method described above may further include and determines from having the 5 ' of highly structural
The amount of the protein of the mRNA translation of untranslated region (5 ' UTR), the most wherein said protein selects free cell bone marrow sample thin
In the group that born of the same parents' tumor gene (c-Myc), X Linked Inhibitor of Apoptosis Protein in Children (XIAP) and ODC Ornithine decarboxylase (ODC1) form.Known
The helicase activity of eIF4B stimulation eIF4A is to untie the secondary structure of the 5 ' UTR of mRNA, and eIF4B is to tool 5 ' UTR structures
Critically important (Dmitrievet al. (2003) Mol.Cell Biol.23:8925-8933 and Shahbazianet of translation of mRNA
al.(2010)Mol.Cell Biol.30 1478-1485).Those skilled in the art easily find to encode the tool 5 ' of carcinogenic protein
The mRNA of UTR structure, such as c-Myc, XIAP (X Linked Inhibitor of Apoptosis Protein in Children), ODC (ODC Ornithine decarboxylase), VEGF, HIF-
1alpha etc. (refer to, at least Bert et al. (2006) RNA 12:1074-1083).
Phosphorylation is a kind of biochemical reaction, wherein a phosphate group is added to the serine of protein, threonine or
On tyrosine residue, and it is catalyzed by protein kinase.Phosphorylation generally modifies the function of target protein, it is common that cause activation.Make
For a part for Cell Homeostasis mechanism, phosphorylation only can be referred to as, by other, the iterative procedures that phosphatase reverses.Therefore, albumen
Matter phosphorylation level changes over, and can be estimated by many known methods, including, such as, by immunity
Method.Such as, corresponding phosphorylation in the amount of the people eIF4B of the 406th serine phosphorylation or people's eIF4B ortholog thing
Amino acid whose amount is to be specifically bound to people eIF4B or the corresponding phosphorylated human of the 406th serine phosphorylation by use
The reagent of eIF4B ortholog thing carries out what immunoassay determined.These immunoassay comprise mode known to many, including
But it is not limited to radioimmunoassay, western blot analysis, immunofluorescence assay, enzyme immunoassay, immunoprecipitation survey
Determine method, chemoluminescence method, Immunohistochemical assay, Dot blot mensuration or slit engram detection method.This area general
The known general technology used in carrying out above-mentioned various immunoassay of technical staff and other modification of technology, such as in situ
Ortho position linking parsing (PLA), fluorescent polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA) (EIA), press down
System immunity scattering turbidometry (NIA), enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), ELISA etc.,
It is independent or same NMR, MALDI-TOF, LC-MS/MS etc. combine or alternatively use.
In one embodiment, enzyme immunoassay is a kind of Sandwich ELISA, and it uses specifically
In conjunction with capture antibody or the fragment of people eIF4B or corresponding people's eIF4B ortholog thing with specifically combine the 406th silk ammonia
The detection antibody of people's eIF4B ortholog thing of people eIF4B or the corresponding phosphorylation of acid phosphoric acid or fragment.The immunity of this enzyme
Measure advantageous particularly, because it can differentiate between associated kinase family member and hypotype the difference at protein level, even if
Between these kinases itself and between its phosphorylation form, there is of a relatively high homology.
For identifying eIF4B phosphorylation and unphosphorylated form and the immunoreagent of detection MELK, it is in this area
Know, and standard technique (such as by with suitable eIF4B fluorophor peptide vaccination host cell) can be used to produce.This
Plant anti-MELK, anti-eIF4B, and/or anti-phosphorylation eIF4B antibody reagent (such as, monoclonal antibody) can be used for separating and/or true
The amount of fixed (such as in cell lysate) respective egg white matter.This reagent can also be used for monitoring cell or tissue (such as, the most carefully
Born of the same parents or lymphocyte) in protein level, as a part for clinical testing procedure, such as, in order to monitor the optimal of inhibitor
Dosage.Monitoring can promote by antibody is coupled (such as, physical connection) with detection material.The example bag of detectable substance
Include multiple enzyme, prothetic group, fluorescent material, luminescent material, bioluminescent material and active material.The suitably example of enzyme includes peppery
Root peroxidase, alkali phosphatase, beta galactosidase or acetylcholinesterase;The suitably example of prosthetic group complexes includes
Streptavidin/biotin and avidin/biotin;The suitably example of fluorescent material includes umbelliferone, fluorescein, different sulfur cyanogen
Acid fluorescein, rhodamine, dichlorotriazine base amine fluorescein, dansyl Cl or phycoerythrin;The example of luminescent material includes Rumi
Promise;The example of bioluminescent material includes luciferase, luciferin and aequorin, the example of suitable active material
Including 125I, 131I, 35S or 3H.
Above-mentioned screening technique is also adapted for identifying that candidate/test compound, the regulation of these compounds (such as, stimulate or press down
System) interaction between eIF4B protein and the target eIF4B protein carcinogenic activity of MELK and eIF4B (most probable be also),
Wherein eIF4B protein and target eIF4B protein normally interact or regulate its with the enzymatic activity of checking MELK mediation with
The reduction of phosphorylation eIF4B level and reduce.The example of these target molecules or material includes as further described herein certain
The protein encoded by the mRNA of lamps structureization 5 ' UTR a bit.
As set forth above, it is possible to be similarly determined to eIF4B phosphorylation, the 3rd threonine phosphorylation of human histone H3,
10 serine phosphorylations and/or the 11st threonine phosphorylation, or human histone H3 ortholog thing corresponding phosphorylation amino
The identification of acid and its regulation (such as, suppression).
In another embodiment, the application provides mutual with MELK and/or eIF4B and/or histone H 3 protein
The screening technique of the candidate/test compound of effect (such as, being attached to)." binding compounds " should refer to binding compositions, such as
Little molecule, antibody, peptide, peptidometic ligands or non-peptide ligand, protein, oligonucleotide, oligonucleotide analogs, such as peptide core
Acid, lectin or other any stable chemical combination that can be which can specifically bind to target protein or molecule or there is purpose analyte
The molecular entity that thing is formed, such as protein complex." bound fraction " refers to that any molecular marker can directly or indirectly combine
Molecule up, it can be which can specifically bind to analyte.Bound fraction including, but not limited to, antibody, antibody binding composition,
Peptide, protein, nucleic acid and having is up to about 1000 Dalton molecular weights and comprises the free hydrogen of choosing, fluorine, carbon, oxygen, nitrogen, sulfur and phosphorus
The organic molecule of the atom of the group of composition.Generally, described mensuration is mensuration based on cell.Described cell, for example, it may be feed
Breast expression MELK and/or eIF4B of animal origin and/or histone H 3, such as cancerous cell.
In other embodiments, mensuration is acellular mensuration, and it includes MELK and/or eIF4B and/or histone
The step that H3 protein or its biologically-active moiety and candidate/test compound combine, such as, is allowing candidate/test chemical combination
Thing and MELK and/or eIF4B and/or histone H 3 protein or its biologically-active moiety interact (such as, combining) formation
Under conditions of complex, and the formation of detection complex, wherein candidate compound and MELK and/or eIF4B and/or histone
The ability that H3 polypeptide or its bioactive fragment interact is present in complex by candidate compound and shows.Can
Carry out quantitatively, such as, making with the complex to MELK and/or eIF4B and/or histone H 3 protein and candidate compound formation
With the immunoassay of standard.These analysis meeting identifications are as the test chemical combination of MELK and/or eIF4B and/or histone H 3 part
Thing.
In order to carry out said medicine screening technique, fixing MELK and/or eIF4B and/or histone H 3 or its target molecule, with
Be easy to isolated complex from the non-complexes form of one or both protein, and the automatization that measures of debugging can compare can
Take.In the case of candidate compound presence or absence, MELK and/or eIF4B and/or histone H 3 are mutual with target molecule
Effect (such as, combine) can realization in any container being suitable to accommodate reactant.The example of these containers includes ELISA Plate, examination
Pipe and microcentrifugal tube.In one embodiment, it is provided that a kind of fused polypeptide, which are added a domain and make described
Polypeptide is attached in substrate.Such as, glutathione-S-transferase MELK ,-histone H 3 and/or-eIF4B fused polypeptide are permissible
It is adsorbed on glutathione sepharose beads the enzyme that (Sigma Chemical, St.Louis, Mo.) or glutathion are derivative
Target, then by it with cell lysate (such as,35S-labeled) combine with candidate compound, formed at beneficially complex
Under conditions of hatch this mixture (such as, under the physiological condition of salt and pH).After hatching, beadlet is cleaned with removing any not
In conjunction with labelling, described substrate is fixed and directly determines in radioactive label, or the supernatant after complex dissociation and determine.
It addition, complex can be dissociated from substrate by SDS-PAGE, use standard electrophoretic techniques on quantitative magnetic bead from gel
MELK-, histone H 3-and/or the level of eIF4B-Binding peptide.
The other technologies being fixed in substrate by polypeptide can also make in the illustrative drug Screening test method of the application
With.Such as, MELK and/or eIF4B and/or histone H 3 or its target molecule can utilize biotin and Streptavidin to couple
And fix.Biotinylated MELK and/or eIF4B and/or histone H 3 molecule can use techniques known in the art raw
Thing element-NHS (N-hydroxy-succinimide) prepare (such as, biotinylation kit, Pierce Chemicals, Roc good fortune
Moral, Illinois), and it is fixed on (Pierce Chemical) in the hole of coated 96 orifice plates of Streptavidin.Additionally, it is anti-
Body reacts with MELK and/or eIF4B and/or histone H 3, but this reaction does not disturb polypeptide and its can be derived from the target in plate hole
Molecule combines, and MELK and/or eIF4B and/or histone H 3 are coupled by antibody and be trapped in hole.As it has been described above, MELK-
And/or the preparation of eIF4B-Binding peptide and candidate compound is at MELK-and/or eIF4B-and/or histone H 3-exist
Plate hole is hatched, and the amount of the complex retained in hole can be quantitative.The method detecting these complex, except above-mentioned
For the method for GST immobilization complex, also include using antibody anti-with MELK and/or eIF4B and/or histone H 3 target molecule
Complex immunoassay should be carried out, or use other reactive polypeptides many with MELK and/or eIF4B and/or histone H 3 and divide with target
The material of son competition carries out immunoassay;And depend on the enzyme connection analysis of the relevant enzymatic activity of detection target molecule.
On the other hand, it is provided that a kind of method is used for assessing reagent and suppresses people MELK or its ortholog thing in subject
The curative effect of kinase activity, including: a) people of the 406th serine phosphorylation in first time point detects experimenter's sample
People's eIF4B ortholog thing of the amount of eIF4B or corresponding aminoacid phosphorylation amount;B) after using described reagent
Individual or multiple subsequent point in time repeat step a);And the c) people of the 406th serine phosphorylation in comparison step a) and step b)
The amount of eIF4B or the amount of people's eIF4B ortholog thing of corresponding aminoacid phosphorylation, wherein the 406th serine phosphorylation
The amount of people eIF4B or corresponding aminoacid phosphorylation people's eIF4B ortholog thing amount in first time point higher than extremely
A few subsequent point in time, shows described reagent suppression MELK or the kinase activity of its ortholog thing.Similarly, it is provided that a kind of
Method suppresses people MELK or the curative effect of its ortholog thing kinase activity for assessing reagent in subject, including: a) exist
3rd threonine phosphorylation, the 10th serine phosphorylation and/or the 11st Soviet Union in first time point detection experimenter's sample
The human histone H3 ortholog thing of the amount of the human histone H3 of propylhomoserin phosphorylation or corresponding aminoacid phosphorylation amount;b)
One or more subsequent point in time after using reagent repeat step a);And c) phosphorylation in comparison step a) and step b)
The amount of human histone H3 or the amount of the human histone ortholog thing of corresponding aminoacid phosphorylation, wherein the 3rd threonine phosphorus
The amount of the human histone H3 of acidifying, the 10th serine phosphorylation and/or the 11st threonine phosphorylation or corresponding aminoacid
The human histone H3 ortholog thing of phosphorylation amount in first time point higher than at least one subsequent point in time, show described
Reagent suppression MELK or the kinase activity of its ortholog thing.
As described herein, " time course " refers to primary event and time quantum between event subsequently.Such as, along with experimenter
The progress of cancer, time course may be relevant to experimenter's disease, and can be surveyed by the notable event in metering lysis
Fixed, wherein primary event can be diagnosis, and event can be propagation, transfer etc. subsequently.
Once combine and be proved, extra algoscopy can be carried out according to methods known in the art, such as, be used for determining
The kinase assay of phosphorylation suppression.Such as, determine that the algoscopy of MELK kinase activity is known in the art (in detail
See, such as, described herein and by quoting the publication being fully incorporated herein).In short, MELK can allow eIF4B or
The buffer of phosphorylated histone H3 use suitable substrate hatch.Substrate phosphorylation can be by using the phosphate of labelling
Group is measured, as used the radioactive label originated in buffer as ATP32P.Live it addition, be specifically used for eIF4B catalysis
Property the antibody of Phosphorylated products can be used to detection activity.As apparent to those of ordinary skill in the art, survey
The method of determining is adapted in use to the high-throughput techniques of robotization and automatic business processing.It addition, MELK kinase activity can use the synthesis end
Thing (such as peptide storehouse) measures.MELK activity also can be measured by detecting kinase whose downstream targets as described herein.
The eIF4B of the 406th serine phosphorylation can be according to any means as herein described and use arbitrary sample (example
As, single experimenter's sample or experimenter's sample of merging) it is analyzed.Can identify and ring at phosphorylation eIF4B dependency
Should (such as, in suppression the 406th serine phosphorylation of people eIF4B or eIF4B ortholog thing, corresponding phosphorylated amino acid be residual
Base) aspect produce statistically significant change candidate compound.The change of this statistically significant can be by multiple standards and/or phase
Multiple comparisons are measured.Such as, compared to matched group, if analyzed output be suppressed 1.1-, 1.2-, 1.3-,
1.4-、1.5-、1.6-、1.7-、1.8-、1.9-、2.0-、2.1-、2.2-、2.3-、2.4-、2.5-、2.6-、2.7-、2.8-、
2.9-、3.0-、3.1-、3.2-、3.3-、3.4-、3.5-、3.6-、3.7-、3.8-、3.9-、4.0-、4.1-、4.2-、4.3-、
4.4-、4.5-、4.6-、4.7-、4.8-、4.9-、5.0-、5.5-、6.0、6.5-、7.0-、7.5-、8.0-、8.5-、9.0-
9.5-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-times or more different (includes contained any
Scope), then can confirm that the notable regulation to the 406th serine phosphorylation.In one embodiment, in the described very first time
Between point and time point subsequently, experimenter has been subjected to treatment of cancer, has completed treatment of cancer, and/or is in cancer remission
Phase.
As set forth above, it is possible to identify similarly and/or analyze eIF4B phosphorylation, the 3rd threonine phosphorylation, the 10th
Serine phosphorylation and/or the histone H 3 of the 11st threonine phosphorylation, or the human histone H3 corresponding phosphorus of ortholog thing
Acidifying aminoacid, and its change (such as, suppression).
Term " cancer " refers to exist the cell with typical carcinogenic cells feature, described feature such as proliferation out of control, immortality,
Metastatic potential and specific characteristic morphologic feature.Cancerous cell is usually presented in tumor, but these cells can be single
Solely it is present in animal body, can be maybe non-tumorigenic cancer cells, such as leukaemia.As described herein, term " cancer " wraps
Include precancerous lesion and malignant tumor.Cancer including, but not limited to, this is special for B cell tumor, such as multiple myeloma, Walden
Human relations macroglobulinemia;Heavy chain disease, such as alpha-chain disease, γ-chain is sick, and mu heavy chain disease;Benign monoclonal gammopathy and immunity are thin
Born of the same parents' amyloidosis, melanoma, breast carcinoma, pulmonary carcinoma, bronchogenic carcinoma, colorectal carcinoma, carcinoma of prostate, cancer of pancreas, gastric cancer,
The cancer of ovarian cancer, bladder cancer, brain or central nervous system, peripheral nervous system cancer, esophageal carcinoma, cervical cancer, uterus or son
Endometrial carcinoma, oral cavity or hypopharyngeal carcinoma, hepatocarcinoma, renal carcinoma, carcinoma of testis, cancer of bile ducts, small intestinal or vermiform appendix cancer, salivary-gland carcinoma, thyroid carcinoma,
Adrenal carcinoma, osteosarcoma, chondrosarcoma, hematology organize cancer etc..Also include coding MELK and/or eIF4B and/or histone
The gene amplification of H3 or any cancer of overexpression, or have the cancer of Activating mutations, or MELK and/or eIF4B and/or group
Albumen H3 is by the overactive cancer of other kinases.In some embodiments, it may be preferred to be that ovarian cancer (includes serosity capsule
Adenocarcinoma), incidence cancer (including nonsmall-cell lung cancer (NSCLC)), squamous cell carcinoma, cancer of pancreas, colon cancer, carcinoma of prostate,
And/or glioma.
" process ", other forms of " treatment " and this word refer to use reagent, and wherein reagent can suppress 1) MELK phosphorylation
EIF4B and/or the ability of histone H 3 and/or 2) MELK phosphorylation eIF4B or the ability of histone H 3, to improve cancer disease
Shape, the expection time-to-live extending experimenter and/or the time etc. of prolongation cancer progression.
Specifically described herein arrives " reaction " treatment " response ", and other forms of this verb refer to that experimenter uses
The reaction occurred after agent therapy, wherein reagent can suppress 1) ability of MELK phosphorylation eIF4B and/or histone H 3 and/
Or 2) MELK phosphorylation eIF4B or the ability of histone H 3.Such as, if condition determination relative to do not use reagent or beyond to
Fix time and change at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, then experimenter rings
The treatment that reply experimenter or its cell reagent are carried out.
C. Therapeutic Method
MELK and/or eIF4B described herein and/or histone H 3 inhibitor may be used for treating cancer.An enforcement
In mode, the method for the patient that treatment suffers from cancer includes the 406th serine phosphorylation or the people using suppression people eIF4B
The amino acid whose reagent of the corresponding phosphorylation of eIF4B ortholog thing, such as specificity regulates the 406th serine phosphorylation, from
And treat the reagent of the patient suffering from cancer.In another embodiment, these MELK and/or eIF4B and/or histone H 3
Inhibitor also may be used to determine the curative effect for the treatment of, toxicity or the side effect carried out with described reagent.As described further below,
The experimenter that these Therapeutic Method generally include to needing this treatment (such as suffers from cancer or faces and suffer from the tested of cancer risk
Person) use the regulator in pharmaceutical composition.
Term administering " it is intended to include route of administration, it allows reagent to play its desired suppression MELK phosphorylation eIF4B
Ability, eIF4B by the ability of MELK phosphorylation, the ability of MELK phosphated lanolin, and/or histone H 3 is by MELK phosphorus
The function of the ability of acidifying.The example of the route of administration that can use include injection (subcutaneous, intravenous, parenteral, intraperitoneal,
In sheath etc.), oral, suck and percutaneous dosing.Injection can be bolus injection can also be continuous infusion.According to route of administration,
Described reagent can be coated with or be in selected material, to protect it from it being played predictive role generation
The effect of natural conditions of adverse effect.Reagent can be administered alone, or is used in combination with pharmaceutically acceptable carrier.Described examination
Agent also can be used as prodrug, and it is converted into its activity form in vivo.
The ability of term suppression MELK phosphorylation eIF4B and/or eIF4B " having by the reagent of the ability of MELK phosphorylation
Effect amount " refer to necessary in experimenter or test crowd or that be enough to suppress MELK phosphorylation eIF4B ability and/or eIF4B quilt
The amount of the ability of MELK phosphorylation, such as, according to the amount of people eIF4B of the 406th serine phosphorylation that said method measures
Or the level of corresponding phosphorylated residue in eIF4B ortholog thing.Same analysis is applicable to suppress MELK phosphated lanolin
Ability, and/or histone H 3 is by the ability of MELK phosphorylation.Effective dose can use type, experimenter's build according to therapeutic agent
The factor such as size or disease severity and change.
It should be appreciated that personal dose may according to attending doctor judge experimenter's demand, treatment disease tight
Weight degree and the specific compound of use and change.When determining therapeutically effective amount or dosage, the doctor in charge may consider perhaps
Other factors many, including, but not limited to: the pharmacodynamic profile of particular agent and administering mode thereof and approach;During required treatment
Between process;The kind of mammal;Its body size, age and general health;The disease being specifically related to;The journey of disease
Degree or complexity or seriousness;The reaction of individual subjects;The specific compound used;Form of medication;The biological utilisation of preparation
Degree characteristic;The dosage regimen selected;The kind of therapeutic alliance;With other correlation circumstances.
Treatment can be from the beginning of smaller dose, and the effective dose of this dose ratio compound is little.Hereafter, an embodiment party
In formula, described dosage should be increased up reaching the optimum efficiency in the case of this by less increment.For convenience, the most always
Daily dosage can separate and local administration.
The effect of the treatment cancer of any particular agent, can be by comparing from the experimenter's acquisition accepting treatment of cancer
Two or more samples are monitored.Generally, first sample accepts to treat front acquisition experimenter, obtains over the course for the treatment of
One or more samples.In such a case, it is possible to determine and accept the baseline that the cell of the front cancer patient for the treatment of is expressed, then may be used
To monitor the state change of the baseline that cancer patient's cell is expressed over the course for the treatment of.Or, it is possible to use therapeutic process obtains
The two or more continuous samples taken are without baseline sample before treatment.In this case, the obtained from experimenter
Whether one sample determines that as baseline the cell of the experimenter of metabolism disorder is expressed and is increased or decreased.
MELK and/or eIF4B and/or histone H 3 inhibitor can be used in pharmaceutically acceptable compositions, pharmacy
Acceptable compositions includes formulated together with one or more pharmaceutically acceptable carriers (additive) and/or diluent
The inhibitor of therapeutically effective amount.Such as, formula can carry out based on known to pharmaceutical field method adjust be applicable to (1) be administered orally to
Medicine, such as, gavages agent (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, paste;(2) intestinal
External administration, such as by subcutaneous, intramuscular or intravenous injection, such as, sterile solution or suspension;(3) topical, such as, makees
For being applicable to the cream of skin, oral cavity or tongue lower surface, ointment or spray;(4) intravaginal or internal rectum, such as vagina
Suppository, cream or foam;Or (5) nasal cavity/aerosol, such as, as one containing water aerosol, Liposomal formulation or containing should
The solid particle of compound.
Phrase " pharmaceutically acceptable " is used for referring to these reagent, material, compositions, and/or dosage form in this article, they
Reasonably in the range of medical judgment, be suitable for humans and animals contact tissue and do not have too much toxicity, zest, anaphylaxis anti-
Should, or other problems or complication, and match with rational interests/risk-ratio.
Phrase " pharmaceutically acceptable carrier " refers to a kind of pharmaceutically acceptable material, compositions or solvent in this article, as
Liquid or solid filler, diluent, excipient, solvent or encapsulating material, relate to object chemicals from an organ or body
Another organ or a part for health is carried or transported to a part for body.Each carrier is compatible at other compositions with preparation
And it is " can accept " in the sense that not damaging experimenter.Some can be as the examples of materials bag of pharmaceutically acceptable carrier
Include: (1) saccharide, such as lactose, dextrose plus saccharose;(2) starch, such as corn starch and potato starch;(3) cellulose,
And derivant, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth;(5) Fructus Hordei Germinatus;
(6) gelatin;(7) Talcum;(8) excipient, such as cocoa butter and suppository wax;(9) oil, such as Oleum Arachidis hypogaeae semen, Oleum Gossypii semen, safflower oil, Semen Sesami
Oil, olive oil, Semen Maydis oil and soybean oil;(10) glycol, such as propylene glycol;(11) polyhydric alcohol, such as glycerol, sorbitol, mannitol
And Polyethylene Glycol;(12) ester, such as ethyl oleate and ethyl laurate;(13) agar;(14) buffer agent, such as magnesium hydroxide and hydrogen
Aluminium oxide;(15) alginic acid;(16) apirogen water;(17) isotonic saline solution;(18) Ringer's mixture;(19) ethanol;(20) phosphoric acid
Salt buffer solution;(21) other non-toxic compatible substances used in pharmaceutical preparation.
Term " pharmaceutically acceptable salt " refers to that what the application comprised can reduce PKC-iota phosphorylation level and/or activity
The inorganic and organic addition salts of relative nontoxic of reagent.These salt being finally recovered and can be prepared during purification in position,
Or individually react preparation by the reagent of purification with its free alkali form and suitable organic or inorganic acid, and separate formation
Salt.Representational salt include hydrobromate, hydrochlorate, sulfate, disulfate, phosphate, nitrate, acetate, valerate,
Oleate, palmitate, stearate, laruate, benzoate, lactate, phosphate, toluene fulfonate, citrate,
Maleate, fumarate, succinate, tartrate, naphthalene sulfonate, mesylate, gluceptate, Lactobionate and
Lauryl sulfonate etc. (see, e.g., Berge et al. (1977) " Pharmaceutical Salts ",
J.Pharm.Sci.66:1-19)。
It addition, method described herein also includes except using one or more other antitumor and anticancer agents and/or making experimenter's
Sample is exposed to outside these antitumor and anticancer agents, with MELK and/or eIF4B and/or histone H 3 inhibitor for treating experimenter.Ability
Antitumor and anticancer agent known to field technique personnel, it is including, but not limited to chemotherapy and radiation, also immunization therapy, hormone therapy and making
The nucleic acid molecules relevant by, progress initial to tumor or cancer and/or pathology and/or the gene therapy of protein.
Chemotherapy includes using chemotherapy agents.These chemotherapy agents may be but not limited to from following compound group selection
Those: platinum-like compounds, cytotoxic antibiotics, antimetabolite, mitotic inhibitor, alkylating agent, arsenic compound, DNA open up
Flutter isomerase inhibitors, taxanes, nucleoside analog, plant alkaloid and toxin;With its synthesis of derivatives.Typical chemical combination
Thing is including, but not limited to, alkylating agent: cisplatin, treosulfan and trofosfamide;Plant alkaloid: vinblastine, paclitaxel, many
Western paclitaxel;DNA topoisomerase enzyme inhibitor: teniposide, crisnatol, mitomycin;Anti-folic acid: methotrexate, mould phenol
Acid, and hydroxyurea;Pyrimidine analogue: 5-fluorouracil, doxifluridine and cytosine arabinoside;Purine analogue: mercaptopurine and sulfur
Guanine;DNA antimetabolite: 2'-Deoxy-5-Floxuridine, aphidicolin glycinate and pyrazolo imidazoles;Silk is had to divide with anti-
Split agent: halichondrins, Colchicine, and rhizomycin.Be used as comprising one or more chemotherapy agents (such as, FLAG,
CHOP) compositions.FLAG includes fludarabine, cytosine arabinoside (Ara-C) and G-CSF.CHOP includes that cyclophosphamide, Changchun are new
Alkali, amycin and prednisone.In another embodiment, (such as, PARP-1 and/or PARP-2) that can use PARP suppresses
Agent, it is known in the art (such as, Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research
Laboratories,Inc.);INO-1001(Inotek Pharmaceuticals Inc.);PJ34(Soriano et al.,
2001;Pacheret al.,2002b);3-AB (Trevigen);4-amino-1,8-naphthalimide;
(Trevigen);6 (5H)-phenanthridones (Trevigen);Benzoylamide (U.S.Pat.Re.36,397);With NU1025 (Bowman
et al.).In another embodiment, chemotherapy agents is platinum compounds, such as cisplatin, carboplatin, oxaliplatin, nedaplatin and different
Third platinum.Other anti-tumor platinum coordination compound is well known in the art, and can carry out according to procedures known in the art
Modify, and be included in U.S. Patent number 4,996,337,4,946,954,5,091,521,5,434,256,5,527,905 and 5,
Compound disclosed in 633,243, all these is all incorporated herein by.Chemotherapy agents in above-mentioned example is exemplary
, and be not intended to limit.
Radiotherapy also comprises other antitumor and anticancer agents.The radiation used in radiotherapy can be ionizing radiation.Radiation
Treatment can also be gamma-rays, X-ray, or proton beam.Radiocurable example including, but not limited to external beam X-ray therapy,
Radiosiotope interventional implantation (iodine-125, palladium-103, Iridium-192 source), intravenous injection radiosiotope (such as strontium-89), breast
Portion's radiotherapy in the treatment, abdominal cavity32P radiotherapy, and/or full abdomen pelvic cavity radiation therapy.Radiocurable General Introduction, sees
Hellman,Chapter 16:Principles of Cancer Management:Radiation Therapy,6th
edition,2001,DeVitaet al.,eds.,J.B.Lippencott Company,Philadelphia.X-ray therapy can
Using as external beam radiation or teletherapy, wherein said radiation protection is derived from remote source.Radiotherapy is alternatively arranged as inside
Treatment or plesioradiotherapy are used, and wherein radioactive source is placed in internal close to cancerous cell or the position of tumor mass.Also include
The photodynamic therapy used containing photosensitizer, as hemoporphyrin and its derivant, Verteporfin (BPD-MA), phthalocyanine, photosensitizer PC4,
Demethoxylation hypocrellin A;And 2BA-2-DMHA.
Other antitumor and anticancer agent includes immunization therapy, hormone therapy and gene therapy.These therapies including, but not limited to,
Use antisense polynucleotides, ribozyme, rnai molecule, the polynucleotide etc. of three spirals, the wherein nucleotides sequence of these compounds
Arrange relevant to the nucleotide sequence of DNA and/or RNA of gene, described gene and tumor or initial, the progress of cancer and/or disease
Reason is relevant.Such as, proto-oncogene, growth factor gene, growth factor receptor gene, cell cycle gene, DNA-repair gene and
Other, they can be as the target spot of these therapies.
Immunization therapy can include, such as, uses cancer vaccine and/or sensitising antigens presenting cell.Immunization therapy is all right
Including checking of inhibitive ability of immunity path, as by targeting PD-L1, PD-L2, PD-1, CTLA-4 etc..Immunization therapy can relate to
And host is carried out the passive immunity of short-term protection, it realizes (example by using the antibody for cancer antigen or disease antigen
As, tumor antigen is used monoclonal antibody, it is optionally connected with chemotherapy agents or toxin).Immunotherapy also can focus on
Use the cytotoxic lymphocyte identifying JEG-3 epi-position.
Hormone therapy can comprise, such as, Hormone agonists, hormone antagonist (such as, flutamide, bicalutamide, he not
Former times sweet smell, raloxifene, leuprorelin acetate (LUPRON), LH-RH antagonist), hormone biosynthesis and the inhibitor of processing, and
Steroid (such as, dexamethasone, retinoic acid, triangular muscle, betamethasone, hydrocortisone, cortisone, prednisone, dehydrogenation testis
Ketone, glucocorticoid, mineralocorticoid, estrogen, androgen, progestogen), vitamin A derivative (such as, total trans dimension first
Acid (ATRA));Vitamin D 3 analogs;Progesterone receptor antagonists (such as, mifepristone, onapristone), or androgen antagonist (example
As, cyproterone acetate).
In one embodiment, determined by the present processes for treatment that the anti-cancer therapies of the cancer of phenotype can wrap
Include one or more therapies described herein, including, but not limited to, chemotherapy agents, immunization therapy, anti-angiogenic agent, cell
The factor, hormone, antibody, polynucleotide, radiation and optical dynamic therapy agent.Such as, combination treatment can include one or more chemotherapy
Reagent and radiation, one or more chemotherapy agents and immunization therapy, or one or more chemotherapy agents, radiation and chemotherapy.
Embodiment
The application is further described by following example, but should not be taken is the restriction to the application.
Embodiment 1: the material of embodiment 2-3 and method
A. plasmid
From human mammary epithelial cell (HMEC), extract total serum IgE, utilize primer (forward: ATGGCGGCCTCAGCAAAAAAG;
Reverse: CTATTCGGCATAATCTTCTC) clone people from the reverse transcription product of this total serum IgE eIF4B.By PCR primer (1.8kb)
As template amplification with the FLAG labelling of restriction site or the eIF4B of HA labelling.By this construct of sequence verification
(pWzl-Flag-eIF4B、pTrex-eIF4B-HA).QuickChange XL (Stratagene) is used to carry out determining of eIF4B
Point mutation, and the construct of all sudden changes is confirmed by order-checking.
In order to produce the pLKO-tet-on shRNA of targeted human eIF4B, the oligonucleotide of synthesis is annealed and uses digestion
PLKO carrier connect.Random sequence, sh-eIF4B-1 sequence, sh-eIF4B-2 sequence are GTGGACTCTTGAAAGTAC respectively
TAT, GGACCAGGAAGGAAAGATGAA and GCGGAGAAACACCTTGATCTT.
B. retrovirus and lentiviral gene deliver
Retrovirus produces by using retroviral vector transfection HEK293T cell and packaging DNA.Ordinary circumstance
Under, use 1.6 μ g pWzl DNA, 1.2 μ g pCG-VSVG and 1.2 μ g pCG-gap/pol, 12 μ l liquid volumes
Metafectene Pro(Biontex).DNA and lipid dilute respectively in the PBS of 300 μ l and mix.Incubation 15 minutes
(min), after, inoculate before adding them to one day in 6 cm dishes of 3,000,000 HEK293T cells.At 48 hours (h) of transfection
Viral supernatants is collected after 72 hours.Supernatant is filtered the thin film of 0.45 μm, and deposits at 8 μ g/ml polybrene (Millipore)
Time add it to target cell.Slow virus is produced by similar path, except HEK293T cell is to use 2 μ g pLKO
DNA, 1.5 μ g pCMV-dR8.91, and 0.5 μ g pMD2-VSVG transfect.Use antibiotic-screening thin after primary infection 72h
Born of the same parents.Puromycin and blasticidin S use when final concentration 1.5 μ g/ml and 4 μ g/ml respectively.
C. immunoblotting
For processing with nocodazole, the culture medium containing nocodazole (200ng/ml) is recovered cell.Process 20 little
Shi Hou, by the mitotic cell that soft method of shaking to cause drop results are floating.For drug treating, by cell at OTSSP167
(ChemExpress,HY15512;The DMSO of 10mM stores liquid) in the presence of be inoculated into porous plate.
Harvesting with being added with protease inhibitor cocktail (Roche) and inhibitors of phosphatases mixture
The RIPA buffer of (Thermo Scientific) (25mM Tris, pH 7.4,150mM sodium chloride, 1%Nonidet P-40,
0.5% NaTDC and 0.1% sodium lauryl sulphate) cracking.Use BCA test kit (Thermo Scientific) point
Analysis cleared lysate is to obtain protein concentration.The protein (10-20 μ g) of equivalent can separate on SDS-PAGE, is turned subsequently
Move on to celluloid or polyvinylidene fluoride film (Amersham).These films are closed with 5% skim milk, subsequently together with one is anti-
4 DEG C of night incubation.After cleaning, film is resisted with fluorescently-labeled two and at room temperature hatches 1 hour.Then by Membrane cleaning, and useInfrared scanner (Li-Cor Biosciences) scans.
Following antibody is used for immunoblotting or immunoprecipitation.MELK(Epitomics,2916)、p-eIF4B(S406)
(Cell Signaling,8151)、p-eIF4B(S422)(Cell Signaling,3591)、eIF4B(Cell
Signaling,3592)、c-Myc(Cell Signaling,5605)、XIAP(Cell Signaling,2045)、p-Akt
(S473) (Cell Signaling, 4060), p-MAPK (T202/Y204) (Cell Signaling, 4370), the PARP sheared
(Asp214) (Cell Signaling, 9541), aurora (Aurora) A (Cell Signaling, 4718), aurora (Aurora)
B(Cell Signaling,3094)、p-Aurora A(T288)/Aurora B(T232)/Aurora C(T198)(Cell
Signaling, 2914), p-histone (Histone) H3 (T3) (Cell Signaling, 13576), p-histone H 3 (S10)
(Cell Signaling, 3377), p-histone H 3 (T11) (Cell Signaling, 9767), p-histone H 3 (S28)
(Cell Signaling, 9713), ODC (Sigma, O1136), vinculin (Vinculin) (Sigma, V9131,
Alpha-tubulin (alpha-tubulin) (Sigma, T9016), anti-HA magnetic bead (Pierce, 88836), anti-Flag magnetic bead
(Sigma,M8823).Two anti-Alexa Fluor 680 goat anti-rabbit igg (Invitrogen, A-21109) and IRDye800-
The anti-Mus IgG (Rockland) connected.
D. vitro kinase measures
EIF4B or Flag-eIF4B (S406A) of Flag labelling is transfected into HEK293T cell (at a 60mm culture dish
Middle add 4 μ g DNA to cell).Transfect latter 36 hours, by cell IP buffer (100mM NaCl, 50mM Tris, pH value
7.5,0.5%NP-40,0.5% NaTDC, it is supplemented with protease/inhibitors of phosphatases mixture) cracking.Lysate leads to
Cross the magnetic bead that anti-mouse IgG connects to hatch (4 DEG C, 30min) and clarify, then use anti-Flag M2 magnetic bead (Sigma) (4
DEG C, 120min) carry out immunoprecipitation.Beadlet IP buffer solution for cleaning 5 times with conjugated antigen.Cleaning process the last time
Middle beadlet is aliquoted in 1.5ml micro tube.After removing IP buffer, by the beadlet 1 times of kinase buffer liquid (5mM without ATP
Tris, pH 7.5,5mM-β-phosphoglycerol, 2mM dithiothreitol, DTT, 0.1mM Na3VO4,10mM MgCl2;Cell
Signaling) clean once.After cleaning, 1 times of kinase buffer liquid of 40 μ l is added in each test tube, then with 200mM ATP
Add the 5 μ l buffer with or without 500ng restructuring MELK.Reaction is hatched 30 minutes, then by adding 40 μ l at 30 DEG C
2 times of SDS sample buffer terminate.Sample is boiled and carries out immunoblotting.The vitro kinase of histone H 3 proceeded as above
Algoscopy, except using the histone H 3 .1 (New England BioLabs, M2503S) (each reaction 50ng) of restructuring.
E. spot scan peptide library selection
Purification activity Full-length people MELK from insect cell.Carry out such as Turk et al. (2006) Nat.Protocol.1:
Point bit scan peptide library selection described in 375..In brief, one group is used to have following sequence of 180 (or 198) individual biology
The peptide of element-connection, sequence is Y-A-X-X-X-X-X-S/T-X-X-X-X-A-G-K-K-biotin.In the sequence, S/T refers to an ammonia
Acid and the equimolar mixture of threonine.For each peptide, one in 9 X position represents one of 20 kinds of all amino acids.Will
Peptide is arranged in the buffer on 384 orifice plates, and wherein buffer comprises 50mM HEPES, pH7.5,20mM MgCl2、0.02mg/
Ml BSA, 0.01%Brij 35,5mM DTT, 0.5mM EGTA and activity MELK, and γ-[32P]-ATP is added in hole (
[peptide]=50 μM, [ATP]=100 μM, every hole 0.025 μ Ci/ μ l eventually).After hatching 2 hours at 30 DEG C, by the decile object point of reaction
Sample is on Streptavidin film.By film cancellation, clean completely, be dried, and be exposed to fluorophor storage screen.
Embodiment 2:eIF4B phosphorylation state is the biomarker of MELK enzymatic activity and carcinogenic activity
For finding the MELK potential molecular mechanism to cancer (such as Basaloid breast carcinoma (BBC)) importance, research
Many experimental techniques, including immunoprecipitation-tandem mass spectrometry and phosphoeptide atlas analysis.When the MELK of Flag labelling is having silk
The cell pyrolysis liquid of division carries out immunoprecipitation, when carrying out mass spectral analysis subsequently, finds translation initiation factor during mitosis
EIF4B Yu MELK has the strongest dependency (Fig. 1).Use some position phosphoeptide atlas analysis, the optimal substrate of MELK can be identified
Motif, compared to serine/threonine, its arginine of p-3 has stronger selectivity (Fig. 2).
People's eIF4B residue has two flanking regions (such as, in Ser406 and Ser422 site), and it includes MELK phosphorylation base
Sequence (Fig. 3 and 4).The restructuring MELK of purification whether at the two site phosphorylation people MELK, can be used for detection MELK and exempt from
Epidemic disease precipitation eIF4B carries out vitro kinase mensuration.406th serine rather than the 422nd serine of finder eIF4B are easy
By the kinase domain phosphorylation of total length MELK or MELK, and the mutant serine of work as people IF4B the 406th is alanine
Or MELK is carried out other operation, see the phosphorylation found and disappear (Fig. 3-5).These result specificitys depend on MELK, because
MTOR inhibitors (such as rapamycin and torins) suppression mitotic cell, it will not produce identical result (Fig. 6;van
Gorpet al.(2009)Oncogene 28:95-106).These data show that MELK is a kind of at S406 phosphorylated human eIF4B
Kinases because the sequence of high conservative and have described phosphorylation site eIF4B polypeptide region structure composition, other things
The eIF4B ortholog thing planted is also by similarly phosphorylation (table 2).
Table 2
Known eIF4B stimulates the helicase activity of eIF4A to untie the secondary structure (Dmitrievet of the 5 ' UTR of mRNA
Al. (2003) Mol.Cell Biol.23:8925-8933 and Shahbazianet al. (2010) Mol.Cell Biol.30
1478-1485).Many mRNA encode carcinogenic protein, such as c-Myc, XIAP (X Linked Inhibitor of Apoptosis Protein in Children), ODC (ornithine
Decarboxylase).The application determines that MELK lowers and reduces the eIF4B (p-eIF4B) of MDA-MB-468 cell during mitosis
The phosphorylation of S406, this also causes c-Myc, XIAP and ODC1 level to be remarkably decreased (Fig. 7 and 8).Therefore, during mitosis
The phosphorylation of the S406 of the eIF4B of MELK mediation, the optimization translation to the mRNA with highly structural 5 ' UTR is functionally
Important, many of which is known which are carcinogenic, such as c-Myc, XIAP and ODC1.In sum, these data show MELK
It is a kind of Azaindole kinase regulating eIF4B during mitosis, thus the translation of the mRNA that mediation is containing structuring 5 '-UTR,
It is for the survival of cancerous cell and breeds particularly significant.Therefore, the eIF4B phosphorylation level of MELK mediation is MELK carcinogenic activity
Target contact biomarker, it is to before clinic and clinical practice is useful.
Embodiment 3: phosphorylated histone H3 state is the biomarker of MELK enzymatic activity and carcinogenic activity
Due to MELK protein abundance during mitosis the highest, this process is also that histone H 3 is by a large amount of phosphorylations
Cell cycle phase, thus MELK and histone H 3 existence contact are proposed.It is true that the 11st of human histone H3 the threonine sites
The flanking region of the residue of point comprises the optimal MELK phosphorylation motif described in embodiment 2.
Whether can be at the 3rd threonine (Thr3), the 10th serine (Ser10) and the 11st threonine for detection MELK
(Thr11) phosphorylated human histone H 3, uses the recombined human MELK kinase domain of purification to carry out external sharp with human histone H3
Enzymatic determination.Thr3, Ser10 and Thr11 of finder's histone H 3 is easily by the kinase domain phosphorylation (Fig. 9) of MELK.
MELK lower the histone H 3 reduced during mitosis in MDA-MB-468 cell Thr3, Ser10 and
Thr11 site phosphorylation, but do not affect the phosphorylation of BTAK, B or C, these kinases be known can be in Ser10 site
The kinases (Figure 10 and 11) of phosphated lanolin.Similarly, MDA-MB-468 uses small-molecule chemical MELK inhibitor
OTSSP167 suppresses MELK, reduces the histone H 3 phosphorylation (Figure 12) in Thr3/Ser10/Thr11 position.Can observe
Thr3, Ser10 and Thr11 site of histone H 3 rather than Ser28 site phosphorylation are that OTSSP167 concentration-dependant reduces
(Figure 13).
These data show that MELK is that one is at least at Thr3, Ser10 and Thr11 site rather than Ser28 site phosphoric acid
Change the kinases of human histone H3, because the sequence of high conservative and there is the knot of polypeptide region of histone H 3 of phosphorylation site
Structure forms, and the histone H 3 ortholog thing of other species is also by similarly phosphorylation (table 3).
Table 3
Histone H 3
In sum, these data show MELK be a kind of regulate during mitosis histone H 3 novel swash
Enzyme, breeds particularly significant thus for cell (such as, cancerous cell).Therefore, the phosphorylated histone H3 level of MELK mediation is
The target contact biomarker of MELK carcinogenic activity, it is to before clinic and clinical practice is useful.
Quote and be incorporated to
All lists of references, patent application, patent and the disclosed patent application that the application quotes, and accompanying drawing and sequence
Table, its content is incorporated herein by reference the application.
Equivalent
Those skilled in the art it will be appreciated that or use no more than the test of routine and can determine, many is equal to this
The specific embodiments of the application that literary composition describes.These equivalents are intended to include in the claims.
Claims (114)
1. one kind identifies suppression people maternal embryo leucine zipper kinases (MELK) or the kinase activity of its ortholog thing or cause
The method of the reagent of cancer activity, including:
A) i) people MELK or its ortholog thing and ii will be comprised) human eukaryote initiation factor 4B (eIF4B) or its ortholog thing
Sample contact with described reagent, and
B) determine in described reagent suppression the 406th serine phosphorylation of people eIF4B or people's eIF4B ortholog thing corresponding
The ability of phosphorylated amino acid, wherein phosphorylation minimizing identifies described reagent suppression people MELK or the kinases of its ortholog thing
Activity or carcinogenic activity.
Method the most according to claim 1, wherein by by described 406th serine phosphorylation of people eIF4B in sample
In the amount changed or people's eIF4B ortholog thing, the amount of corresponding phosphorylated amino acid compares with compareing, and it is described right to determine
The suppression of corresponding phosphorylated amino acid in 406th serine phosphorylation of people eIF4B or people's eIF4B ortholog thing.
Method the most according to claim 2, wherein said comparison is, compared in the case of unused described reagent or
The amount of the relatively early time point after described sample is contacted with described reagent, the people of the 406th serine phosphorylation in described sample
The described amount of corresponding phosphorylated amino acid in the amount of eIF4B or people's eIF4B ortholog thing.
Method the most according to claim 1, wherein by by the 406th serine phosphorylation of people eIF4B in sample
Total relative to people E1F4B or its ortholog thing of the amount of corresponding phosphorylated amino acid in amount or people's eIF4B ortholog thing
The ratio of amount compares with compareing, and determines that described the 406th serine phosphorylation to people eIF4B or people eIF4B are lineal
The suppression of corresponding phosphorylated amino acid in congener.
Method the most according to claim 4, wherein said comparison is, compared in the case of unused described reagent or general
Described sample contact with described reagent after the ratio of relatively early time point, the people of the 406th serine phosphorylation in described sample
The described ratio of the amount of corresponding phosphorylated amino acid in the amount of eIF4B or people's eIF4B ortholog thing.
6., according to the method according to any one of claim 1-5, also include determining the RNA from having highly structural 5 ' UTR
The amount of the protein of translation, the most wherein said protein selects free bone marrow cell carcinoma oncogene (c-Myc), X chromosome even
The group that lock IAP (XIAP) and ODC Ornithine decarboxylase (ODC1) form.
Method the most according to claim 1, also includes determining that described reagent is whether directly in conjunction with described people eIF4B or it is straight
It is congener, or described people MELK or the step of its ortholog thing.
Method the most according to claim 1, the group of wherein said sample choosing freely external, in vitro and internal sample composition.
Method the most according to claim 1, wherein said sample packages contains cell.
Method the most according to claim 9, wherein said cell is cancerous cell.
11. methods according to claim 10, wherein said cancer choosing free MELK or eIF4B amplification or overexpression
Arbitrarily any by other kinase activations of cancer, any cancer with MELK or the eIF4B sudden change of activation and MELK or eIF4B
The group of cancer composition.
12. methods according to claim 9, wherein said cell obtains from experimenter.
13. methods according to claim 1, the choosing of wherein said sample is freely organized, whole blood, serum, blood plasma, cheek are scraped, saliva
Liquid, cerebrospinal fluid, urine, feces and the group of bone marrow composition.
14. according to the method according to any one of claim 1-5, the wherein amount of the people eIF4B of the 406th serine phosphorylation
Or the amount of corresponding phosphorylated amino acid is by using specificity and the 406th serine phosphorus in people's eIF4B ortholog thing
The reactant that people eIF4B or the corresponding phosphorylated human eIF4B ortholog thing of acidifying combines carries out what immunoassay determined.
15. methods according to claim 14, wherein said immunoassay are that radioimmunoassay, western blot are divided
Analysis, proximity ligation assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemoluminescence method, immune group
Weave chemistry algoscopy, Dot blot measure or slit engram detection method.
16. methods according to claim 15, wherein said enzyme immunoassay is a kind of Sandwich ELISA,
Described Sandwich ELISA uses the capture of specific binding people eIF4B or corresponding people's eIF4B ortholog thing to resist
The people eIF4B of body or its fragment and the people eIF4B of specific binding 406th serine phosphorylation or corresponding phosphorylation is lineal
The detection antibody of congener or its fragment.
17. according to the method according to any one of claim 1-5, wherein said people eIF4B or its ortholog thing, and/or
Described people MELK or its ortholog thing, including nucleotide sequence as listed in table 1 or aminoacid sequence.
18. according to the method according to any one of claim 1-5, and wherein said reagent is little molecule, or antibody or its antigen
Binding fragment.
19. according to the method according to any one of claim 1-5, and wherein said reagent is by the 406th serine phosphorylation
In the amount of people eIF4B or people's eIF4B ortholog thing, the amount of corresponding phosphorylated amino acid reduces at least 50%.
20. 1 kinds of sides being used for assessing the effect of the kinase activity that reagent suppresses people MELK or its ortholog thing in experimenter
Method, including:
A) amount or corresponding of the people eIF4B of the 406th serine phosphorylation in first time point detects experimenter's sample
The amount of people's eIF4B ortholog thing of aminoacid phosphorylation;
B) the one or more subsequent point in time after using described reagent repeat step a);With
C) amount of the people eIF4B of the phosphorylation of detection or the amount of its ortholog thing in comparison step a) and step b),
The wherein amount of the people eIF4B of the 406th serine phosphorylation or people's eIF4B ortholog of corresponding aminoacid phosphorylation
The amount of thing higher than at least one subsequent point in time, shows described reagent suppression people MELK or its ortholog thing in first time point
Kinase activity.
21. methods according to claim 20, the wherein amount of the people eIF4B of the 406th serine phosphorylation or people eIF4B
In ortholog thing, the amount of corresponding phosphorylated amino acid is determined by immunoassay, and described immunoassay use specific binding
The reactant of people's eIF4B ortholog thing of people eIF4B or the corresponding phosphorylation of the 406th serine phosphorylation.
22. methods according to claim 21, wherein said immunoassay are that radioimmunoassay, western blot are divided
Analysis, proximity ligation assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemoluminescence method, immune group
Weave chemistry algoscopy, Dot blot measure or slit engram detection method.
23. methods according to claim 22, wherein said enzyme immunoassay is a kind of Sandwich ELISA,
Described Sandwich ELISA uses the capture of specific binding people eIF4B or corresponding people's eIF4B ortholog thing to resist
The people eIF4B of body or its fragment and the people eIF4B of specific binding 406th serine phosphorylation or corresponding phosphorylation is lineal
The detection antibody of congener or its fragment.
24. methods according to claim 20, the group of wherein said sample choosing freely in vitro and internal sample composition.
25. methods according to claim 20, wherein said sample packages contains cancerous cell.
26. methods according to claim 25, wherein said cancerous cell is to select free MELK or eIF4B amplification or excessive table
Any cancer that any cancer reached, MELK or eIF4B having activation suddenly change and MELK or eIF4B appointing by other kinase activations
The cell of the group of meaning cancer composition.
27. methods according to claim 20, the choosing of wherein said sample is freely organized, whole blood, serum, blood plasma, cheek are scraped, saliva
Liquid, cerebrospinal fluid, urine, feces and the group of bone marrow composition.
Sample in 28. methods according to claim 20, wherein said step a) and/or step b) is to be subject to available from described
A part for the single sample of examination person.
Sample in 29. methods according to claim 20, wherein said step a) and/or step b) is to be subject to available from described
The part merging sample of examination person.
30. methods according to claim 20, wherein between described first time point and described subsequent point in time, described
Experimenter has accepted treatment of cancer, has completed treatment of cancer and/or be in the cancer remission phase.
31. methods according to claim 20, wherein said people eIF4B or its ortholog thing, and/or described people MELK
Or its ortholog thing, including nucleotide sequence as listed in table 1 or aminoacid sequence.
32. methods according to claim 20, wherein said reagent is little molecule, or antibody or its Fab.
33. methods according to claim 20, wherein said reagent is by the people eIF4B's of the 406th serine phosphorylation
In amount or people's eIF4B ortholog thing, the amount of corresponding phosphorylated amino acid reduces at least 50%.
34. 1 kinds of methods of patients suffering from cancer for treatment, including using the of suppression people eIF4B to described experimenter
The reagent of corresponding phosphorylated amino acid in 406 serine phosphorylations or people's eIF4B ortholog thing, thus treat described trouble
There is the patient of cancer.
35. methods according to claim 34, wherein said reagent is used with pharmaceutically acceptable dosage form.
36. methods according to claim 34, wherein said reagent is little molecule, or antibody or its Fab.
37. methods according to claim 34, wherein said reagent is directly in conjunction with described people eIF4B or its ortholog
Thing, or described people MELK or its ortholog thing.
38. methods according to claim 34, wherein said cancer choosing free MELK or eIF4B amplification or overexpression
Any cancer, any cancer having MELK or eIF4B of activation to suddenly change and MELK or eIF4B are by any cancer of other kinase activations
The group of disease composition.
39. methods according to claim 34, wherein said people eIF4B or its ortholog thing, and/or described people MELK
Or its ortholog thing, including nucleotide sequence as listed in table 1 or aminoacid sequence.
40. methods according to claim 34, wherein said reagent is little molecule, or antibody or its Fab.
41. methods according to claim 34, wherein said reagent is by the people eIF4B's of the 406th serine phosphorylation
In amount or people's eIF4B ortholog thing, the amount of corresponding phosphorylated amino acid reduces at least 50%.
42. methods according to claim 34, also include using one or more other anticarcinogen.
43. 1 kinds are used for determining people MELK or the function of its ortholog thing or the method for activity, including:
A) amount or the people eIF4B of corresponding aminoacid phosphorylation of the people eIF4B of the 406th serine phosphorylation in sample are detected
The amount of ortholog thing;
B) after operating described sample and/or after operation same sample or test sample, in same sample or test sample
One or more subsequent point in time repeat step a);With
C) amount of the people eIF4B of the 406th serine phosphorylation of detection or corresponding amino in comparison step a) and step b)
The amount of people's eIF4B ortholog thing of acid phosphoric acid,
The wherein people of the corresponding aminoacid phosphorylation of the amount of the people eIF4B of the 406th serine phosphorylation or people eIF4B
Compared at least one subsequent point in time and/or at least one is follow-up right in first time point for the amount of eIF4B ortholog thing
The operation of same sample or test sample changes, and shows that people MELK or the function of its ortholog thing or activity change
Become.
44. methods according to claim 43, the wherein amount of the people eIF4B of the 406th serine phosphorylation or people eIF4B
In ortholog thing, the amount of corresponding phosphorylated amino acid is determined by immunoassay, and this immunoassay use specifically
The reactant of people's eIF4B ortholog thing in conjunction with people eIF4B or the corresponding phosphorylation of the 406th serine phosphorylation.
45. methods according to claim 44, wherein said immunoassay are that radioimmunoassay, western blot are divided
Analysis, proximity ligation assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemoluminescence method, immune group
Weave chemistry algoscopy, Dot blot measure or slit engram detection method.
46. methods according to claim 45, wherein said enzyme immunoassay is a kind of Sandwich ELISA,
Described Sandwich ELISA uses the capture of specific binding people eIF4B or corresponding people's eIF4B ortholog thing to resist
The people eIF4B of body or its fragment and the people eIF4B of specific binding 406th serine phosphorylation or corresponding phosphorylation is lineal
The detection antibody of congener or its fragment.
47. methods according to claim 43, wherein said sample choosing freely external, in vitro and internal sample composition
Group.
48. methods according to claim 43, wherein said sample includes that cell or described method use based on cell
Algoscopy.
49. methods according to claim 48, wherein said cell is to select free MELK or eIF4B amplification or overexpression
Any cancer, have any by other kinase activations of any cancer and MELK or eIF4B that MELK or eIF4B of activation suddenly change
The cancerous cell of the group of cancer composition.
50. methods according to claim 43, wherein sample choosing freely organize, whole blood, serum, blood plasma, cheek are scraped, saliva,
Cerebrospinal fluid, urine, feces and the group of bone marrow composition.
Described same sample in 51. methods according to claim 43, wherein said step a) and/or step b) or survey
Sample is originally a part for the single sample available from described experimenter.
Described same sample in 52. methods according to claim 43, wherein said step a) and/or step b) or survey
Sample is originally the part merging sample available from described experimenter.
53. methods according to claim 43, wherein between described first time point and described subsequent point in time, described
Experimenter has accepted treatment of cancer, has completed treatment of cancer and/or be in the cancer remission phase.
54. methods according to claim 43, wherein said people eIF4B or its ortholog thing, and/or described people MELK
Or its ortholog thing, including nucleotide sequence as listed in table 1 or aminoacid sequence.
55. methods according to claim 43, wherein the operation to described sample is selected freely by described sample and test examination
Agent contacts, and the stream signal of described sample with MELK signal path is contacted, contacts group with by described sample with MELK inhibitor
The group become.
56. methods according to claim 55, wherein said test agent is little molecule, or antibody or its antigen binding fragment
Section.
57. according to the method described in claim 55 or 56, and wherein said test agent is by the people of the 406th serine phosphorylation
In the amount of eIF4B or people's eIF4B ortholog thing, the amount of corresponding phosphorylated amino acid reduces at least 50%.
58. 1 kinds for the method identifying the reagent of suppression people MELK or its ortholog thing kinase activity or carcinogenic activity, bag
Include:
A) i) people MELK or its ortholog thing and ii will be comprised) sample and the described examination of human histone H3 or its ortholog thing
Agent contacts;And
B) corresponding phosphoric acid in described reagent suppression the 3rd threonine phosphorylation of human histone H3 or its ortholog thing is determined
Change aminoacid;And/or corresponding phosphorylation amino in the 10th serine phosphorylation of human histone H3 or its ortholog thing
Acid;And/or the energy of corresponding phosphorylated amino acid in the 11st threonine phosphorylation of human histone H3 or its ortholog thing
Power, the phosphorylation wherein reduced identifies suppression people MELK or the kinase activity of its ortholog thing or the reagent of carcinogenic activity.
59. methods according to claim 58, wherein by the human histone H3 by the in sample the 3rd threonine phosphorylation
And/or the amount of the human histone H3 of the human histone H3 of the 10th serine phosphorylation and/or the 11st threonine phosphorylation, or
In human histone H3 ortholog thing, the amount of corresponding phosphorylated amino acid compares with compareing, and determines human histone H3
Described 3rd threonine phosphorylation and/or the 10th serine phosphorylation and/or the 11st threonine phosphorylation, or people organizes egg
The suppression of corresponding phosphorylated amino acid in white H3 ortholog thing.
60. methods according to claim 59, wherein said comparison is, compared in the case of unused described reagent or
The amount of the relatively early time point after described sample is contacted with described reagent, in described sample the 3rd threonine phosphorylation and/or
The described amount of the human histone H3 of the 10th serine phosphorylation and/or the 11st threonine phosphorylation or human histone H3 are lineal
The described amount of corresponding phosphorylated amino acid in congener.
61. methods according to claim 58, are wherein by by the in sample the 3rd threonine phosphorylation and/or the 10th
Phase in the amount of the human histone H3 of position serine phosphorylation and/or the 11st threonine phosphorylation or histone H 3 ortholog thing
The amount of the phosphorylated amino acid answered compares with compareing relative to the ratio of histone H 3 or the total amount of its ortholog thing, comes
Determine the described in human histone H3 the 3rd threonine phosphorylation and/or the 10th serine phosphorylation and/or the 11st threonine
The suppression of corresponding phosphorylated amino acid in phosphorylation, or human histone H3 ortholog thing.
62. methods according to claim 61, wherein comparison is, compared in the case of unused described reagent or by institute
State the ratio of the relatively early time point after sample contacts, the 3rd threonine phosphorylation and/or the 10th in described sample with described reagent
The described ratio of the human histone H3 of position serine phosphorylation and/or the 11st threonine phosphorylation or human histone H3 are lineal same
The described ratio of corresponding phosphorylated amino acid in the thing of source.
63., according to the method according to any one of claim 58-62, also include the amount determining mitosis specific proteins.
64. methods according to claim 58, also include determining that whether described reagent is directly in conjunction with described human histone H3
Or described its ortholog thing, or the step of described its ortholog thing of people MELK or described.
65. methods according to claim 58, wherein said sample choosing freely external, in vitro and internal sample composition
Group.
66. methods according to claim 58, wherein said sample packages contains cell.
67. methods according to claim 66, wherein said cell is cancerous cell.
68. methods according to claim 67, wherein said cell is to select free MELK or histone H 3 amplification or excessive
Any cancer expressed, the MELK having activation or any cancer of histone H 3 sudden change and MELK or histone H 3 are by other kinases
The cancerous cell of the group of any cancer composition of activation.
69. methods according to claim 66, wherein said cell is to obtain from experimenter.
70. methods according to claim 58, the choosing of wherein said sample is freely organized, whole blood, serum, blood plasma, cheek are scraped, saliva
Liquid, cerebrospinal fluid, urine, feces and the group of bone marrow composition.
71. according to the method according to any one of claim 58-62, wherein the 3rd threonine phosphorylation and/or the 10th silk
In the amount of the human histone H3 of propylhomoserin phosphorylation and/or the 11st threonine phosphorylation or human histone H3 ortholog thing accordingly
The amount of phosphorylated amino acid be by using specific binding 3rd threonine phosphorylation and/or the 10th serine phosphorylation
Change or the reactant of the human histone H3 of the 11st threonine phosphorylation or corresponding phosphorylated human histone H 3 ortholog thing
Carry out what immunoassay determined.
72. according to the method described in claim 71, and wherein said immunoassay are that radioimmunoassay, western blot are divided
Analysis, proximity ligation assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemoluminescence method, immune group
Weave chemistry algoscopy, Dot blot measure or slit engram detection method.
73. according to the method described in claim 72, and wherein said enzyme immunoassay is a kind of Sandwich ELISA,
Described Sandwich ELISA uses specific binding human histone H3 or corresponding human histone H3 ortholog thing
Capture antibody or its fragment and specific binding 3rd threonine phosphorylation and/or the 10th serine phosphorylation or the 11st
The detection antibody of the human histone H3 ortholog thing of human histone H3 or the corresponding phosphorylation of threonine phosphorylation or its sheet
Section.
74. according to any method described in claim 58-62, wherein said human histone H3 or its ortholog thing, and/or
Described people MELK or its ortholog thing, including nucleotide sequence as listed in table 1 or aminoacid sequence.
75. according to the method according to any one of claim 58-62, and wherein said reagent is little molecule, or antibody or its resist
Former binding fragment.
76. according to the method according to any one of claim 58-62, wherein said reagent by the 3rd threonine phosphorylation and/
Or the amount of the human histone H3 of the 10th serine phosphorylation and/or the 11st threonine phosphorylation and/or human histone H3 straight
It it is the amount reduction at least 50% of corresponding phosphorylated amino acid in congener.
77. 1 kinds for assessing the side that reagent suppresses the effect of people MELK or its ortholog thing kinase activity in subject
Method, including:
A) the 3rd threonine phosphorylation and/or the 10th serine phosphorylation in first time point detects experimenter's sample
And/or the human histone H3 of the amount of the human histone H3 of the 11st threonine phosphorylation or corresponding aminoacid phosphorylation is lineal same
The amount of source thing;
B) the one or more subsequent point in time after using described reagent repeat step a);Then
C) in comparison step a) and step b), the amount of the human histone H3 of phosphorylation or the people of corresponding aminoacid phosphorylation organize egg
The amount of white ortholog thing,
Wherein the 3rd threonine phosphorylation and/or the 10th serine phosphorylation and/or people's group of the 11st threonine phosphorylation
The amount of the amount of albumen H3 or the human histone H3 ortholog thing of corresponding aminoacid phosphorylation is higher than at least in first time point
One subsequent point in time, shows described reagent suppression people MELK or the kinase activity of its ortholog thing.
78. according to the method described in claim 77, wherein the 3rd threonine phosphorylation and/or the 10th serine phosphorylation
And/or corresponding phosphorylation ammonia in the amount of the human histone H3 of the 11st threonine phosphorylation or human histone H3 ortholog thing
Base acid amount determined by immunoassay, described immunoassay use be specifically bound to the 3rd threonine phosphorylation and/or
The human histone H3 of the 10th serine phosphorylation or the human histone H3 of the 11st threonine phosphorylation, or corresponding phosphorylation
The reagent of human histone H3 ortholog thing.
79. according to the method described in claim 78, and wherein said immunoassay are that radioimmunoassay, western blot are divided
Analysis, proximity ligation assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemoluminescence method, immune group
Weave chemistry algoscopy, Dot blot measure or slit engram detection method.
80. according to the method described in claim 79, and wherein said enzyme immunoassay is a kind of Sandwich ELISA,
Described Sandwich ELISA uses specific binding human histone H3 or corresponding human histone H3 ortholog thing
Capture antibody or its fragment and specific binding 3rd threonine phosphorylation and/or the 10th serine phosphorylation or the 11st
The detection antibody of the human histone H3 ortholog thing of human histone H3 or the corresponding phosphorylation of threonine phosphorylation or its sheet
Section.
81. according to the method described in claim 77, the group of wherein said sample choosing freely in vitro and internal sample composition.
82. contain cancerous cell according to the method described in claim 77, wherein said sample packages.
83. methods described in 2 according to Claim 8, wherein said cell is to select free MELK or histone H 3 amplification or excessive
Any cancer expressed, the MELK having activation or any cancer of histone H 3 sudden change and MELK or histone H 3 are by other kinases
The cancerous cell of the group of any cancer composition of activation.
84. according to the method described in claim 77, and the choosing of wherein said sample is freely organized, whole blood, serum, blood plasma, cheek are scraped, saliva
Liquid, cerebrospinal fluid, urine, feces and the group of bone marrow composition.
85. according to the method described in claim 77, wherein the described sample in step a) and/or step b) be taken from described in be subject to
A part for the single sample of examination person.
86. according to the method described in claim 77, wherein the described sample in step a) and/or step b) be taken from described in be subject to
The part merging sample of examination person.
87. according to the method described in claim 77, wherein between described first time point and described subsequent point in time, described
Experimenter has accepted treatment of cancer, has completed treatment of cancer and/or be in the cancer remission phase.
88. according to the method described in claim 77, wherein said human histone H3 or its ortholog thing, and/or described people
MELK or its ortholog thing, including nucleotide sequence as listed in table 1 or aminoacid sequence.
89. according to the method described in claim 77, and wherein said reagent is little molecule, or antibody or its Fab.
90. according to the method described in claim 77, and wherein said reagent is by the 3rd threonine phosphorylation and/or the 10th silk
In the amount of the human histone H3 of propylhomoserin phosphorylation and/or the 11st threonine phosphorylation or human histone H3 ortholog thing accordingly
Phosphorylated amino acid amount reduce at least 50%.
The method of 91. 1 kinds of patients suffering from cancer for treatment, uses suppression human histone H3 the 3rd Soviet Union including to experimenter
In propylhomoserin phosphorylation and/or the 10th serine phosphorylation and/or the 11st threonine phosphorylation or its ortholog thing corresponding
The reagent of phosphorylated amino acid, thus described in treatment, suffer from the patient of cancer.
92. use with pharmaceutically acceptable dosage form according to the method described in claim 91, wherein said reagent.
93. according to the method described in claim 91, and wherein said reagent is little molecule, or antibody or its Fab.
94. according to the method described in claim 91, and wherein said reagent is same directly in conjunction with described human histone H3 or its direct line
Source thing, or described people MELK or its ortholog thing.
95. according to the method described in claim 91, and wherein said cancer is to select free MELK or histone H 3 amplification or excessive
Any cancer expressed, the MELK having activation or any cancer of histone H 3 sudden change and MELK or histone H 3 are by other kinases
The group of any cancer composition of activation.
96. according to the method described in claim 91, wherein said human histone H3 or its ortholog thing, and/or described people
MELK or its ortholog thing, including nucleotide sequence as listed in table 1 or aminoacid sequence.
97. according to the method described in claim 91, and wherein said reagent is little molecule, or antibody or its Fab.
98. according to the method described in claim 91, and wherein said reagent is by the 3rd threonine phosphorylation and/or the 10th silk
In the amount of the human histone H3 of propylhomoserin phosphorylation and/or the 11st threonine phosphorylation or human histone H3 ortholog thing accordingly
Phosphorylated amino acid amount reduce at least 50%.
99. according to the method described in claim 91, also includes using one or more other anticarcinogen.
100. 1 kinds are used for determining people MELK or the function of its ortholog thing or the method for activity, including:
A) the 3rd threonine phosphorylation and/or the 10th serine phosphorylation and/or the 11st threonine phosphoric acid in detection sample
The amount of the human histone H3 changed or the amount of the human histone H3 ortholog thing of corresponding aminoacid phosphorylation;
B) after operating described sample and/or after operation same sample or test sample, at one or more subsequent point in time
Step a) is repeated in same sample or test sample;With
C) comparison step a) and described b) in the amount of human histone H3 of phosphorylation or the people of corresponding aminoacid phosphorylation organize egg
The amount of white H3 ortholog thing,
Wherein with at least one subsequent point in time and/or to compared with same sample or test sample subsequent operation at least one times,
The 3rd threonine phosphorylation of first time point and/or the 10th serine phosphorylation and/or the people of the 11st threonine phosphorylation
The quantity of the human histone H3 ortholog thing of histone H 3 quantity or corresponding aminoacid phosphorylation changes, and shows the mankind
MELK or its ortholog thing function or activity are changed.
101. according to the method described in claim 100, wherein the 3rd threonine phosphorylation and/or the 10th serine phosphorylation
Corresponding phosphorylation in the amount of the human histone H3 of change and/or the 11st threonine phosphorylation or human histone H3 ortholog thing
Amino acid whose amount is determined by immunoassay, this immunoassay use specific binding 3rd threonine phosphorylation and/or
10th serine phosphorylation or the human histone H3 of the human histone H3 of the 11st threonine phosphorylation or corresponding phosphorylation
The reagent of ortholog thing.
102. according to the method described in claim 100, and wherein said immunoassay are radioimmunoassay, western blot
Analysis, proximity ligation assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemoluminescence method, immunity
Histochemistry, Dot blot measure or slit engram detection method.
103. according to the method described in claim 102, and wherein said enzyme immunoassay is that a kind of sandwich enzyme-linked immunosorbent measures
Method, described Sandwich ELISA uses specific binding human histone H3 or corresponding human histone H3 ortholog thing
Capture antibody or its fragment and specific binding 3rd threonine phosphorylation and/or the 10th serine phosphorylation or the 11st
The detection antibody of the human histone H3 ortholog thing of human histone H3 or the corresponding phosphorylation of position threonine phosphorylation or its
Fragment.
104. according to the method described in claim 100, wherein said sample choosing freely external, in vitro and internal sample composition
Group.
105. according to the method described in claim 100, and wherein said sample includes that cell or described method use based on cell
Algoscopy.
106. according to the method described in claim 105, and wherein said cell is to select free MELK or histone H 3 amplification or mistake
Any cancer that degree is expressed, the MELK having activation or any cancer of histone H 3 sudden change and MELK or histone H 3 are by its separate excitation
The cancerous cell of the group of any cancer composition of enzyme activation.
107. according to the method described in claim 100, the choosing of wherein said sample is freely organized, whole blood, serum, blood plasma, cheek are scraped,
Saliva, cerebrospinal fluid, urine, feces and the group of bone marrow composition.
108. according to the same sample in the method described in claim 100, wherein said step a) and/or step b) or test
Sample is available from a part for the single sample of described experimenter.
109. according to the same sample in the method described in claim 100, wherein said step a) and/or step b) or test
Sample is available from the part merging sample of described experimenter.
110. according to the method described in claim 100, wherein between described first time point and described subsequent point in time, and institute
State experimenter to have accepted treatment of cancer, completed treatment of cancer and/or be in the cancer remission phase.
111. is according to the method described in claim 100, wherein said human histone H3 or its ortholog thing and/or described
People MELK or its ortholog thing, including nucleotide sequence as listed in table 1 or aminoacid sequence.
112. according to the method described in claim 100, and wherein the operation to described sample is selected described sample and test freely
Reagent contacts, and the stream signal of described sample with MELK signal path is contacted, contacts with MELK inhibitor with by described sample
The group of composition.
113. according to the method described in claim 112, and wherein said test agent is little molecule, or antibody or its antigen combine
Fragment.
114. according to the method described in claim 112 or 113, wherein said test agent by the 3rd threonine phosphorylation and/
Or the amount of the human histone H3 of the 10th serine phosphorylation and/or the 11st threonine phosphorylation or human histone H3 direct line are together
In the thing of source, the amount of corresponding phosphorylated amino acid reduces at least 50%.
Applications Claiming Priority (5)
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US201361902877P | 2013-11-12 | 2013-11-12 | |
US61/902,877 | 2013-11-12 | ||
US201461954046P | 2014-03-17 | 2014-03-17 | |
US61/954,046 | 2014-03-17 | ||
PCT/US2014/065173 WO2015073509A2 (en) | 2013-11-12 | 2014-11-12 | Biomarker for melk activity and methods of using same |
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EP (1) | EP3068930A4 (en) |
CN (1) | CN106232878A (en) |
AU (1) | AU2014348780B2 (en) |
CA (1) | CA2928464A1 (en) |
WO (1) | WO2015073509A2 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080293044A1 (en) * | 2003-08-14 | 2008-11-27 | Lisa Kadyk | Melks as Modifiers of the Rac Pathway and Methods of Use |
CN103173527A (en) * | 2011-12-22 | 2013-06-26 | 上海生物芯片有限公司 | Application of MELK gene |
Family Cites Families (2)
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EP2574929A1 (en) * | 2011-09-28 | 2013-04-03 | IMG Institut für medizinische Genomforschung Planungsgesellschaft M.B.H. | Marker in diagnosing prostate cancer (PC) |
KR101920109B1 (en) * | 2012-01-19 | 2018-11-19 | 온코세라피 사이언스 가부시키가이샤 | 1,5-naphthyridine derivatives and melk inhibitors containing the same |
-
2014
- 2014-11-12 CN CN201480072848.3A patent/CN106232878A/en active Pending
- 2014-11-12 AU AU2014348780A patent/AU2014348780B2/en not_active Ceased
- 2014-11-12 WO PCT/US2014/065173 patent/WO2015073509A2/en active Application Filing
- 2014-11-12 CA CA2928464A patent/CA2928464A1/en not_active Abandoned
- 2014-11-12 EP EP14862279.8A patent/EP3068930A4/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20080293044A1 (en) * | 2003-08-14 | 2008-11-27 | Lisa Kadyk | Melks as Modifiers of the Rac Pathway and Methods of Use |
CN103173527A (en) * | 2011-12-22 | 2013-06-26 | 上海生物芯片有限公司 | Application of MELK gene |
Non-Patent Citations (2)
Title |
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JIANLING YANG ET AL: "eIF4B Phosphorylation by Pim Kinases Plays a Critical Role in Cellular Transformation by Abl Oncogenes", 《CANCER RES》 * |
张汉骞: "猪母源胚胎亮氨酸拉链激酶MELK基因的克隆", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
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EP3068930A2 (en) | 2016-09-21 |
AU2014348780A1 (en) | 2016-06-02 |
WO2015073509A8 (en) | 2016-05-26 |
CA2928464A1 (en) | 2015-05-21 |
AU2014348780A8 (en) | 2016-06-30 |
WO2015073509A3 (en) | 2016-01-14 |
WO2015073509A2 (en) | 2015-05-21 |
AU2014348780B2 (en) | 2018-12-13 |
EP3068930A4 (en) | 2017-10-04 |
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