CN114136941A

CN114136941A - SARM1 enzyme inhibitor and screening method and application thereof

Info

Publication number: CN114136941A
Application number: CN202111420360.3A
Authority: CN
Inventors: 赵永娟; 李汉璋; 黎婉华
Original assignee: Peking University Shenzhen Graduate School
Current assignee: Peking University Shenzhen Graduate School
Priority date: 2020-11-27
Filing date: 2021-11-26
Publication date: 2022-03-04
Anticipated expiration: 2041-11-26
Also published as: CN114136941B

Abstract

The application discloses a SARM1 enzyme inhibitor, a screening method and application thereof. The screening method of the SARM1 enzyme inhibitor comprises the steps of mixing SARM1 enzyme, a candidate inhibitor, nicotinamide adenine dinucleotide and a fluorescent probe, reacting at normal temperature, detecting the rate of fluorescence generation in the reaction process, and judging that the candidate inhibitor has the activity of the SARM1 enzyme inhibitor if the fluorescence rate is less than the standard fluorescence rate; the standard fluorescence rate is the rate of fluorescence generation during the reaction detected under the same conditions without the addition of a candidate inhibitor; the fluorescent probe is formed by coupling a pyridine ring and a phenyl group or a phenyl alkenyl derivative. The screening method can simply obtain a large amount of active SARM1 enzyme inhibitors which can effectively inhibit SARM1 enzyme, thereby providing a new scheme and approach for inhibiting axonal degeneration and disease process and treating axonal degeneration-related neurodegenerative diseases.

Description

SARM1 enzyme inhibitor and screening method and application thereof

Technical Field

The application relates to the technical field of SARM1 enzyme inhibitors, in particular to a SARM1 enzyme inhibitor and a screening method and application thereof.

Background

SARM1 is a newly discovered signaling enzyme that acts as a performer in the process of axonal degeneration, an early event in many neurological disorders such as traumatic brain injury, parkinson's disease, and chemotherapy-induced peripheral neuropathy. In various neurodegenerative diseases, SARM1 is activated, leading to NAD and ATP depletion, mitochondrial dysfunction, and thus initiating a novel cell death mechanism; the knockout of SARM1 can inhibit axonal degeneration and disease progression, and therefore is considered to be a potential drug target for treating axonal degeneration related diseases.

Although, knockout of SARM1 inhibits axonal degeneration and disease progression; however, in clinical applications, it is difficult to effectively perform the SARM1 gene knockout technique for individuals with diseases, or the effect of treatment by gene knockout of SARM1 is limited. Therefore, how to effectively block the axonal degeneration-related neurodegenerative diseases performed by the SARM1 so as to achieve the effect of effective treatment still remains the focus and difficulty of the current SARM1 research.

Disclosure of Invention

The application aims to provide a novel method for screening a SARM1 enzyme inhibitor, and the SARM1 enzyme inhibitor obtained by screening and application thereof.

The following technical scheme is adopted in the application:

one aspect of the present application discloses a method for screening for an inhibitor of the SARM1 enzyme, comprising:

mixing SARM1 enzyme, a candidate inhibitor, Nicotinamide Adenine Dinucleotide (NAD) and a fluorescent probe to prepare a reaction system, reacting at normal temperature, detecting the rate of fluorescence generation in the reaction process, and marking as a first fluorescence rate;

determining that the candidate inhibitor has SARM1 enzyme inhibitor activity if the first fluorescence rate is less than the standard fluorescence rate; the greater the extent to which the first fluorescence rate is less than the standard fluorescence rate, the greater the inhibitory activity of the corresponding candidate inhibitor;

wherein, the standard fluorescence rate refers to the rate of fluorescence generation in the reaction process detected under the same condition without adding a candidate inhibitor, that is, the same amount of SARM1 enzyme, nicotinamide adenine dinucleotide and a fluorescent probe are mixed to prepare a reaction system, the reaction is carried out under the same reaction condition, the rate of fluorescence generation in the reaction process is detected and marked as the standard fluorescence rate; the fluorescent probe is used for detecting the enzymatic activity of SARM1 and is formed by coupling a pyridine ring and a phenylalkenyl or phenylalkenyl derivative.

The key to the screening method of the present application is to utilize the base exchange activity of NAD by the SARM1 enzyme. In the presence of the fluorescent probe, the fluorescent probe exchanges with nicotinamide of NAD to form a substance with fluorescence, namely PADs, SARM1 enzyme which can promote base exchange of NAD. That is, the enzymatic activity of SARM1 can be characterized by detecting the absorption spectrum kinetics of the substrate PCs and the product PADs and the fluorescence spectrum kinetics of the product PADs by a microplate reader; that is, the intensity of the activity of the SARM1 enzyme can be judged based on the rate of fluorescence generation. Thus, if a candidate inhibitor is capable of inhibiting SARM1 enzyme activity, the rate of fluorescence generation will be reduced; the stronger the inhibitor effect, the greater the degree of reduction. It is the screen of SARM1 enzyme inhibitors that was conducted in this application based on the above study.

It is also noted that the screening method for SARM1 enzyme inhibitors of the present application is key to the detection of SARM1 enzyme activity; that is, the accuracy and effectiveness of the screening method of the SARM1 enzyme inhibitor is based on the detection of SARM1 enzyme activity, which has strong specificity and high sensitivity. The fluorescent probe formed by coupling the pyridine ring and the phenyl or phenyl derivative is a novel detection substance provided by the inventor of the application in the previous patent application, has the advantages of strong specificity, high sensitivity, strong membrane permeability and the like, and can detect the enzymatic activity of the SARM1 or the activation process thereof in vitro or in living cells in real time. Reference may be made to patent application 202010489831.5 for fluorescent probes of the present application, which is incorporated herein by reference in its entirety for its full disclosure in connection with the detection of SARM1 enzymatic activity.

In one implementation of the present application, the fluorescent probe is at least one of a structure shown by PC1 to a structure shown by PC 15;

preferably, the fluorescent probe has a structure represented by PC5, PC6, PC7, PC8, PC10 or PC11, and more preferably PC6 and PC 11.

It should be noted that the fluorescent probes from the structure shown in PC1 to the structure shown in PC15 are fluorescent probes that have been confirmed by the inventors of the present application to be capable of detecting the enzymatic activity of SARM1, and on the basis of the fluorescent probes, more fluorescent probes can be designed and synthesized, for example, the fluorescent probes are modified on the basis of pyridine ring or benzene base, so as to prepare and obtain more new fluorescent probes.

It should be further noted that PC5, PC6, PC7, PC8, PC10, and PC11 are fluorescent probes that demonstrate better activity in one implementation of the present application, and that other fluorescent probes have relatively poor activity; thus, PC5, PC6, PC7, PC8, PC10 or PC11 are preferred in preferred embodiments. Of course, other nine fluorescent probes may be used where the requirements are less stringent.

In one implementation mode of the application, the method further comprises adding an activator into the reaction system to promote the reaction.

Preferably, the activator is nicotinamide mononucleotide or an analogue thereof.

It is noted that the activator acts to release the intramolecular self-inhibition of SARM1 and activate the enzymatic activity; it is to be understood that the compound can be used as an activator or activation method of the present application as long as the compound has the above-mentioned effect. Specifically employed in one implementation of the present application is nicotinamide mononucleotide or CZ-48. Of course, if a cloned SARM1 enzyme that has been released from inhibition is used, an activator may not be required.

In one implementation manner of the present application, the reaction system and the reaction conditions of the first fluorescence rate specifically include that the SARM1 enzyme and the candidate inhibitor are incubated in Tris-HCl solution for at least 10 minutes, then nicotinamide adenine dinucleotide and the fluorescent probe are added into the solution, the reaction is performed for at least 30 minutes at normal temperature, and the rate of fluorescence generation in the reaction process, that is, the first fluorescence rate, is detected. If it is desired to add an activator of nicotinamide mononucleotide or an analogue thereof, it is added together with the fluorescent probe.

Similarly, the reaction system and reaction conditions of the standard fluorescence rate specifically include that SARM1 enzyme is firstly incubated in Tris-HCl solution for at least 10 minutes, then nicotinamide adenine dinucleotide and a fluorescent probe are added into the SARM1 enzyme, the reaction is carried out for at least 30 minutes at normal temperature, and the rate of fluorescence generation in the reaction process, namely the standard fluorescence rate, is detected. If it is desired to add an activator of nicotinamide mononucleotide or an analogue thereof, it is added together with the fluorescent probe.

As for the concentration of the SARM1 enzyme, the candidate inhibitor, the nicotinamide adenine dinucleotide, the fluorescent probe and the like in the reaction system, the concentration of the examples of the present application can be referred to or adjusted according to the detection requirements, so as to be able to efficiently generate fluorescence.

Another aspect of the present application discloses a SARM1 enzyme inhibitor obtained using the screening method of the present application.

The SARM1 enzyme inhibitor obtained by the screening method can screen the activity of the required inhibitor according to the requirement, and better meets the clinical application of neurodegenerative diseases related to axonal degeneration.

In an implementation manner of the present application, the SARM1 enzyme inhibitor obtained by the screening method of the present application includes Nifedipine (NFDP), Nisoldipine (NSDP), Nicardipine (NCDP), Nimodipine (NMDP), Amlodipine (Amlodipine), Felodipine (Felodipine), Clevidipine butyrate (Clevidipine butyrate), Isradipine (Isradipine), diltiazem

(dintizem), Verapamil (Verapamide), dehydronitrosonifedipine (dHN-Nifedipine, dHN-NFDP), dehydronitrosonidipine (dHN-Nisodipine, dHN-NSDP), dehydronitrosonicardipine (dHN-Nicardipine, dHN-NCDP), dehydronitrosonidipine (dHN-Nimodipine, dHN-NMDP), dehydroamlodipine (dH-Amlodipine), dehydrofelodipine (dH-Felodipine), dehydrobutyric acid Clevidipine (dH-Clevidipine butyrate), dehydroepirubicin (dH-Isradipine), Epirubicin hydrochloride (Epirubicin hydrochloride), Oritavancin diphosphate (Oritanancine diphosphate), benzalkonium chloride (Benzethimide), Berberine hydrochloride (Berberine), dihydronilotinib (Beloridinil), Sunitinib (Estradiol), Sunitinib (Merunethimide), Sunitinib (Merunidipine (mercuridine), and Merunidin (mercuridinil), Merunib (mercuridinil) Malate (mercuridine), and Merunib (mercuridinil) salts (Merunimide), and their salts (R, L, menadione (Menadione), Bismuth subsalicylate (Bismuth subsalicylate), Phenolphthalein (phenophtalin), Bismuth Subgallate (Bismuth Subgallate), Sanguinarine hydrochloride (Sanguinarine chloride), Evans blue (Evans blue), methyl Violet (Crystal Violet), cetrimide (Cetrimonium bromide), Zinc pyrithione (Zinc pyrithionone), Carboplatin (Carboplatin), amphotericin B (Ampelothricin B), Omeprazole (Omeprazole), Diethylstilbestrol (Diethylstilbestrol), Tigecycline (Tigecycline), Zinc undecylenate (Zinc monocyclopyralate), Lapatinib diglycolate (Lapatinib dimorpholine hydrochloride), hydrochloric acid (doxycycline), Tetracycline (Tetracycline hydrochloride), Tetracycline (Tetracycline), Chlortetracycline (Chlortetracycline), chlorfenadine (Tetracycline), chlorfenapyr (hydrochloride), chlorfenadine (Tetracycline), chlorfenadine (Tetracycline (hydrochloride), chlorfenadine (Tetracycline), chlorfenapyr (chlorfenapyr), chlorfenadine (chloride), chlorfenapyr), chlorfenadine (e (Tetracycline), chlorfenapyr), and chlorfenapyr (e), and chlorfenapyr), and so-D, such as (e, such as a salt), such as a salt, such as a compound, such as an extract, such as a compound, such as a compound, such as a compound, a, Butylbenzoic acid (Bufexamac), Epalrestat (Epalrestat), Entacapone (Entacapone), Reserpine (Reserpine), Tannic acid (Tannic acid), Carbazochrome Sodium Sulfonate (carbachome Sodium Sulfonate), Mefloquine hydrochloride (Mefloquine hydrochloride), Phenazopyridine hydrochloride (Phenazopyridine hydrochloride), and axineTinib (Axitinib), Otilonium Bromide (Otilonium Bromide), Thiostrepton (Thiostrepton), Montelukast sodium (Montelukast sodium), Panobinostat (Panobinostat), Levosimendan (Levosimendan), Lapatinib Ditosylate xylenesulfonate (Lapatinib Ditosylate), Ceftiofur hydrochloride (Ceftiofur hydrochloride), Daunorubicin hydrochloride (daunomycin hydrochloride), Mecobalamin (Mecobalamin), Afatinib (Afatinib), dimercaptozel (Surccimer), Crocin (Crocin), Povidone iodine (Povidone iodide), sodium Tanshinone A sulfonate (Tanshinone ilfonato sodium), dimecycline hydrochloride (Decylcociline hydrochloride), luteolin (bismuth subclinic), silvicine (bismuth), bismuth (bismuth subcarbonate), bismuth (bismuth subclinic), bismuth (bismuth), bismuth (bismuth), bismuth (bismuth), bismuth (bismuth), bismuth (bismuth), bismuth (bismuth), bismuth (bismuth), sodium (bismuth), sodium (bismuth), sodium, bismuth), bismuth (bismuth), bismuth (bismuth), sodium, bismuth), bismuth (bismuth), sodium (bismuth), sodium, Idarubicin hydrochloride (Idarubicin HCL), Pirarubicin (Pirarubicin), afatinib maleate (Afatinib dimaleate), Rifabutin (Rifabutin), Octenidine hydrochloride (Octenidine dihydrate), Dantrolene sodium salt heptahydrate (Dantrolene sodium hydrate), Mitoxantrone (Mitoxantrone), Fondaparinux sodium (Fondaparinux sodium), Fidaxomicin (Fidaxomicin), Visimitin (E/Z), Endoxifen (Elbasvir), Thielamine (fursutine), Cefotiam (Cefotiam hydrochloride), Malathion (Malathion), Strychnine (Strycyline), Thimerosin (Thixoside), thimerosin (Tetrafosinate), pyrazosin (Tetrafolin), pyraclostrobin (Tetramethylosine), pyrazosin (Tetrafosmin A), pyrazosin (Tetramethylosine), pyrazosin (Tetrafosmin (Tetramethyle), pyrazosin (Tetramethylosine), pyrazosin (Tetrafosmin (Tetradoxine), Tetrafosmin (Tetramethylosine (A), Tetramethylosine (Tetrafosmin (Tetramethylosine), Tetrafosmin (Tetramethylosine (A), Tetrafosmin, Tetramethylosine (Tetramethylosine), Tetrafosmin (Tetramethylosine, Tetradoxine (E, Tetramethylosine, Tetramethyle, Tetramethylosine, Tetramethyle, Tetramethylosine, Tetramethyle, Tetramethylbenomycoside, Tetramethyle, Tetramethylosine, Tetramethyle, Tetramethylbenomyl, Tetramethyle, dihydropyrazozine, Tetramethyle, and/Z, Tetramethyle, or Tetramethyle, or Tetramethyle, E, or Tetramethyle, E, Tetramethyle, E, or Tetramethylbenomyl, and/L-L, or Tetramethylbenomyl, E, and/Z, E, or Tetramethyle, E, and/L-L, Berberine (berberberine), Anidulafungin (Anidulafungin), and Dovitinib (Dovitinib).

It is to be noted that the specific SARM1 enzyme inhibitors described above are only compounds screened for their ability to inhibit SARM1 enzymatic activity in one implementation of the present application; under the same inventive concept, the screening method of the present application can also screen more SARM1 enzyme inhibitors, which is not specifically limited herein.

In yet another aspect, the present application discloses the use of an inhibitor of the SARM1 enzyme of the present application in the manufacture of a medicament for the treatment of a neurodegenerative disease associated with axonal degeneration.

It should be noted that, the study of the present application found that these compounds have the activity of SARM1 enzyme inhibitor, and based on this, the skilled person would understand that the SARM1 enzyme inhibitor of the present application can be used to prepare the drugs for treating the neurodegenerative diseases related to axonal degeneration without any error. As for the pharmaceutical dosage form, it may be determined according to the requirements, and is not particularly limited herein.

It will be appreciated that the present application uses an inhibitor of the SARM1 enzyme in the manufacture of a medicament for the treatment of axonal degeneration-related neurodegenerative disorders including, but not limited to, parkinson's disease, alzheimer's disease, glaucoma, amyotrophic lateral sclerosis, chemotherapy-induced peripheral neuropathy, diabetes-induced peripheral neuropathy and stroke-induced axonal degeneration.

In yet another aspect, the present application discloses a medicament for treating a neurodegenerative disease associated with axonal degeneration, comprising an inhibitor of the SARM1 enzyme of the present application.

Likewise, the agents of the present application are capable of treating neurodegenerative diseases associated with isometric mutations including, but not limited to, Parkinson's disease, Alzheimer's disease, glaucoma, amyotrophic lateral sclerosis, chemotherapy-induced peripheral neuropathy, diabetes-induced peripheral neuropathy, and stroke-induced axonal degeneration.

In one implementation of the present application, the medicament of the present application further comprises a pharmaceutically acceptable carrier.

It should be noted that the key point of the present application lies in the research and discovery that the SARM1 enzyme inhibitor of the present application can be used for treating neurodegenerative diseases related to axonal degeneration, and the carrier and the added auxiliary components used for preparing the drug can be determined according to the required dosage form, and are not limited herein. In addition, other active ingredients can be added into the medicine according to the requirements, so that a synergistic treatment effect is achieved; specifically, reference may be made to the existing pharmaceutical knowledge, which is not specifically limited herein.

The beneficial effect of this application lies in:

the screening method of the SARM1 enzyme inhibitor can simply and effectively screen and obtain a large number of SARM1 enzyme inhibitors with different activities; in addition, the obtained SARM1 enzyme inhibitor can effectively inhibit the activity of SARM1 enzyme, thereby providing a new scheme and approach for inhibiting axon degeneration and disease process and treating neurodegenerative diseases related to axon degeneration.

Drawings

FIG. 1 is a graph of the results of a Western blot of dN-SARM1 protein in an example of the present application;

FIG. 2 is a statistical graph of the inhibition efficiency of high throughput screening of drug libraries for inhibition of SARM1 activity by PC6 probe fluorescence assay in the examples of the present application;

FIG. 3 is a graph showing the results of half inhibitory concentration (IC50) of an inhibitor measured by PC6 fluorescence and HPLC analysis in examples of the present application;

FIG. 4 is a graph showing the results of inhibition of SARM1 activity by dehydronitrosonisoldipine, dehydronitrosonifedipine, dehydronitrosonimodipine, dehydronitrosonicardipine, and dehydroisradipine in the examples of the present application;

FIG. 5 is an intracellular inhibition curve of 9 compounds of the present example, including nifedipine, nicardipine, nimodipine, felodipine, amlodipine, clevidipine butyrate, isradipine, verapamil, diltiazem

；

FIG. 6 is a graph demonstrating that inhibition of SARM1 by dHNN is time-dependent in the examples of the present application, where nicotinamide (Nam) is the control, and inhibition is time-independent;

FIG. 7 is a graph demonstrating irreversible inhibition of SARM1 by dHNN in the examples of the present application, where nicotinamide (Nam) is the control, which inhibition is irreversible;

FIG. 8 shows that the inhibition of SARM1 by dHNN mainly inhibits the activation of protein, and the inhibition of fully activated truncated SAM-TIR is weaker in the present application example;

FIG. 9 is a graph of the results of the inhibition of inducible iSARM1 and iSAM-TIR by dHNN in cells of the present application;

FIG. 10 is a mass spectrum secondary polypeptide spectrum of a modification of SARM1 cysteine by dHNN in an example of the present application;

FIG. 11 is a graph of the results of dHNN inhibition of cADPR production by vincristine-induced SARM1 activation of primary dorsal root neurons in the examples of the present application;

FIG. 12 is a graph of the results of dHNN inhibition of vincristine-induced axonal degeneration caused by SARM1 activation of primary dorsal root neurons in the examples of the present application;

FIG. 13 is a statistical plot of the time profile of inhibition of vincristine-induced axonal degeneration by SARM1 activation of primary dorsal root neurons by dHNN in the examples of the present application;

FIG. 14 is a graph of the results of inhibition of axonal degeneration caused by SARM1 activation of injury-induced primary dorsal root neuronal cells by dHNN in the examples of the present application;

fig. 15 is a statistical graph of the time profile of inhibition of axonal degeneration by dHNN on injury-induced activation of SARM1 in primary dorsal root neuronal cells in the examples of the present application.

Detailed Description

The pathogenesis of neurodegenerative diseases is very complex and needs to be studied intensively. It is generally considered that neurodegenerative diseases are mainly caused by intracellular gene mutations, resulting in aggregation and accumulation of a large number of misfolded proteins or polypeptides in the brain or spinal cord, loss of synaptic connections, and various signaling pathway disorders including degradation pathway disorder of proteins, autophagosomal dysfunction, mitochondrial dysfunction, etc., further causing a large number of nerve cell deaths, thereby disrupting neural networks to cause serious neurological diseases. With the research on the molecular mechanism of neurodegenerative diseases, more and more mechanisms are known and perfected. Axial degeneration is increasingly recognized as an early event and important driver of the development and progression of neurodegenerative diseases including Alzheimer's Disease (AD), Parkinson's Disease (PD), Traumatic Brain Injury (TBI), Chemotherapy-induced peripheral neuropathy (CIPN), and muscular Atrophy (ALS). Axonal degeneration is also considered a potential target for alleviating neuropathy since it precedes extensive nerve damage.

SARM1 is a multifunctional signal enzyme, which can catalyze various substrates NAD, NADP and NA, etc. to generate signal molecules cADPR, ADPR and NAADP, etc. In various neurodegenerative diseases, SARM1 is activated, leading to NAD depletion, which in turn initiates a novel cell death mechanism; knockout of SARM1 inhibits axonal degeneration and disease progression and is considered to be a potential drug target for related neurological diseases, including TBI, CIPN, ASL, and the like. The function of the SARM1 in axonal mutation is inseparable from the enzyme activity, so that screening inhibitors against the enzyme activity is an effective strategy for treating the SARM 1-dependent axonal mutation.

SARM1 is composed of three domains, namely, an ARM (Armadillo/HEAT repeat) domain at the nitrogen terminal, two SAM (Sterile alpha motif) domains in series, and a TIR (Toll/Interleukin Receptor) domain at the carbon terminal, and further has a mitochondrial localization signal peptide at the nitrogen terminal. SARM1 is a multifunctional protease that reacts with a variety of substrates, including NAD, NADP, NA and cADPR, possibly catalyzed by E642 as active site to produce reaction intermediates that are eventually attacked by various nucleophilic molecules (e.g., water, adenine N1, nicotinic acid) to form ADPR, cADPR or NAADP, among others.

The SARM 1-mediated axonal degeneration is enzyme activity dependent. Axonal injury activates SARM1, resulting in NAD depletion and axonal degeneration. In SARM1 knockout neurons, overexpression of the SARM1 truncation without the TIR-active domain failed to cause injury-induced axonal degeneration. The E642 mutation on TIR to alanine resulted in loss of enzymatic activity of SARM1 with loss of its mediated axonal degeneration function. In cell lines overexpressing SARM1, cell death can be induced by the addition of the membrane permeability activator CZ-48 to activate SARM 1.

In the normal state, the SARM1 is in a self-inhibiting state. Upon stimulation, the self-inhibitory ARM domain is released to change the conformation of SARM1, TIR dimerizes, and SARM1 is activated to catalyze the hydrolysis and cyclization of NAD to ADPR and cADPR and release Nam. More and more evidence supports this model. First, removal of the ARM domain constitutively activates SAM-TIR, leading to axonal degeneration of uninjured neurons, suggesting that ARM inhibits enzymatic activity of SARM1 under normal conditions. Expression of only the TIR domain with no detectable axonal degeneration suggests that SAM and TIR are essential for SARM1 enzymatic activity. TIR is expressed by fusing with FKBP/Frb, and rapamycin treatment induces dimerization, can activate NAD + hydrolytic activity of the TIR, leads to axonal degeneration, and indicates that the TIR is an enzyme-active structural domain of SARM1, and SAM mediates protein dimerization to activate the TIR.

Reduced levels of NMNAT2 in axonal degeneration can activate SARM 1. NMNAT2 may modulate the activity of SARM1 by modulating its metabolite levels. When the content of NMNAT2 is reduced, the NAD of a product is reduced, the NMN of a substrate is accumulated, and the ratio of NMN/NAD is increased to promote axonal degeneration. These results suggest that NMN/NAD levels may modulate the activation of SARM 1. The membrane permeability activator CZ-48 further demonstrated that NMN was able to directly activate SARM1, while multiple SARM1 full-length structures showed that high concentrations of NAD were able to effectively inhibit SARM1 protein activity, immobilizing it in a self-inhibiting structure.

Calcium ion release can promote axonal degeneration, and knocking out SARM1 can affect a calcium ion signal path in the axonal degeneration, and the action mechanism of the SARM1 can be related to metabolism of small molecules, namely cADPR and ADPR. Knock-out of SARM1 inhibited axmutation and cell death induced by pro-oxidant treatment. The process of induction of SARM1 activation and cell death by the activator CZ-48 is accompanied by an increase in mitochondrial ROS, enhanced depolarization, and ATP depletion. These results indicate that the axonal degeneration and cell death process induced by SARM1 is a novel death mechanism, and may involve calcium signaling pathway and mitochondrial dysfunction.

In summary, SARM1 is a multifunctional signaling enzyme capable of catalyzing a variety of substrates, NAD, NADP, NA, etc., to produce signaling molecules, cADPR, ADPR, NAADP, etc. In various neurodegenerative diseases, SARM1 is activated, leading to NAD depletion, which in turn initiates a novel cell death mechanism; knock-out of SARM1 inhibits axonal degeneration and disease progression and is therefore considered a potential drug target for related neurological diseases, including TBI, CIPN, ASL, and others. Therefore, the development of the inhibitor aiming at the activity of the SARM1 can promote the treatment and drug development of neurodegenerative diseases and is helpful to reveal the specific enzyme activity mechanism, protein structure analysis and signal path of the SARM 1.

Part of the english abbreviations of this application are explained as follows:

cADPR: cyclic ADP-ribose, i.e., cyclic adenosine diphosphate ribose.

ADPR (adenosine triphosphate): ADP-ribose, i.e., adenosine diphosphate ribose.

NAADP: nicotinic acid adenosine dinucleotide phosphate, i.e., nicotinic acid adenine dinucleotide phosphate.

DRG: dorsal root ganglia, dorsal root ganglia.

NMN: nicotinamide mononuleotide, nicotinamide mononucleotide.

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Examples

Expression and purification of SARM1 protein

(1) Plasmid construction

In this example, the gene sequence of dN-SARM1 was PCR-amplified, the N-terminal mitochondrial localization signal peptide of SARM1 was removed, and the PCR-amplified product was constructed into pLenti-CMV-puro-dest plasmid (adddge catalog #17452) as follows:

the BC2T-TEV polypeptide gene fragment, dN-SARM1-F and dN-SARM1-R were synthesized by Shanghai Producer. Wherein, the BC2T-TEV polypeptide gene segment is a sequence shown by Seq ID No.1, the dN-SARM1-F is a sequence shown by Seq ID No.2, and the dN-SARM1-R is a sequence shown by Seq ID No. 3.

Seq ID No.1：

5’-CTCATGccagacagaaaagcggctgttagtcactggcagcaaGATATCGGCGGAGGCGGATCTGGCGGAGGCGGATCTGGCGGAGGCGGATCTgagaatttgtattttcagggtGGCGGAGGCGGAGGTACCCTG-3’

Seq ID No.2：5’-GGTACCCTGGCGGTGCCTGGGCCAG-3’

Seq ID No.3：5’-GCGGCCGCCTAGGTTGGACCCATGGGTGCAGCACCC-3’

The synthesized BC2T-TEV polypeptide gene fragment was ligated to pENTR vector pENTR1A-GFP-N2 (addge: catalog #19364) using HindIII/KpnI enzymatic cleavage sites. The dN-SARM1 gene fragment is amplified by using primers dN-SARM1-F and dN-SARM1-R, and the amplified dN-SARM1 gene fragment is constructed on a pENTR vector with BC2T-TEV through KpnI and NotI enzyme cleavage sites. All endonucleases of this example were purchased from ThermoFisher.

The dN-SARM1 gene fragment obtained by PCR amplification is the sequence shown in Seq ID No. 4.

Seq ID No.4：

GGTACCCTGGCGGTGCCTGGGCCAGATGGGGGCGGTGGCACGGGCCCATGGTGGGCTGCGGGTGGCCGCGGGCCCCGCGAAGTGTCGCCGGGGGCAGGCACCGAGGTGCAGGACGCCCTGGAGCGCGCGCTGCCGGAGCTGCAGCAGGCCTTGTCCGCGCTGAAGCAGGCGGGCGGCGCGCGGGCCGTGGGCGCCGGCCTGGCCGAGGTCTTCCAACTGGTGGAGGAGGCCTGGCTGCTGCCGGCCGTGGGCCGCGAGGTAGCCCAGGGTCTGTGCGACGCCATCCGCCTCGATGGCGGCCTCGACCTGCTGTTGCGGCTGCTGCAGGCGCCGGAGTTGGAGACGCGTGTGCAGGCCGCGCGCCTGCTGGAGCAGATCCTGGTGGCTGAGAACCGAGACCGCGTGGCGCGCATTGGGCTGGGCGTGATCCTGAACCTGGCGAAGGAACGCGAACCCGTAGAGCTGGCGCGGAGCGTGGCAGGCATCTTGGAGCACATGTTCAAGCATTCGGAGGAGACATGCCAGAGGCTGGTGGCGGCCGGCGGCCTGGACGCGGTGCTGTATTGGTGCCGCCGCACGGACCCCGCGCTGCTGCGCCACTGCGCGCTGGCGCTGGGCAACTGCGCGCTGCACGGGGGCCAGGCGGTGCAGCGACGCATGGTAGAGAAGCGCGCAGCCGAGTGGCTCTTCCCGCTCGCCTTCTCCAAGGAGGACGAGCTGCTTCGGCTGCACGCCTGCCTCGCAGTAGCGGTGTTGGCGACTAACAAGGAGGTGGAGCGCGAGGTGGAGCGCTCGGGCACGCTGGCGCTCGTGGAGCCGCTTGTGGCCTCGCTGGACCCTGGCCGCTTCGCCCGCTGTCTGGTGGACGCCAGCGACACAAGCCAGGGCCGCGGGCCCGACGACCTGCAGCGCCTCGTGCCGTTGCTCGACTCTAACCGCTTGGAGGCGCAGTGCATCGGGGCTTTCTACCTCTGCGCCGAGGCTGCCATCAAGAGCCTGCAAGGCAAGACCAAGGTGTTCAGCGACATCGGCGCCATCCAGAGCCTGAAACGCCTGGTTTCCTACTCTACCAATGGCACTAAGTCGGCGCTGGCCAAGCGCGCGCTGCGCCTGCTGGGCGAGGAGGTGCCACGGCCCATCCTGCCCTCCGTGCCCAGCTGGAAGGAGGCCGAGGTTCAGACGTGGCTGCAGCAGATCGGTTTCTCCAAGTACTGCGAGAGCTTCCGGGAGCAGCAGGTGGATGGCGACCTGCTTCTGCGGCTCACGGAGGAGGAACTCCAGACCGACCTGGGCATGAAATCGGGCATCACCCGCAAGAGGTTCTTTAGGGAGCTCACGGAGCTCAAGACCTTCGCCAACTATTCTACGTGCGACCGCAGCAACCTGGCGGACTGGCTGGGCAGCCTGGACCCGCGCTTCCGCCAGTACACCTACGGCCTGGTCAGCTGCGGCCTGGACCGCTCCCTGCTGCACCGCGTGTCTGAGCAGCAGCTGCTGGAAGACTGCGGCATCCACCTGGGCGTGCACCGCGCCCGCATCCTCACGGCGGCCAGAGAAATGCTACACTCCCCGCTGCCCTGTACTGGTGGCAAACCCAGTGGGGACACTCCAGATGTCTTCATCAGCTACCGCCGGAACTCAGGTTCCCAGCTGGCCAGTCTCCTGAAGGTGCACCTGCAGCTGCATGGCTTCAGTGTCTTCATTGATGTGGAGAAGCTGGAAGCAGGCAAGTTCGAGGACAAACTCATCCAGAGTGTCATGGGTGCCCGCAACTTTGTGTTGGTGCTATCACCTGGAGCACTGGACAAGTGCATGCAAGACCATGACTGCAAGGATTGGGTGCATAAGGAGATTGTGACTGCTTTAAGCTGCGGCAAGAACATTGTGCCCATCATTGATGGCTTCGAGTGGCCTGAGCCCCAGGTCCTGCCTGAGGACATGCAGGCTGTGCTTACTTTCAACGGTATCAAGTGGTCCCACGAATACCAGGAGGCCACCATTGAGAAGATCATCCGCTTCCTGCAGGGCCGCTCCTCCCGGGACTCATCTGCAGGCTCTGACACCAGTTTGGAGGGTGCTGCACCCATGGGTCCAACCTAG

The PCR amplification reaction system is as follows: 5 × PrimeSTAR Buffer (Mg)²⁺plus) 10. mu.L, dNTP mix (2.5mM each) 4. mu.L, dN-SARM1-F added to a final concentration of 0.2. mu. mol/L, dN-SARM1-R, DNA template 100ng added to a final concentration of 0.2. mu. mol/L, PrimeSTAR HS DNA Polymerase (2.5U/. mu.L) 0.5. mu.L, and finally sterilized ddH₂O to 50. mu.L. The full-length SARM1 was synthesized into a pUC57 plasmid by Wittig Bio Inc., and PCR was performed using pUC57-SARM1 as a DNA template.

The PCR amplification conditions were: denaturation at 98 ℃ for 4 min, then 30 cycles: 10s at 98 ℃, 5s at 60 ℃ and 2 minutes at 72 ℃, extending for 5 minutes at 72 ℃ after the circulation is finished, and standing by at 4 ℃.

The PCR amplification product is subjected to agarose gel electrophoresis, then is recovered and purified by an Omega gel recovery kit D2500-02, and the specific steps of gel cutting recovery refer to the kit specification. The purified PCR amplification product was recovered for construction into pENTR vector with BC 2T-TEV.

The construction system of the recombinant plasmid comprises the following steps:

an enzyme digestion reaction system: 800ng of the recovered PCR product or plasmid, 1. mu.L of each endonuclease (Fastduest), and 1. mu.L of a buffer solution were added to the recovered PCR product or plasmid, and sterilized water was added thereto to a volume of 10. mu.L. The digestion reaction is carried out at 37 ℃ for 30 minutes.

Plasmid ligation: after the enzyme digestion reaction is finished, 300ng of the enzyme-digested PCR amplification recovery product and 50ng of the enzyme-digested plasmid are uniformly mixed with 1 mu L of T4 DNA ligase 1 mu L, T4 DNA ligase buffer solution, and sterile water is supplemented until the volume is 20 mu L. The ligation conditions were thermostated at 16 ℃ overnight.

The ligation product was subjected to agarose gel electrophoresis, and then recovered and purified using an Omega gel recovery kit D2500-02, and the recovered and purified product, i.e., the recombinant plasmid of this example, was designated pENTR1A-BC2T-dN-SARM 1.

After the plasmid construction of pENTR1A-BC2T-dN-SARM1 was completed, dN-SARM1 was recombined to pLenti-CMV-puro-dest by LR reaction.

A recombination reaction system: 150ng of pENTR1A-BC2T-dN-SARM1, 50ng of pLenti-CMV-puro-dest, 1. mu.L of 5 XL clonaseaction buffer, and sterile water to a total volume of 5. mu.L.

(2) Transfection

In this example, a virus carrying a dN-SARM1 reading frame was prepared by co-transfecting pLenti-CMV-puro-dest constructed with the virus packaging plasmids psPAX2, pMD2.G (addge psPAX2: #12260, pMD2.G: #12259) into HEK293T cells (ATCC) by liposome lipofectamine 2000(Life Technologies). The method comprises the following specific steps:

spreading in a 3.5cm dish at a height of 1X 10⁶Individual cells, transfected the next day.

Plasmid mixture: mu.g of pLenti-dN-SARM1, 1.7. mu.g of psPAX2, 0.6. mu.g of pMD2.G, and 8. mu.L lipofectamine 2000 transfection reagents, were transfected according to the instructions, and after 8 hours, the solution was changed and virus was collected for 48 hours.

(3) Cell screening

The HEK293T cell obtained in the step of (2) transfection is infected by dN-SARM1 virus, and the cell which stably expresses the dN-SARM1 protein is obtained by adding puromycin for screening. The method comprises the following specific steps:

virus: 80 μ L/3.5cm infection 2X 10⁵After 48 hours of infection, puromycin 2. mu.g/mL was added for selection, and 48 hours after selection, cells not infected with the virus were completely killed. The virus-infected cells were mostly viable and screened again for 48 hours with the addition of puromycin 2. mu.g/mL.

(4) Protein extraction

Culturing and collecting the cells stably expressing the dN-SARM1 protein obtained in the step of (3) cell screening, and obtaining the dN-SARM1 protein expressed in cytoplasm by a digitonin cracking mode for in vitro activity determination experiments. The method comprises the following specific steps:

cell culture DMEM was cultured in 10cm dishes, cells were digested with trypsin-EDTA, centrifuged at 1000rpm for 5 minutes, washed once with PBS, resuspended in PBS containing 100. mu.M digitonin, 0.6mL PBS/10cm cells, and lysed for 5 minutes. Cells were taken and observed under a trypan blue microscope, and more than 90% of the cells had been lysed. The supernatant of dN-SARM1 protein was collected by centrifugation at 5000rpm for 10 minutes.

The collected dN-SARM1 protein was detected by Western blotting, and the results are shown in FIG. 1. In FIG. 1, the SARM1-dN protein samples are shown in the first and second lanes, and BSA standards are shown in different amounts in the third to seventh lanes. The results in FIG. 1 show that 10. mu.g of SARM1-dN was contained per mg of total protein to meet the subsequent use requirements.

Secondly, adopting a PC6 fluorescence method to screen the inhibitor of SARM1 in vitro

dN-SARM1 protein obtained by expression and purification of 'I' and SARM1 protein 'and' 4 'protein extraction' is adopted, and a 'marketed drug' is adopted as a drug (ceramic biological, L1000) for screening a compound library.

The screening method of the embodiment comprises the steps of mixing SARM1 enzyme, a candidate inhibitor, nicotinamide adenine dinucleotide and a fluorescent probe to prepare a reaction system, reacting at normal temperature, detecting the rate of fluorescence generation in the reaction process, and marking as a first fluorescence rate; determining that the candidate inhibitor has SARM1 enzyme inhibitor activity if the first fluorescence rate is less than the standard fluorescence rate; the greater the extent to which the first fluorescence rate is less than the standard fluorescence rate, the greater the inhibitory activity of the corresponding candidate inhibitor; wherein, the standard fluorescence rate refers to the rate of fluorescence generation in the reaction process detected under the same condition without adding a candidate inhibitor, that is, the same amount of SARM1 enzyme, nicotinamide adenine dinucleotide and a fluorescent probe are mixed to prepare a reaction system, the reaction is carried out under the same reaction condition, the rate of fluorescence generation in the reaction process is detected and marked as the standard fluorescence rate; the fluorescent probe is used for detecting the enzymatic activity of SARM1 and is formed by coupling a pyridine ring and a phenylalkenyl or phenylalkenyl derivative. The present example specifically used a PC6 fluorescent probe.

The specific screening method of this example is as follows:

reaction conditions are as follows: mu.g/mL dN-SARM1 and 50. mu.M of the candidate inhibitor compound were first incubated in 50mM Tris-HCl (pH 7.5) solution for 10 minutes, then 50. mu.M NAD, 20. mu.M PC6 as substrate and 50. mu.M NMN as activator were added to the drug-incubated dN-SARM1 protein and reacted at room temperature for 30 minutes. Wherein the concentration of each component is the final concentration in the reaction system.

As a control, 1.5. mu.g/mL dN-SARM1 was incubated in 50mM Tris-HCl (pH 7.5) solution for 10 minutes, and then 50. mu.M NAD, 20. mu.M PC6 as a substrate and 50. mu.M NMN as an activator were added to the dN-SARM1 protein after incubation with the drug, and reacted at room temperature for 30 minutes.

During the reaction, the fluorescence spectrum dynamics of the product PAD6 were detected by a microplate reader, wherein the detection excitation wavelength and the emission wavelength were 390nm and 520nm, respectively. Finally, the reaction rate is adopted to represent the activity of the protein, the higher the reaction rate is, the stronger the activity of the protein is, and the lower the inhibition efficiency of the compound is; the activity of the candidate inhibitor can be determined by comparing the rate of fluorescence generation with and without the addition of the candidate inhibitor.

2015 compounds in L1000 were screened in this example, and the results are shown in FIG. 2. In fig. 2, the abscissa represents different inhibitors and the ordinate represents inhibition efficiency. The results in figure 2 show that 34 compounds with inhibitor activity up to 80%. In this example, 120 compounds with better inhibitor activity were finally determined according to the use requirements, and specifically include: nifedipine (NFDP), Nisoldipine (NSDP), Nicardipine (NCDP), Nimodipine (Nimodipine, NMDP), Amlodipine (Amlodipine), Felodipine (Felodipine), Clevidipine butyrate (Clevidipine butyrate), Isradipine (Isradipine), diltiazem

(dintizem), Verapamil (Verapamide), dehydronitrosonifedipine (dHN-Nifedipine, dHN-NFDP), dehydronitrosonidipine (dHN-Nisodipine, dHN-NSDP), dehydronitrosonicardipine (dHN-Nicardipine, dHN-NCDP), dehydronitrosonidipine (dHN-Nimodipine, dHN-NMDP), dehydroamlodipine (dH-Amlodipine), dehydrofelodipine (dH-Felodipine), dehydrobutyric acid Clevidipine (dH-Clevidipine butyrate), dehydroepirubicin (dH-Isradipine), Epirubicin hydrochloride (Epirubicin hydrochloride), Oritavancin diphosphate (Oritanancine diphosphate), benzalkonium chloride (Benzethimide), Berberine hydrochloride (Berberine), dihydronilotinib (Beloridinil), Sunitinib (Estradiol), Sunitinib (Merunethimide), Sunitinib (Merunidipine (mercuridine), and Merunidin (mercuridinil), Merunib (mercuridinil) Malate (mercuridine), and Merunib (mercuridinil) salts (Merunimide), and their salts (R, L, menadione (Menadione), Bismuth subsalicylate (Bismuth subsalicylate), Phenolphthalein (phenophtalin), Bismuth Subgallate (Bismuth Subgallate), Sanguinarine hydrochloride (Sanguinarine chloride), Evans blue (Evans blue), methyl Violet (Crystal Violet), cetrimide (Cetrimonium bromide), Zinc pyrithione (Zinc pyrithionone), Carboplatin (Carboplatin), amphotericin B (Ampelothricin B), Omeprazole (Omeprazole), Diethylstilbestrol (Diethylstilbestrol), Tigecycline (Tigecycline), Zinc undecylenate (Zinc monocyclopyralate), Lapatinib diglycolate (Lapatinib dimorpholine hydrochloride), hydrochloric acid (doxycycline), Tetracycline (Tetracycline hydrochloride), Tetracycline (Tetracycline), Chlortetracycline (Chlortetracycline), chlorfenadine (Tetracycline), chlorfenapyr (hydrochloride), chlorfenadine (Tetracycline), chlorfenadine (Tetracycline (hydrochloride), chlorfenadine (Tetracycline), chlorfenapyr (chlorfenapyr), chlorfenadine (chloride), chlorfenapyr), chlorfenadine (e (Tetracycline), chlorfenapyr), and chlorfenapyr (e), and chlorfenapyr), and so-D, such as (e, such as a salt), such as a salt, such as a compound, such as an extract, such as a compound, such as a compound, such as a compound, a, Butylbenzoic acid (Bufexamac), Epalrestat (Epalrestat), Entacapone (Entacapone), Reserpine (Reserpine), Tannic acid (Tannic acid), Carbazochrome Sodium Sulfonate (Carbazochrome Sodium sulfate), Mefloquine hydrochloride (Mefloquine hydrochloride), phenazopyridine hydrochloride (Phenazo)pyridine hydrochloride, amoxicillin (Axitinib), Otilonium bromide (Otilonium bromide), Thiostrepton (Thiostrepton), Montelukast sodium (Montelukast sodium), Panobinostat (Panobinostat), Levosimendan (Levosimendan), Lapatinib Ditosylate (Lapatinib dimosylate), Ceftiofur hydrochloride (Ceftiofur hydrochloride), Daunorubicin hydrochloride (Daunorubicin hydrochloride), Mecobalamin (Mecobalamin), Afatinib (Afatinib), dimercaptosuccinic acid (Succimer), Crocin (Crocin), Povidone (Povidone-iodine), sodium Tanshinone sulfonate (Tahinolide A sulbactione), Demeclocycline hydrochloride (tetracycline), luteolin (bismuth subclinic), siluroxime hydrochloride (bismuth), siluroxime hydrochloride), siluroxime (bismuth), siluroxime hydrochloride (bismuth), silurocortin (bismuth), siluroxime (bismuth subcitrate (bismuth), bismuth (bismuth subcarbonate (bismuth), bismuth (bismuth subcitrate), bismuth (bismuth subcarbonate (bismuth), bismuth (bismuth subcitrate), bismuth (bismuth subcatelate), bismuth (bismuth subcitrate), sodium Tanshinone A (bismuth subcatelate), sodium Tanshinone (calcium chloride), sodium sulfate), sodium (calcium chloride), sodium (calcium chloride), calcium (calcium chloride), calcium (calcium chloride), calcium (calcium chloride), calcium (calcium chloride), calcium (calcium chloride), calcium (calcium chloride), calcium chloride (calcium chloride), calcium (calcium chloride), calcium chloride), calcium (calcium chloride, calcium (calcium chloride), calcium chloride, domiphen Bromide (Domiphen Bromide), Idarubicin hydrochloride (Idarubicin HCl), Pirarubicin (Pirarubicin), alfasin maleate (Afatinib dimaleate), Rifabutin (Rifabutin), Octenidine hydrochloride (Octenidine dihydrate), Dantrolene sodium salt heptahydrate (Dantrolene sodium hydrate), Mitoxantrone (Mitoxantrone), Fondaparinux sodium (Fondaparinux sodium), Fidaxomicin (Fidaxomicin), visimitin, (E/Z) endoxifene, albuterovir (Elbasvir), furathiamine (furtiaspirine), Cefotiam hydrochloride (Cetitide), Malathion (Malathion), Strychnine (Strychnine), thifluzine (sodium chloride), Thimerosal (sodium chloride), nervone (Chlorpyrifos), pyrazosin (Methyl salicylate), pyrazothion (Chlorpyrifos), pyrazosin (Chlorpyrifos (Methyl), Chlorpyrifos (Methyl salicylate), pyrazothiopyrazothion (Methyl salicylate), pyrazothion (Chlorpyrifos (Methyl salicylate), fluazulene (Methyl salicylate), pyrazothion, Chlorpyrifos (Methyl salicylate), fluazulene (sodium chloride, Chlorpyrifos (sodium chloride), fluazulene (sodium chloride), fluazulene (sodium chloride), Chlorpyrifos (sodium chloride), flubenazolidone (sodium chloride, Chlorpyrifos (sodium chloride), benazolidone (sodium, sodium chloride), benazolidone (sodium chloride, sodium, Chlorpyrifos (sodium chloride), benazolidone (sodium chloride, Chlorpyrifos (sodium chloride), flubenazolidone (sodium, Chlorpyrifos (sodium chloride), fluazulene), and (sodium, Chlorpyrifos (sodium chloride), flubenazolidone (sodium chloride, Chlorpyrifos (sodium chloride), flubenazolidone (sodium chloride, Chlorpyrifos (sodium chloride), flubenazolidone (sodium chloride), flubenazolidone), fluazulene), fluben), flubenazolidone (sodium, Chlorpyrifos (sodium chloride), flubenazolidone (sodium, Chlorpyrifos), Chlorpyrifos (sodium, Chlorpyrifos), flubenazolidone, Chlorpyrifos (sodium, Chlorpyrifos), Chlorpyrifos, velpatasvir (Velpatasvir), Berberine (Berberine), Anidulafungin (Anidulafungin), and Dovitinib (Dovitinib).

The structural formula of the 120 SARM1 enzyme inhibitors is as follows:

thirdly, detecting the half inhibitory concentration of the SARM1 inhibitor in vitro by adopting a PC6 fluorescence method and HPLC

The compound of 'two, in vitro screening inhibitor of SARM 1' and its SAR compound are used as candidate inhibitors, and the half inhibitory concentration (IC50) of drug to SARM1 in vitro is detected by PC6 fluorescence method.

Reaction conditions are as follows: mu.M of the compound was first added to a 50mM Tris-HCl (pH 7.5) solution containing 0.4. mu.g/mL dN-SARM1, half of which was mixed with an equal volume of 50mM Tris-HCl (pH 7.5) solution containing 0.4. mu.g/mL dN-SARM1, and so on to dilute the drug 6 times to final concentrations of 200. mu.M, 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, respectively, and the control was incubated at room temperature for 10 minutes without inhibitor.

Then 50. mu.M NAD, 50. mu.M PC6 as substrate and 50. mu.M NMN as activator were added to dN-SARM1 protein incubated with inhibitor, and reacted at room temperature for 30 minutes. Wherein the concentration of each component is the final concentration in the reaction system.

During the reaction, the fluorescence spectrum dynamics of the product PAD6 were detected by a microplate reader, wherein the detection excitation wavelength and the emission wavelength were 390nm and 520nm, respectively. Finally, the reaction rate is used to express the activity of the protein and calculate the half inhibitory concentration, and the higher the reaction rate is, the stronger the activity of the protein is, and the lower the inhibitory efficiency of the compound is.

The compound of 'two, in vitro screening inhibitor of SARM 1' and its SAR compound are adopted as candidate inhibitors, and the half inhibitory concentration (IC50) of the drug to SARM1 in vitro is detected by High Performance Liquid Chromatography (HPLC), which is concretely as follows:

reaction conditions are as follows: the compounds at different concentrations were first added to a 50mM Tris-HCl (pH 7.5) solution containing 0.015. mu.g/mL dN-SARM1, a control without inhibitor added, and incubated at room temperature for 10 minutes.

mu.M NAD as substrate and 100. mu.M NMN as activator were added to dN-SARM1 protein incubated with inhibitor and reacted at 37 ℃ for 0 min, 15 min and 30 min, respectively, and the reaction was terminated by protein removal by means of a 10K 96-Well Filter plate (Millipore SigmaMultiScreen HTS 96-Well Filter Plates, catalog no MSIPN 4510). Wherein the concentration of each component is the final concentration in the reaction system.

Then separating the reactant and the product by adopting a C-18 column; mobile phase is as follows, phase a: 0.1M KH₂PO₄pH 6.0, phase B: 0.1M KH₂PO₄pH 6.0: MeOH ═ 7: 3. the isolation procedure was as follows: from 0 to 3 minutes, 1% B increased to 6% B; 3 to 6 minutes, held at 30% B; 6 to 10 minutes, hold to 1% B. Sample retention time: NMN (3.295 min), cADPR (3.833 min), ADPR (6.570 min), NAD (7.542 min), NM (9.543 min); the final calculated amount of the product ADPR represents the activity of dN-SARM1, and the half inhibitory concentration of the inhibitor was calculated by plotting a correlation curve of the inhibitor concentration with the amount of ADPR produced in a 30-minute reaction.

This example tested the inhibition of the concentrations of disulfiram, tannic acid, auranofin, zinc pyrithione, evans blue, Visomitin, methylene blue, sanguinarine hydrochloride, oritavancin diphosphate, bismuth subgallate, bismuth potassium citrate, procyanidins, nisoldipine, cetylpyridinium chloride, silver sulfadiazine, bismuth subsalicylate, tigecycline, dimercaptosuccinic acid, Nifedipine, cetrimide, octenidine hydrochloride, thiobischlorophenol, phenolphthalein, thimerosal, anidulafungin, zinc undecylenate, domiphen, cisplatin, dehydronitrosonisoldipine (dHN-Nisodipine, dHNN), dehydronitrosonifedipine (dHN-Nifedipine, dHN-NFDP), dehydronimodipine (dHN-Nimodipine, dHN-NMDP), dehydronitrosonicardipine (dHN-Nicardipine, dHN-NCDP), dehydroisradipine (dnitroso-IsdH-radipine), the results are shown in FIGS. 3 and 4. In FIG. 3, the abscissa represents the half inhibitory concentration measured by the PC6 fluorescence method, and the ordinate represents the half inhibitory concentration measured by the HPLC analysis method. In FIG. 4, the drug concentration is plotted on the abscissa as a tenth log and the activity of SARM1 is plotted on the ordinate. The results in FIG. 3 show that the half inhibitory concentrations of the two are basically within 5 times, which indicates that the inhibitory efficiency obtained by the two methods is comparable, while the PC6 fluorescence method has more drug screening advantages due to its simplicity. Fig. 4 shows that the compounds dehydronitrosonisoldipine, dehydronitrosonifedipine, dehydronitrosonimodipine, dehydronitrosonicardipine and dehydroisradipine all have good inhibitory effect on SARM 1.

Fourthly, detecting EC50 of drug inhibition in cell line of inducible overexpression of SARM1

(1) Preparation of the iSARM1 cell line

In this example, the gene sequence of SARM1 was amplified by PCR and constructed into pInducer20-neo plasmid. HEK293 was infected with liposome-packaged pInducer20-SARM1 virus to obtain an inducible SARM1 overexpressing cell line, labeled iSARM1(HEK 293). The preparation method comprises the following steps:

in this example, primers having sequences shown in Seq ID No.5 and Seq ID No.6 were used to carry out PCR amplification of the SARM1 gene sequence, and the recovery of PCR amplification products, digestion, recombinant plasmid construction, transfection and cell selection were all identical to dN-SARM1 in "purification of expression of a first SARM1 protein", except that "2. mu.g/mL puromycin" was replaced with 2mg/mL neomycin "in" (3) cell selection ", and the rest were the same, which is not described herein.

Seq ID No.5：5’-TCTAGAGCCACCATGGTCCTGACGCTGCTTC-3’

Seq ID No.6：5’-GAATTCTTAGGTTGGACCCATGGGTG-3’

(2) EC50 for detecting inhibition of inhibitor activity on SARM1 protein in cell lines

Selecting a compound for detecting the activity of the SARM1 protein in a cell line; the method comprises the following specific steps:

96 well plates were first treated with 0.05mg/mL polylysine for 5 minutes and washed once with PBS. Will be 3X 10⁴The iSARM1(HEK293) was plated in 96-well plates and incubated overnight at 37 ℃ in a 5% incubator. The next day, the inhibitor was added to the cells at final concentrations of 30, 7.5 and 1.87, 0.47 μ M and incubated for 1.5 hours in an incubator; then, 100. mu.M of the activator CZ-48 was added to the cells, and the cells were incubated for 16 hours while a control group without CZ-48 or drug was set. Intracellular cADPR levels were finally examined to show SARM1 activity, cADPR levels and inhibitor concentration were plotted, and the median effective concentration of inhibitor in cells was finally calculated (EC 50).

The cADPR assay is specifically as follows: cells were first washed once with PBS, and 150 μ L of pre-cooled 0.6M perchloric acid (PCA) was added to rapidly lyse and precipitate proteins. The PCA supernatant was transferred to a 1.5mL centrifuge tube and the protein in the medium was re-solubilized with 100. mu.L of 1M NaOH. The supernatant was added to 0.5mL of an organic reagent mixture (trioctylamine: chloroform: 1: 3), and the PCA was extracted from the water. After sufficient shaking, centrifugation at 12000rpm for 10 minutes, the solution was divided into 3 layers: an upper aqueous phase comprising a small molecule of interest; the lower organic phase, in which the PCA is dissolved; and a thin protein layer is arranged between the upper layer and the lower layer, and the upper layer is taken and transferred into a new centrifugal tube. According to the following steps: 100 ratio 1M Tris-Mg (1M Tris (pH 8.0): 1M MgCl) was added to the solution₂9: 1) according to the formula 1: NADase was added at a rate of 250 and treated overnight at 37 ℃ to remove NAD + from the mixture. After completion of the treatment, NADase was removed by filtration through a Millipore 10K 96-well filter plate.

The content of cADPR in the solution is determined by a Cycling analysis method, and the specific operation is as follows, 20 mu L of sample to be detected or cADPR standard substance is added into a 96-hole opaque white board. Preparing a reaction solution: 9.6mL PBS (pH 7.4), 200. mu.L ethanol, 150. mu.L 1mg/mL AD, 10. mu.L 10mM FMN, 5. mu.L 18mg/mL Diaphorase, 10. mu.L 10mM Resazurin, 100. mu.L 1M Nam. Half of the reaction solution was separated and 0.2. mu.g/mL of cyclese was added, and the reaction solution without cyclese was used as a control experiment. Each sample was divided into two groups of 3 replicates, and the reaction was started by adding the reaction solution with or without the cyclese, and the kinetic curve was recorded over 30 minutes (Ex: Em. 544/599). Calculating the average reaction slope, and converting the cADPR standard substance to obtain the accurate cADPR content.

Nifedipine, nicardipine, nimodipine, felodipine, Amlodipine (Amlodipine), Clevidipine butyrate (Clevidipine butyrate), isradipine, Verapamil (Verapamul), diltiazem

The results of the intracellular inhibition curve of (dintizem) are shown in fig. 5. In figure 5, drug concentration is plotted on the ten log abscissa and SARM1 activity is plotted on the ordinate. The results in FIG. 5 show that except Clevidipine butyrate and diltiazem

(dintizem), other compounds all inhibited SARM1 significantly.

Fifthly, protein mass spectrum identification of dHNN covalent inhibition SARM1 activity

In this example, dehydronitrosonisoldipine (dHN-Nisodipine, dHN-NSDP or dHNN) was selected to detect the modification of SARM1 protein inhibition.

(1) Construction of dtSARM1-dN expression vector

the Shanghai Producer company synthesizes a flag-strep tag II polypeptide gene fragment, dN-SARM1-F and dN-SARM 1-R. Wherein, the flag-strep tag II polypeptide gene fragment is a sequence shown by Seq ID No.7, the dN-SARM1-F is a sequence shown by Seq ID No.8, and the dN-SARM1-R is a sequence shown by Seq ID No. 9.

Seq ID No.7：

5’-AAGCTTATGGACTACAAGGATGACGATGACAAAGAATTCTCGGCGTGGAGCCACCCGCAGTTCGAGAAAGGTGGAGGTTCCGGAGGTGGATCGGGAGGTTCGGCGTGGAGCCACCCGCAGTTCGAAAAATCGGCGGGTACC GGATCC-3’

Seq ID No.8：5’-CCGGATCCCTGGCGGTGCCTGGGC-3’

Seq ID No.9：5’-ATAGCGGCCGCTTAGGTTGGACCC-3’

The synthesized flag-strep tag II polypeptide gene fragment was ligated to pENTR vector pENTR1A-GFP-N2 (addge: catalog #19364) using HindIII/EcoRI enzymatic cleavage sites. The SARM1-dN gene fragment is amplified by primers seq ID No.8 and seq ID No.9, and the amplified dN-SARM1 gene fragment is constructed on a pENTR vector with flag-strep tag II through BamHI and NotI enzyme cleavage sites. All endonucleases of this example were purchased from thermo. The other steps adopt a plasmid construction method in 'I, SARM1 protein expression and purification'.

(2) Expression of dtSARM1-dN protein

The virus packaging process adopts a virus packaging method of 'expression and purification of the SARM1 protein'. The packaged virus infected HEK293F cells, using 1 u g/L puromycin screening to obtain the expression of dtSARM1-dN stable expression cell line.

(3) Purification of dtSARM1-dN protein

HEK293F cells were harvested, washed once with PBS and then lysed for five minutes using digitonin containing two hundred micromoles per liter. After centrifugation at 13,000 for ten minutes, the supernatant was removed and incubated overnight with Streptactin agarose beads.

The next day, the supernatant was discarded, and the agarose beads were washed four times with buffer W containing 100mM Tris, 150mM NaCl and 1mM EDTA, and dtSARM1-dN was eluted from the beads by adding buffer W containing 2mM biotin. The activity of the samples was determined by the PC6 reaction.

(4) Mass spectrum sample preparation of dHNN and dtSARM1-dN proteins

50 μ M of dHNN and dtSARM1-dN protein was incubated for 30 minutes at room temperature, followed by addition of protein loading buffer and boiling for 10 minutes in boiling water. And finally, running the gel, carrying out in-gel enzyme digestion to extract polypeptide and carrying out mass spectrum identification. The specific implementation method comprises the following steps:

first, a polyacrylamide gel was prepared, with the following formulation, 10mL of 12% separation gel: 3.4mL of water, 4mL of 30% polyacrylamide, 2.5mL of 1.5M tris (pH 8.8), 100. mu.L of 10% SDS, 10% APS 100. mu. L, TEMED 10. mu.L; 4mL of 6% concentrated gum: 2.7mL of water, 0.8mL of 30% polyacrylamide, 0.5mL of 1M tris (pH 6.8), 40. mu.L of 10% SDS, and 4. mu.L of 10% APS 100. mu. L, TEMED 4.

The protein was concentrated in a polyacrylamide gel and the electrophoresis was stopped at 1 cm run into the separation gel. The gel was then cut out and placed in an incubation box and rinsed 3 times for 5 minutes each with ultrapure water. Then adding Simplystain Blue dye solution to incubate for 1 hour on a horizontal shaker; simplystain Blue dye solution was discarded, and ultra pure water was added thereto to decolorize 3 times for 2 hours.

In the process of preparing mass spectrum samples, the used blades and glass plates need to be washed once by using methanol with chromatographic grade in advance and then washed 3 times by using water with mass spectrum grade, and the used reagents and consumables are both in the chromatographic grade or the mass spectrum grade. After the decolorization was completed, the albumin glue was placed on a glass plate, and the albumin strips were cut with a blade, and then the albumin glue was cut into 1mm pieces with a flat blade³Into a 1.5mL centrifuge tube. 100% acetonitrile was added for dehydration for 10 minutes, the acetonitrile was removed, and the residual acetonitrile was drained in a vacuum concentrator. 100 μ L of 10mM DTT was added and heated at 56 ℃ for 30 minutes. After the liquid was allowed to return to room temperature, 100. mu.L of 55mM IAA was added and the solution was left to stand for 30 minutes at room temperature to alkylate the free thiol groups on the proteins. The liquid was aspirated off and 25mM NH added₄HCO₃And 50% acetonitrile on a shaker at 80rpm to decolorize the protein 3 times, each time for 15 minutes. After the liquid was blotted dry, the residual acetonitrile and water were drained in a vacuum concentrator. Add 100. mu.L of 3.3. mu.g/mL Trypsin for enzymolysis overnight.

The next day, 10. mu.L of 5% formic acid was added to stop the enzymatic reaction, and the liquid was aspirated into a new centrifuge tube. Subsequently, 5% formic acid and 50% acetonitrile were added, and the mixture was shaken in an 80rpm shaker to extract the enzymatically hydrolyzed polypeptide, and shaking was repeated three times for 15 minutes each, and the extracted polypeptide was collected in the same centrifuge tube. The samples were frozen with liquid nitrogen and lyophilized with a vacuum lyophilizer. After re-solubilization of the polypeptides with 0.1% formic acid, primary and secondary mass spectrometric identification was performed using a Q active HF-X instrument from Saimerfin. Finally, the modified polypeptides were analyzed using Protein resolver. Possible molecular weight increases of the polypeptide after dHNN modification include: 370.15287Da, 354.15796Da, 402.14270Da, 386.14779 Da.

The results of the dHNN inhibition test on SARM1 of this example are shown in fig. 6-10, fig. 6 is a time plot of dHNN inhibition on SARM1 activity, and fig. 7 is a graph of the results of ultrafiltration removal of free dHNN inhibition on SARM1 activity. The results in fig. 6 show that inhibition of SARM1 by dHNN is time dependent, with nicotinamide (Nam) being the negative control. The results in fig. 7 show that inhibition of SARM1 by dHNN is irreversible, with nicotinamide (Nam) being the negative control. The results in fig. 8 show that inhibition of SARM1 by dHNN mainly inhibits protein activation, and has weaker inhibitory ability for fully activated truncated SAM-TIR. The results in fig. 9 show that dHNN has inhibitory effect on both inducible iSARM1 and iSAM-TIR in cells, but has weaker inhibitory ability on iSAM-TIR, suggesting that its primary effect is inhibition of SARM1 activation.

Fig. 10 is a polypeptide of SARM1 protein modified by dHNN, and the results in fig. 10 show that dHNN modification can be detected in the polypeptide map, indicating that dHNN inhibits SARM1 activity through cysteine covalent modification. Further studies showed that 13 polypeptides on SARM1 protein were able to detect dHNN modification.

Sixthly, the activity and the axon mutation of SARM1 protein in neuron inhibition by medicine

In this example, dHNN was selected to detect the activity and the inhibitory effect of the peripheral mutation type of SARM1 protein in neurons, as follows:

(1) culture dish handling

In this example, 24-well adherent cell culture dishes were treated with 300. mu.l of 0.1mg/mL poly-D-lysine +0.02mg/mL lamin at 37 ℃ for 24 hours, aspirated, washed once with PBS, treated with 300. mu.l of 5% FBS at 37 ℃ for 24 hours, washed 1 time with PBS, and air-dried.

(2) Dorsal root ganglion neuron acquisition

In this example, dorsal root ganglion neurons were isolated from mouse embryos E12.5-E14.5, plated on culture dishes, and 3 days later, 5-fluoro-2 '-deoxyuridine (5-fluoro-2' -deoxyuridine) and 5. mu.M uridine (uridine) were added to a final concentration of 5. mu.M inhibitor. Wherein, the mouse embryo E12.5-E14.5 is bred by the laboratory, and the mouse is from C57/BL6J of the center of the experimental animal of Guangdong province.

Wherein the culture dish is a culture dish subjected to the culture dish treatment of (1). The components of the culture medium are as follows: neuronal basal medium (neuronabasal plus medium) and added to it a final concentration of 2% B27 plus, 1% glutamine (GlutaMax), 1% penicillin/streptavidin solution (penicilin/streptavidin solution), and a final concentration of 50ng/mL NGF.

(3) Drug treatment inhibits activation of SARM1 by vincristine, a chemotherapeutic drug

Inhibitors of dHNN were selected in this example to examine the effect of inhibitors on the activity of SARM1 protein in dorsal root neuronal cells. After 9-13 days of in vitro culture of dorsal root neuronal cells, neuronal cells were incubated with DMSO and dHNN at various concentrations for 1.5 hours, respectively, followed by the addition of 50nM of vincristine to activate the activity of intracellular SARM1 protein. After 16 hours of treatment, the intracellular cADPR content was measured by the cADPR extraction assay in EC50 for drug inhibition in inducible SARM1 overexpressing cell lines, which reflects the activity of the SARM1 protein in neuronal cells.

The results of the tests are shown in fig. 11, with cADPR levels on the ordinate and Vincristine (VCR) or dHNN treatment groups on the abscissa in fig. 11. The results in fig. 11 show that dHNN was able to significantly inhibit VCR induced SARM1 activation in DRG neurons, resulting in a decrease in cADPR levels.

(4) Drug treatment inhibits vincristine-induced SARM 1-dependent axonal degeneration

In this example, inhibitors of dHNN were selected to examine the effect of the inhibitor on vincristine-induced axonal degeneration of dorsal root neurons. After the dorsal root neuron cells are cultured in vitro for 9-13 days, the dorsal root neuron cells are treated by DMSO or dHNN with different concentrations and vincristine with the concentration of 50 nM. At 0, 24, 48, 72 hours post-treatment, bright field pictures were taken with an inverted fluorescence microscope under a 20-fold lens and the progression of axonal degeneration recorded.

The axonal injury quantification method specifically comprises the following steps: the pictures were first cropped to 147 x 147 pixel pictures, with 60 pictures cropped for each drug treatment. Then, the picture is converted into a binary picture by using threshold, all axon pixel points (the size is 16 to infinity) and pixel points (the pixel size is 16 to 10,000) of the damaged axon in the picture are calculated by using an analyze particle, and finally, the amount of axon damage/total axon amount is calculated, namely the degree of axon damage.

The test results are shown in fig. 12 and fig. 13, the axonal degeneration imaging graph after vincristine treatment for 72 hours in fig. 12, the abscissa of fig. 13 is the treatment time of vincristine, and the ordinate is the axonal degeneration process statistical graph. The results in fig. 12 show that dHNN was able to significantly inhibit vincristine-induced axonal degeneration, with DRG knocked out by SARM1 being a control group; the results in fig. 13 show that vincristine-induced axonal degeneration increased with time, whereas dHNN significantly inhibited the axonal degeneration process, essentially consistent with the negative control (DMSO group).

(5) Drug treatment inhibits injury-induced SARM 1-dependent axonal degeneration

Inhibitors of dHNN were selected in this example to test the effect of the inhibitor on injury-induced axonal degeneration of the rootstock neurons. After the dorsal root neuron cells are cultured in vitro for 5-7 days, the cells are respectively incubated with DMSO and dHNN with different concentrations for 0.5 hour, then axons are damaged by a flat blade with the diameter of 3mm under a microscope, and the axons are separated from cell bodies while the cell bodies are removed to prevent the regeneration of the neurons. At 0, 24, 48, 72 hours post-treatment, bright field pictures were taken with an inverted fluorescence microscope under a 20-fold lens and the progression of axonal degeneration recorded. Finally, the extent of axonal damage was quantified using ImageJ. The degree of axonal damage was calculated using "(4) drug treatment to inhibit vincristine-induced SARM 1-dependent axonal degeneration".

The test results are shown in fig. 14 and 15, in fig. 14, axonometric graphs at 24 and 48 hours after the lesion excision are shown, in fig. 15, the abscissa is the time after the lesion excision, and the ordinate is the axonometric progression statistical chart. The results in fig. 14 show that dHNN was able to significantly inhibit axonal degeneration processes. The results in figure 15 show that 24 hours post injury resulted in complete axonal degeneration, while dHNN was able to delay axonal degeneration progression.

Seven, conclusion

According to the above experimental protocol, the following compounds were finally screened for their ability to inhibit the SARM1 enzyme, inhibit axonal degeneration, maintain intracellular NAD levels, and inhibit the production of the metabolite cADPR: nifedipine, nisoldipine, nicardipine, nimodipine, amlodipine, felodipine, clevidipine butyrate, isradipine and diltiazem

Verapamil, dehydronitrosonifedipine, dehydronitrosonisoldipine, dehydronitrosonicardipine, dehydronitrosonimodipine, dehydroamlodipine, dehydrofelodipine, dehydrobutyric acid clevidipine, dehydroeladipine, epirubicin hydrochloride, oritavancin diphosphate, benzethonium chloride, berberine hydrochloride, dienestrol, pasecinib, estradiol valerate, merbromin, sunitinib malate, menadione, bismuth subsalicylate, phenolphthalein, bismuth subgallate, sanguinarinate, evans blue, methyl violet, cetrimide, zinc pyrithione, carboplatin, amphotericin B, omeprazole, diethylstilbestrol, tigecycline, zinc undecylenate, lapatinib disaccharide monohydrate, doxorubicin hydrochloride, aurantifene, chlortetracycline hydrochloride, isoliquiritigenin, rifampicin, cetylpyridinium chloride, magnolol, honokiol, flukiol, doxycycline, dehydronitrosodipine, dehydrogefitinib, dehydronifedipine, benzethonidipicoline, benzethol, bismerclin, dihydrogeine, dihydrogefitinib, and a, Procyanidins, tetracycline hydrochloride, cefsulodin sodium, thiodicofol, bufexamic acid, epalrestat, entacapone, reserpine, tannic acid, carbazochrome sodium, mefloquine hydrochloride, phenazopyridine hydrochloride, axitinib, otilonium bromide, thiostrepton, montelukast sodium, panobinostat, levosimendan, lapatinib ditosylate, ceftiofur hydrochloride, daunorubicin hydrochloride, mecobalamin, afatinib, dimercaptosuccinic acid, crocin, povidone iodine, tanshinone ILA sodium sulfonate, demeclocycline hydrochloride, lapatinib, gamma oryzanol, bismuth potassium citrate, tetrabenazine, ossification glycol, teicoplanin, rifapentine, troglitazone, sulfadiazinonSilver pyridine, tolcapone, domiphen bromide, idarubicin hydrochloride, pirarubicin, afatinib maleate, rifabutin, octenidine hydrochloride, dantrolene sodium salt hemiheptahydrate, mitoxantrone, fondaparinux sodium, fidaxomycin, Visomitin, (E/Z) endoxifen, elvavir, fursultiamine, cefotiam hydrochloride, malathion, strychnine, thimerosal, methylene blue, zotarolimus, ceforanide, cisplatin, neratinib, methobardoxolone, Pracinostat, retinol, sennoside a, chlorpyrifos, vepatavir, berberine, anidulafungin, and doxertinib.

The above compounds can be used for treating axonal degeneration related neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, glaucoma, amyotrophic lateral sclerosis, chemotherapy drug induced peripheral neuropathy, diabetes induced peripheral neuropathy, and apoplexy induced axonal degeneration. The inhibitors provide a new scheme and a new way for inhibiting axon degeneration and disease process and treating neurodegenerative diseases related to axon degeneration.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

SEQUENCE LISTING

<110> Shenzhen institute of university of Beijing

<120> SARM1 enzyme inhibitor and screening method and application thereof

<130> 20I31015

<160> 9

<170> PatentIn version 3.3

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ctcatgccag acagaaaagc ggctgttagt cactggcagc aagatatcgg cggaggcgga 60

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gcggccgcct aggttggacc catgggtgca gcaccc 36

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<213> dN-SARM1 Gene fragment

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ggtaccctgg cggtgcctgg gccagatggg ggcggtggca cgggcccatg gtgggctgcg 60

ggtggccgcg ggccccgcga agtgtcgccg ggggcaggca ccgaggtgca ggacgccctg 120

gagcgcgcgc tgccggagct gcagcaggcc ttgtccgcgc tgaagcaggc gggcggcgcg 180

cgggccgtgg gcgccggcct ggccgaggtc ttccaactgg tggaggaggc ctggctgctg 240

ccggccgtgg gccgcgaggt agcccagggt ctgtgcgacg ccatccgcct cgatggcggc 300

ctcgacctgc tgttgcggct gctgcaggcg ccggagttgg agacgcgtgt gcaggccgcg 360

cgcctgctgg agcagatcct ggtggctgag aaccgagacc gcgtggcgcg cattgggctg 420

ggcgtgatcc tgaacctggc gaaggaacgc gaacccgtag agctggcgcg gagcgtggca 480

ggcatcttgg agcacatgtt caagcattcg gaggagacat gccagaggct ggtggcggcc 540

ggcggcctgg acgcggtgct gtattggtgc cgccgcacgg accccgcgct gctgcgccac 600

tgcgcgctgg cgctgggcaa ctgcgcgctg cacgggggcc aggcggtgca gcgacgcatg 660

gtagagaagc gcgcagccga gtggctcttc ccgctcgcct tctccaagga ggacgagctg 720

cttcggctgc acgcctgcct cgcagtagcg gtgttggcga ctaacaagga ggtggagcgc 780

gaggtggagc gctcgggcac gctggcgctc gtggagccgc ttgtggcctc gctggaccct 840

ggccgcttcg cccgctgtct ggtggacgcc agcgacacaa gccagggccg cgggcccgac 900

gacctgcagc gcctcgtgcc gttgctcgac tctaaccgct tggaggcgca gtgcatcggg 960

gctttctacc tctgcgccga ggctgccatc aagagcctgc aaggcaagac caaggtgttc 1020

agcgacatcg gcgccatcca gagcctgaaa cgcctggttt cctactctac caatggcact 1080

aagtcggcgc tggccaagcg cgcgctgcgc ctgctgggcg aggaggtgcc acggcccatc 1140

ctgccctccg tgcccagctg gaaggaggcc gaggttcaga cgtggctgca gcagatcggt 1200

ttctccaagt actgcgagag cttccgggag cagcaggtgg atggcgacct gcttctgcgg 1260

ctcacggagg aggaactcca gaccgacctg ggcatgaaat cgggcatcac ccgcaagagg 1320

ttctttaggg agctcacgga gctcaagacc ttcgccaact attctacgtg cgaccgcagc 1380

aacctggcgg actggctggg cagcctggac ccgcgcttcc gccagtacac ctacggcctg 1440

gtcagctgcg gcctggaccg ctccctgctg caccgcgtgt ctgagcagca gctgctggaa 1500

gactgcggca tccacctggg cgtgcaccgc gcccgcatcc tcacggcggc cagagaaatg 1560

ctacactccc cgctgccctg tactggtggc aaacccagtg gggacactcc agatgtcttc 1620

atcagctacc gccggaactc aggttcccag ctggccagtc tcctgaaggt gcacctgcag 1680

ctgcatggct tcagtgtctt cattgatgtg gagaagctgg aagcaggcaa gttcgaggac 1740

aaactcatcc agagtgtcat gggtgcccgc aactttgtgt tggtgctatc acctggagca 1800

ctggacaagt gcatgcaaga ccatgactgc aaggattggg tgcataagga gattgtgact 1860

gctttaagct gcggcaagaa cattgtgccc atcattgatg gcttcgagtg gcctgagccc 1920

caggtcctgc ctgaggacat gcaggctgtg cttactttca acggtatcaa gtggtcccac 1980

gaataccagg aggccaccat tgagaagatc atccgcttcc tgcagggccg ctcctcccgg 2040

gactcatctg caggctctga caccagtttg gagggtgctg cacccatggg tccaacctag 2100

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gaattcttag gttggaccca tgggtg 26

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aagcttatgg actacaagga tgacgatgac aaagaattct cggcgtggag ccacccgcag 60

ttcgagaaag gtggaggttc cggaggtgga tcgggaggtt cggcgtggag ccacccgcag 120

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<210> 8

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ccggatccct ggcggtgcct gggc 24

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Claims

1. A method of screening for an inhibitor of the SARM1 enzyme, comprising: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

mixing SARM1 enzyme, a candidate inhibitor, nicotinamide adenine dinucleotide and a fluorescent probe to prepare a reaction system, reacting at normal temperature, detecting the rate of fluorescence generation in the reaction process, and marking as a first fluorescence rate;

the standard fluorescence rate refers to the rate of fluorescence generation in the reaction process detected under the same condition without adding a candidate inhibitor, namely, the same amount of SARM1 enzyme, nicotinamide adenine dinucleotide and a fluorescent probe are mixed to prepare a reaction system, the reaction is carried out under the same reaction condition, the rate of fluorescence generation in the reaction process is detected and marked as the standard fluorescence rate;

the fluorescent probe is used for detecting the enzymatic activity of SARM1 and is formed by coupling a pyridine ring and a phenyl group or a phenyl group derivative.

2. The screening method according to claim 1, wherein: the fluorescent probe is at least one of a structure shown by PC1 to a structure shown by PC 15;

preferably, the fluorescent probe has a structure shown by PC5, PC6, PC7, PC8, PC10 or PC 11;

more preferably, the fluorescent probe has a structure shown in PC6 or PC 11.

3. Screening method according to claim 1 or 2, characterized in that: adding an activator into the reaction system to promote the reaction;

4. A SARM1 enzyme inhibitor obtainable by the screening method according to any one of claims 1 to 3.

5. The SARM1 enzyme inhibitor according to claim 4, characterized in that: the SARM1 enzyme inhibitor comprises nifedipine, nisoldipine, nicardipine, nimodipine, amlodipine, felodipine, clevidipine butyrate, isradipine and diltiazem

Verapamil, dehydronitrosonifedipine, dehydronitrosonisoldipine, dehydronitrosonicardipine, dehydronitrosonimodipine, dehydroamlodipine, dehydrofelodipine, dehydrobutyric acid clevidipine, dehydroeladipine, epirubicin hydrochloride, oritavancin diphosphate, benzethonium chloride, berberine hydrochloride, dienestrol, pasinib, estradiol valerate, merbromin, sunitinib malate, menadione, bismuth subsalicylate, phenolphthalein, bismuth subgallate, sanguinarine hydrochloride, evans blue, methyl violet, cetrimide, zinc pyrithione, carboplatin, amphotericin B, omeprazole, diethylstilbestrol, tegafungin, lapatinib diglycosyl monohydrate, doxorubicin hydrochloride, aurantifin hydrochloride, aureomycin hydrochloride, isoliquiritigenin, rifampin, cetylpyridinium chloride, magnolol, fluazifop, and the like, Procyanidins, tetracycline hydrochloride, cefsulodin sodium, thiodicofol, bufexamic acid, epalrestat, entacapone, reserpine, tannic acid, carbazochrome sodium sulfonate, mefloquine hydrochloride, phenazopyridine hydrochloride, axitinib, otilonium bromide, thiostrepton, montelukast sodium, panobinostat, levosimendan, lapatinib ditosylate, ceftiofur hydrochloride, rouges hydrochlorideMycin, mecobalamin, afatinib, dimercaptosuccinic acid, crocetin, povidone iodine, tanshinone ILA sodium sulfonate, demecycline hydrochloride, lapatinib, oryzanol, bismuth potassium citrate, tetrabenazine, calcifediol, teicoplanin, rifapentine, troglitazone, silver sulfadiazine, tolcapone, domiphen, idarubicin hydrochloride, pirarubicin, afatinib maleate, rifabutin, octenidine hydrochloride, sodium dantrolene hemihydrate, mitoxantrone, fondaparinux sodium, fidaxomicin, Visomitin, (E/Z) endoxifene, elvavine, fursultiamine, cefotiam hydrochloride, malathion, strychnine, thimerosal, zotarolimus, ceforanide, cisplatin, lenatinib, methylprednisolone, pracinostatt, retinol, sennoside a, chlorpyrifos, berberidazine, berberidactinomycin, berrubine, trovaglipizide, trovaglitazone, trovir, troglitazobactam, troglitazone, trexatin, pracins, tretinostat, pracins, tretinomycin, pracins, berberin, berberidazole, berberin, berberidazole, berrubicin, berberin, berberidazole, berberine, berberidazole, berrubicin, berberidazole, and berberidazole, berrubicin, and so, Anidulafungin and dovirtinib.

6. Use of the SARM1 enzyme inhibitor according to claim 4 or 5 in the manufacture of a medicament for the treatment of axonal degeneration-related neurodegenerative disease.

7. Use according to claim 6, characterized in that: the axonal degeneration-related neurodegenerative disease comprises at least one of Parkinson's disease, Alzheimer's disease, glaucoma, amyotrophic lateral sclerosis, chemotherapy-induced peripheral neuropathy, diabetes-induced peripheral neuropathy and stroke-induced axonal degeneration.

8. A medicament for treating axon degeneration related neurodegenerative diseases, which is characterized in that: comprising the SARM1 enzyme inhibitor of claim 4 or 5.

9. The medicament of claim 8, wherein: the axonal degeneration-related neurodegenerative disease comprises at least one of Parkinson's disease, Alzheimer's disease, glaucoma, amyotrophic lateral sclerosis, chemotherapy-induced peripheral neuropathy, diabetes-induced peripheral neuropathy and stroke-induced axonal degeneration.

10. The medicament according to claim 8 or 9, characterized in that: also comprises a pharmaceutically acceptable carrier.