WO2019169131A1 - Compositions peptidiques et méthode de traitement d'une infection par le cytomégalovirus humain - Google Patents

Compositions peptidiques et méthode de traitement d'une infection par le cytomégalovirus humain Download PDF

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Publication number
WO2019169131A1
WO2019169131A1 PCT/US2019/020052 US2019020052W WO2019169131A1 WO 2019169131 A1 WO2019169131 A1 WO 2019169131A1 US 2019020052 W US2019020052 W US 2019020052W WO 2019169131 A1 WO2019169131 A1 WO 2019169131A1
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hcmv
hebtron
peptide
infected
cells
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PCT/US2019/020052
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English (en)
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Yulia A. Komarova
Derek F. WALSH
Michael Abecassis
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The Board Of Trustees Of The University Of Illinois
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the present disclosure relates to peptides for treating human cytomegalovirus infections.
  • the present disclosure also relates to methods of treating human cytomegalovirus infections using the peptides disclosed herein.
  • HCMV Human Cytomegalovirus
  • HCMV can cross the placenta with devastating effects. Every year in the US alone, HCMV induces hundreds of cases of perinatal mortality and leaves thousands with serious physical and developmental defects, yet awareness of this is alarmingly low (Manicklal et al., 2013).
  • HCMV replicates slowly and extensively uses the host's cell systems over the course of infection.
  • the disclosure provides experimental data demonstrating that HCMV actively upregulates the expression of microtubule accessory factor End Binding protein (EB)3, which in turn, mediates host cell remodeling of Golgi into a unique structure called the assembly compartment (AC) that accompanies the formation and maturation of HCMV particles.
  • EB microtubule accessory factor End Binding protein
  • AC assembly compartment
  • EB3 is a novel druggable target to suppress HCMV infection.
  • HEBTRON 16-mer
  • Myr-RSMKRSLIPRWIGNKR SEQ ID NO: 1
  • 8-mer HEBTRON short; Myr-KRSLIPRF-NH2; SEQ ID NO: 2
  • 12-mer HEBTRON-ASLIP
  • RSMKRRWIGNKR SEQ ID NO: 3 peptides that targets EB3 and exhibits antiviral activity in primary human fibroblasts (NHDFs). These peptides are effective against HCMV but not against other viruses tested (Herpes Simplex Virus Type 1 (HSV-1) or Vaccinia Virus (VacV)). Mechanistically, both peptides modulate microtubule dynamics and interfere with the formation and maturation of the HCMV assembly compartment. As a result, cells treated with the peptides do not shed new virus efficiently. Hence these peptides provide novel and effective anti-viral therapy against HCMV.
  • HSV-1 Herpes Simplex Virus Type 1
  • VacV Vaccinia Virus
  • the disclosure provides for isolated peptides comprising the amino acid sequence of RSMKRSLIPRWIGNKR (SEQ ID NO: 1), KRSLIPRF (SEQ ID NO: 2), RSMKRRWIGNKR (SEQ ID NO: 3) or a fragment or variant thereof.
  • the disclosure provides for isolated peptides consisting of the amino acid sequence of RSMKRSLIPRWIGNKR or KRSLIPRF or RSMKRRWIGNKR (SEQ ID NO: 3) or a fragment or variant thereof.
  • the provided variant peptides comprise a conservative substitution or a deletion of one or more amino acids.
  • the peptides may be conjugated to a fatty acid or linked to a myristoyl group, i.e. myristoylated.
  • the peptides may be linked to a carrier peptide.
  • the carrier peptide may be antennapedia peptide (AP), cell -penetrating peptides (CPPs) including
  • Penetratin petpide, TAT peptide, transportan or polyarginine peptides may be modified from the amine- or the carboxy- termini.
  • the amine terminus of the peptide is myristolyated and/or the carboxy terminus of the peptide is aminated.
  • compositions comprising a pharmaceutically acceptable excipient and one or more of the disclosed isolated peptide.
  • the provided compositions are used to treat human cytomegalovirus infection in newborn humans, infant humans and adult humans.
  • the provided pharmaceutical compositions are formulated for oral administration.
  • the disclosure also provides for method of treating human cytomegalovirus infection comprising the step of administering to a subject in need thereof an isolated peptide comprising the amino acid sequence of RSMKRSLIPRWIGNKR (SEQ ID NO: 1), KRSLIPRF (SEQ ID NO: 2), RSMKRRWIGNKR (SEQ ID NO: 3) or a fragment or variant thereof.
  • the disclosure further provides for method of treating human cytomegalovirus infection comprising the step of administering to a subject in need thereof an isolated peptide consisting of the amino acid sequence of RSMKRSLIPRWIGNKR (SEQ ID NO: 1), KRSLIPRF (SEQ ID NO: 2), RSMKRRWIGNKR (SEQ ID NO: 3) or a fragment or variant thereof.
  • the peptides used in the provided methods are linked to a myristoyl group (or the peptide is myristoylated) or a carrier peptide. In some embodiments, the peptide or composition is administered orally.
  • the methods include administering a therapeutically effective amount of a disclosed peptide or disclosed composition, such as an amount effective to inhibit or suppress HCMV infection.
  • a disclosed peptide or disclosed composition such as an amount effective to inhibit or suppress HCMV infection.
  • the subject is an adult human, a new born human or an infant human.
  • the subject is infected with HIV, pregnant or immune-comprised.
  • the disclosure also provides for use of any of the provided peptides or compositions for the preparation of a medicament for treating human cytomegalovirus infection in a subject in need.
  • the medicament comprises a therapeutically effective amount of a disclosed peptide or disclosed composition, such as an amount effective to inhibit or suppress HCMV infection.
  • the medicament is formulated for oral administration.
  • the medicament may be administered to an adult human, a new born human or an infant human.
  • the subject is infected with HIV, pregnant or immune-comprised.
  • compositions comprise a therapeutically effective amount of a disclosed peptide, such as an amount effective to inhibit or suppress HCMV infection.
  • the composition is formulated for oral administration.
  • the composition may be administered to an adult human, a new born human or an infant human.
  • the subject is infected with HIV, pregnant or immune- comprised.
  • Figures 1A-F demonstrate AC dynamics and nuclear rotation during HCMV infection.
  • A NHDFs were infected with TB40/E-UL99-Egfp at MOI 0.5 for 4d. Fixed cells were stained for GFP, TGN46 and Gb. Nuclei were stained with Hoechst. Note, both Golgi structures in each cell stain for UL99-Egfp and Gb and appear to have weak connections.
  • B Time lapse images of NHDFs infected with TB40E-UL99-Egfp at MOI 0.5 imaged 3-5d.p.i. illustrating AC merging.
  • C Time lapse images of NHDFs co-infected with TB40/E-UL99-Egfp and TB40/E-UL32-mCherry at MOI 0.5 imaged 3-5d.p.i. UL99 labels the AC. UL32 domains form in the nucleus (ND) prior to the appearance of virus particles in the AC (acVPs) and then in the cytoplasm (Cvp).
  • D Still showing virus particles in the AC and cytoplasm (cyto) co-labeled with UL99-Egfp and UL32- mCherry.
  • E Distribution of fluorescent intensity for UL32-mCherry and UL99-Egfp particles in D. measured using line-scan analysis.
  • N 5 (64 particles). Both peaks are in the size range of HCMV particles (indicated) and drop rapidly outside this size-range.
  • F NHDFs expressing NLS- mCherry mock infected or infected with TB40E-UL99-Egfp at MOI 0.5 and imaged 3-5d.p.i. Displacement of NLS-mCherry-labeled nuclei was measured. 2 representative examples per group are shown.
  • Figure 2A-H demonstrate that the HCMV AC is a Golgi-derived MTOC.
  • A-B NHDFs were mock infected or infected with TB40/E at MOI 3.
  • A Cells were fixed 5.d.p.i. and stained for g-tubulin, pericentrin and Gb. Nuclei were stained with Hoechst.
  • B Cell lysates were prepared at the indicated times and analyzed by WB. Lower: Densitometry analysis of g-tubulin relative to oc-tubulin.
  • N B; unpaired two-tailed t-test, *p ⁇ 0.05.
  • C-F NHDFs were mock- infected or infected with AD169 at MOI 3 for 3d. Cells were treated with IOmM nocodazole for 8h before washout for the indicated time.
  • C Samples were stained for tyrosinated tubulin, TGN46 and pericentrin. Red arrows indicate centrosomes, white arrows indicate Golgi fragments. Insets show non-centrosomal nucleation sites.
  • E-F Tyrosinated MTs at centrosomes 10 min post- nocodazole washout imaged using confocal microscopy.
  • E Area occupied by tyrosinated MTs nucleated at centrosomes was measured.
  • F Representative examples of MTs at centrosomes, including a bright g- tubulin merge used to identify centrosomes.
  • G NHDFs were infected with TB40/E at MOI 1 for 5d. Fixed cells were stained for acetylated tubulin and TGN46, along with Hoechst .
  • Figure 3A-3F demonstrates that HCMV increases EB protein levels.
  • A Growth- arrested NHDFs were mock-infected or infected with TB40/E at MOI 3 for the indicated times. Cell lysates were analyzed by WB.
  • C Growth-arrested NHDFs were mock-infected or infected at MOI 3 for 4d. EB transcript levels were measured using Qrt- PCR.
  • D NHDFs were treated with control or I El/2 siRNA prior to infection with AD169 at MOI 3 for 3d. Cell lysates were analyzed by WB for the indicated proteins.
  • E NHDFs were transduced with retroviruses encoding GFP or IE proteins. Samples were analyzed by WB.
  • F NHDFs were infected with AD169 at MOI 3 in the presence of DMSO or CDK1 inhibitor (JNJ-770662), re-dosing daily, for the indicated times. Samples were analyzed by WB.
  • FIG. 4A-4G demonstrate that EB1 and EBB play distinct roles in HCMV replication.
  • A NHDFs were treated with siRNA 30h prior to infection (+) or with a second siRNA treatment at 3d.p.i. (++). Cultures were infected at MOI 3 for 5d. Lysates were analyzed by WB for the indicated proteins. Note, IE1/2 are expressed because early infection is not affected using this siRNA treatment strategy.
  • B-D NHDFs were treated with siRNAs as in A. and infected with TB40/E-Egfp at MOI 0.001 for 14d.
  • B Cells were lysed and analyzed by WB for the indicated proteins.
  • C Phase and fluorescent images of plaques for TB40/E or AD169.
  • Figure 5A-5F demonstrate that EB3 regulates acetylated MTs, nuclear rotation and AC structure.
  • A-D NHDFs were treated with siRNAs and infected with TB40/E at MOI 1 for 5d.
  • A Fixed samples were stained for acetylated tubulin and TGN46. Nuclei were stained with
  • C Fixed samples were stained for EB1, EB3 and TGN46. Enlarged insets show EB1 and EB3 comets.
  • D Line-scan analysis of EB1 or EB3 comet intensity and distribution in samples in C. Note, loss of one EB increases MT tip binding by the other. Top: Distributions of EB1 (red) or EB3 (green) in control siRNA (solid) or EB1 siRNA (dashed) samples.
  • Figure 6A-6J provide characterization of HEBTRON.
  • A ITC of HEBTRON binding to purified EB3 C-terminus (200-281aa). KD, binding enthalpy, and stoichiometry were calculated from changes in heat upon binding of HEBTRON to the protein using the "one set of sites" binding model.
  • B Thermal unfolding of full-length EB3 alone (blue) or with HEBTRON (red) using NanoDSF. The first derivative of the 350/330 nm (peak) defines transitions between folded (left) to unfolded (right) states. Magnified insets show 0.20C temperature shift (green lines) for the complex. This suggests HEBTRON stabilizes dimers.
  • DMSO or HEBTRON-treated cells in F containing acetylated MTs. N as indicated.
  • H Sequence of HEBTRON, MutN or MutN-MutC.
  • II NHDFs treated with DMSO or 25mM HEBTRON, Mut-N or MutN-MutC were infected with TB40/E-Egfp at MOI 0.001 for 12d. Representative phase and fluorescent images of plaques.
  • Figure 7A-7G demonstrate that HEBTRON blocks recruitment of CDK5RAP2, nuclear rotation, and HCMV replication.
  • A-B NHDFs treated with DMSO or 25mM HEBTRON or MutN- MutC were infected at MOI 1 for 5d.
  • A Fixed cells were stained for TGN46 and CDK5RAP2. Example of measurements of CDK5RAP2-positive area (based on fluorescence above intensity threshold).
  • C HEBTRON does not affect CDK5RAP2 or EB3 abundance.
  • NHDFs treated with DMSO or 25mM HEBTRON, MutN or MutN- MutC were infected with AD169 at MOI 3 for 5d. Lysates were analyzed by WB.
  • D-E NHDFs treated with DMSO or 25mM HEBTRON were infected with TB40/E-UL99-Egfp at MOI 0.5.
  • D Time lapse imaging was performed 3-5 d.p.i and nuclear displacement was measured.
  • Figure 8A-8D provide AC dynamics and function as an MTOC.
  • A UL99-eGFP labels TGN46-positive regions early in infection. NHDFs were infected with TB40/E-UL99-eGFP at M010.5. Cells were fixed at the indicated times and stained for GFP and TGN46. Nuclei were stained with Hoechst. The kinetics of Golgi remodeling into an AC and the localization pattern of UL99-eGFP with TGN46 are in line with prior fixed imaging studies, validating UL99-eGFP as a means to label the AC from its earliest stages of formation for live cell imaging.
  • B Nucleating material localizes to Golgi-based sites throughout the AC (related to Figure 2A).
  • NHDFs were infected with AD169 at M013 for 3d. Fixed cells were stained for pericentrin and TGN46.
  • C-D HCMV induces the formation of a subpopulation of MTs containing acetylated tubulin. NHDFs were mock infected or infected with TB40/E at MOI 1 for 5d.
  • C Cells were fixed and stained for detyrosinated (Glu) or acetylated tubulin, along with TGN46. Typical examples of detyrosinated and acetylated tubulin networks are shown. White arrows point to singular asters of acetylated tubulin emanating from centrosomes in uninfected cells.
  • Figure 9A-9F demonstrate the effects of HCMV infection on the levels and localization of EB family members.
  • A NHDFs were infected with laboratory (AD169) or clinical (FIX) strains of HCMV at MOI 3 for the indicated time in d.p.i. Cell lysates were analyzed by WB using the indicated antibodies. Although different HCMV strains have different kinetics of early gene expression and replication, in all three HCMV stains tested the increase in EB protein abundance coincided with the expression of 1E2, but not 1E1.
  • B IE1/2 siRNAs do not indirectly affect EB protein levels in mock-infected cells. NHDFs were treated with control or I El/2 siRNA for 3d.
  • C-E Localization of EB proteins in HCMV-infected cells. Blue arrows point to examples of EB1 or EB3 comets at MT plus-ends, and the weaker plus-end accumulation of EB2. NHDFs were mock infected or infected with TB40/E at MOI 3 for 5d. Samples were stained for EB1 (C), EB2 (D) or EB3 (E). In addition, depending on antibody compatibility with each EB, samples were co-stained with antibodies against either tyrosinated-tubulin or a-tubulin to detect MTs, and HCMV gB or TGN46 to label the AC.
  • EB3 localizes at and around y-tubulin-positive regions of the AC.
  • NHDFs were infected with TB40/E at MOI 3 for 5d.
  • Fixed cells were stained for y-tubulin and EB3.
  • Nuclei were stained with Hoechst. Samples were imaged using confocal microscopy. A representative maximum projection through the AC is shown.50
  • Figure 10A-10H provide the characterization of HEBTRON.
  • A The DARTS method for drug target identification. Recombinant full-length human EB3 was incubated with or without the indicated peptides targeting EB1 or EB3 and digested with thermolysin. The products of digests were analyzed by WB for EB3.
  • B-C Control experiments related to the results in Figure 6A. ITC of EBB C-terminus (200-281aa) alone (B) and HEBTRON alone (C). This data shows that changes in heat during formation of the EB3-HEBTRON complex were not the result of dilution of the protein or peptide alone.
  • HEBTRON does not affect MT dynamics or recruitment of CLIP170 to MT tips, similar to EB3 depletion in Figure 4F-G.
  • E-G HEBTRON does not affect Vaccinia Virus replication or spread. NHDFs were treated with DMSO or 25pM HEBTRON, MutN or MutN-MutC, followed by infection with Vaccinia Virus expressing a GFP-tagged B5 protein (VacV-B5-GFP) at MOI 0.01 for 36h.
  • E Phase and fluorescence images of plaques were acquired. 50-pixel background subtraction was performed using Fiji to reduce autofluorescence from plate. Representative images a re shown.
  • Figure 11A-D demonstrate displacement of SxIP-interacting proteins by HEBTRON and the role of EB3 in non-centrosomal MT formation in HCMV-infected cells.
  • A NHDFs were treated with DMSO or 25pM HEBTRON and infected with AD169 at MOI 3 for 4d. Fixed samples were stained for EB1, tastin and Hoechst. Zooms show EB1 comets and trailing tastin on MT plus-ends.
  • B Line-scan analysis of EB1 and tastin intensity and distribution in samples in A.
  • NHDFs were treated with the indicated siRNAs for 30 h (C) or treated with DMSO or 25 pM HEBTRON (D) and then infected with AD169 at MOI 3 for 3 d. Cells were then treated with 10 pM nocodazole for 8h before washout for 0- or 10- min. Samples were stained for TGN46, a-tubulin and acetylated-MTs. White arrows point to examples of TGN46-positive sites that nucleate new acetylated MTs in control samples, or poorly nucleate new MTs in EB3 depleted or HEBTRON-tread cells.
  • Figure 12 demonstrates that treatment with 25 mM of HEBTRON and HEBTRON short interferes with HCMV replication.
  • NHDFs were infected with TB40/E at MOI 1.
  • the cells were treated with HEBTRON and HEBTRIN-short every 2nd day for the duration of the experiment. Infectious virus in culture supernatant has being tittered at 120 hours post-infection.
  • Figure 13 demonstrates the treatment with HEBTRON and HEBTRON ASLIP inhibits HCMV spreading.
  • HEBTRON suppresses HCMV spreading.
  • Primary human fibroblasts were infected with clinical (TB40/E) HCMV strains expressing GFP reporters at low the multiplicity of infection (MOI) for 14 days in the presence of DMSO solvent (control), HEBTRON, HEBTRON- ASLIP or HEBTRON-mutN-mutC (negative control).
  • the cells were treated with HEBTRON and HEBTRON-ASLIP or HEBTRON-mutN-mutC peptides every 2nd day for the duration of the experiment.
  • Phase and fluorescence images show the suppression of HCMV spread by
  • AC Human Cytomegalovirus
  • HCMV Human Cytomegalovirus
  • AC assembly Compartment
  • the experiments described herein demonstrate that the AC also acts as a microtubule-organizing center (MTOC) wherein centrosome activity is suppressed, and Golgi-based MT nucleation is enhanced.
  • MTOC microtubule-organizing center
  • EBB but not EB1 or EB2 was recruited to the AC and was required to nucleate MTs that were rapidly acetylated.
  • HCMV offers new insights into the regulation and functions of Golgi-derived MTs, and the therapeutic potential of targeting EB3.
  • HEBTRON and its shorter forms provide a revolutionary strategy of treating HCMV infection by targeting the host protein EB3.
  • the disclosed peptides are highly specific against HCMV but not against other tested herpesviruses (Herpes Simplex or Vaccinia Virus).
  • Another advantage of HEBTRON is its low toxicity; and is less toxic than marketed drugs. Thus, the peptides are expected to be well tolerated by individuals and result in less adverse side effects.
  • HEBTRON is likely to overcome the limitations of current antiviral therapies including virus evolution and drug resistance. Hence, HEBTRON would fulfill an unmet need in the marketplace for a novel therapeutic strategy in treating HCMV infection in both newborn children and adults.
  • Peptide" or "polypeptide” as used herein may refer to a linked sequence of amino acids and may be natural, synthetic, or a modification or combination of natural and synthetic.
  • “Fragment” as used herein may mean a portion of a reference peptide or polypeptide or nucleic acid sequence.
  • a " variant” means a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity.
  • Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity.
  • a conservative substitution of an amino acid i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and
  • hydropathic index of amino acids is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of .+-.2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function.
  • the peptide may comprise the amino acid sequence of RSMKRSLIPRWIGNKR (SEQ ID NO: 1; referred to HEBTRON), KRSLIPRF (SEQ ID NO: 2), RSMKRRWIGNKR (SEQ ID NO: S), a fragment thereof, or a variant thereof, wherein the peptide or a polypeptide comprising the peptide is 7 amino acid residues, 8 amino acid residues, 9, amino acid residues, 10, amino acid residues, 11, amino acid residues, 12 amino acid residues, 13 amino acid residues, 14 amino acid residues, 15 amino acid residues, 16 amino acid residues, 17 amino acid residues, 18 amino acid residues, 19, amino acid residues, 20 amino acid residues, 21 amino acid residues, 22 amino acid residues, 23 amino acid residues, 24 amino acid residues, 25 amino acid residues, 26 amino acid residues, 27 amino acid residues, 28 amino acid residues, 29 amino acid residues, 30 amino acid residues,
  • the peptide may be modified in that the amino acid sequence has one or more amino acid substitutions, amino acid insertions, amino acid deletions, carboxy terminal truncation, or an amino terminal truncation.
  • the peptides might also be glycosylated, phosphorylated, sulfated, glycosylated, animated, carboxylated, acetylated.
  • the C-terminal may be modified with amidation, addition of peptide alcohols and aldehydes, addition of esters, addition of p- nitroaniline and thioesters and multiple antigens peptides.
  • the N-terminal and side chains may be modified by PEGylation, acetylation, formylation, addition of a fatty acid, addition of benzoyl, addition of bromoacetyl, addition of pyroglutamyl, succinylation, addition of tert- butoxycarbonyl and addition of 3-mercaptopropyl, acylations (e.g. lipopeptides), biotinylation, phosphorylation, sulfation, glycosylation, introduction of maleimido group, chelating moieties, chromophores and fluorophores.
  • PEGylation e.g. lipopeptides
  • the peptides may be conjugated to a fatty acid, e.g. the peptides are myristoylated.
  • a fatty acid may be conjugated to the N-terminus of the peptides, such fatty acids include caprylic acid (C8), capric acid (CIO), lauric acid (C12), myristic acid (C14), palmitic acid (C16) or stearic acid (C18) etc.
  • cysteines in peptides can be palmitoylated.
  • the peptides may be conjugated or linked to another peptide, such as a carrier peptide.
  • the carrier peptide may facilitate cell-penetration, such as antennapedia peptide, penetratin peptide, TAT, transportan or polyarginine peptides.
  • the peptides may be cyclic or stapled.
  • the peptides disclosed herein may be cyclized by adding a single or multiple disulfide bridges, adding a single or multiple amide bonds between the N- and C-terminus, heat to tail cyclization, side chain cyclization (e.g. lactam bridge, thioester), hydrocarbon-stabled peptides.
  • the peptides disclosed herein may be stapled with a single, synthetic multiple, tandem braces ("staples”) to enhance pharmacologic performance of the peptides.
  • the peptides may be labeled with heavy isotope labeling, e.g. 15N, 13C, FITC, conjugation to a carrier protein, conjugation to imaging agent, FRET substrates with a fluorophore/quencher pair, peptide-DNA conjugation, peptide-RNA conjugation and peptide- enzyme labeling.
  • heavy isotope labeling e.g. 15N, 13C, FITC
  • conjugation to a carrier protein conjugation to imaging agent
  • FRET substrates with a fluorophore/quencher pair conjugation to a carrier protein
  • peptide-DNA conjugation peptide-RNA conjugation
  • peptide-RNA conjugation peptide- enzyme labeling.
  • the peptides may be within a fusion protein such as fused to a polypeptide or peptide which promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability or half-life, such as an immunoglobulin constant region, and a polypeptide which has a therapeutic activity different from disclosed peptides.
  • Fusions can be made either at the amine- or at the carboxy-termini of the
  • the fusion proteins may be direct with no linker or adapter molecule or indirect using a linker or adapter molecule.
  • a linker or adapter molecule may be one or more amino acid residues, typically up to about 20 to about 50 amino acid residues.
  • a linker or adapter molecule may also be designed with a cleavage site for a protease to allow for the separation of the fused moieties.
  • the peptide may be fused to one or more domains of an Fc region of human IgG to increase the half-life of the peptide or the addition of a Fab variable domain to shorten the half-life of the peptide.
  • HCMV is a b-herpesvirus with an unusually protracted replication cycle spanning several days (Mocarski et al., 2007). During this time, HCMV forms a unique yet poorly understood cytoplasmic Assembly Compartment (AC) (Alwine, 2012). Only recently has fixed cell imaging revealed that the AC is a restructured Golgi surrounded by several other host organelles (Das and Pellett, 2011; Das et al., 2007; Rebmann et al., 2016; Sanchez et al., 2000a). The AC forms intimate connections with the nucleus resulting in a pinched "kidney bean” shape characteristic of infection. This connectivity allows virus particles to bud from the nucleus into the AC to mature.
  • AC cytoplasmic Assembly Compartment
  • microtubules emanate from the AC and maintain its structure (Sanchez et al., 2000a), while the MT motor dynein recruits specific host and viral components to the AC (Clippinger and Alwine, 2012; Indran et al., 2010).
  • MTs microtubules
  • MTs form through the assembly of a/b-tubulin subunits into polarized filaments through minus-end seeding at MTOCs such as the centrosome (Petry and Vale, 2015).
  • MTOCs such as the centrosome (Petry and Vale, 2015).
  • y-tubulin proteins Central to MT nucleation are y-tubulin proteins, which assemble into higher-order g-Tubulin Ring
  • g-TuRCs complexes that bind a-tubulin. While the centrosome is the primary MTOC in many cell types, several non-centrosomal nucleation sites exist. These include the Golgi that contribute to the broader complexity of MT networks in different cell types (Sanders and Kaverina, 2015). After nucleating, MT plus-ends undergo rapid phases of growth, pause and de polymerization, generating dynamic arrays that radiate from MTOCs. The dynamic behavior and function of MTs is regulated by specialized end-binding proteins (EBs), comprising three family members (Akhmanova and Steinmetz, 2015).
  • EBs end-binding proteins
  • EB1 and EBB are structurally similar and can form hetero- or homo-dimers, while the more divergent EB2 forms only homodimers (Komarova et al., 2009).
  • EB dimers specifically recognize GTP-tubulin that is transiently present at growing MT plus-ends, and thereby track growing MT tips (Maurer et al., 2011; Morrison et al., 1998;
  • +TIPs plus-end tracking proteins
  • +TI Ps can mediate the capture and stabilization of MTs. While dynamic MTs typically have half-lives lasting minutes, stable MTs persist for several hours allowing them to accumulate distinguishing post-translation modifications (PTMs) (Janke and Bulinski, 2011). This includes tubulin detyrosination on the outer filament surface, which does not impart stability but allows stable MTs to be recognized by specific motors. In contrast, tubulin acetylation in the inner lumen confers mechanical strength (Portran et al., 2017; Xu et al., 2017).
  • PTMs post-translation modifications
  • live-cell imaging revealed the dynamic behavior of the HCMV AC and its ability to control host cell remodeling by acting as an MTOC whose nucleating activity is predominantly Golgi-based.
  • HCMV posttranscriptional control of different EBs
  • EBB is recruited to the AC to form acetylated MTs that control both nuclear rotation and AC structure.
  • siRNAs or myristoylated peptides targeting EB3 highlight its specific role in these events, revealing novel functions and regulatory mechanisms for the AC and Golgi-derived MTs, and identifying new targets to suppress infection.
  • HCMV impairs centrosome function and as others report (Hertel and Mocarski, 2004), appears to cause centrosome "splitting".
  • HCMV blocks cell division but pushes the cell into a "pseudo-mitotic" state that promotes viral DNA replication (Hertel et al., 2007; Hertel and Mocarski, 2004).
  • Deregulation of the cell cycle and centrosome organization may release nucleating factors such as g-tubulin, pericentrin and CDK5RAP2, and these factors enriched at Golgi regions during infection.
  • the data provided herein also suggests that other processes beyond centrosome deregulation are involved. These include increased abundance and availability of proteins such as g-tubulin.
  • CDK5RAP2 is known to bind and translocate with EB1 (Fong et al., 2009), but has >4-fold higher affinity for EB3 (Jiang et al., 2012).
  • CDK5RAP2 is particularly noteworthy as it enhances the nucleating activity of g- TuRCs and may be particularly important at weaker, non-centrosomal sites (Choi et al., 2010; Wang et al., 2010; Wu et al., 2016; Yang et al., 2017).
  • the data provided show that HCMV specifically exploits EB3 to enrich factors at Golgi regions.
  • HCMV specifically exploited EB3, but not EB1 to generate acetylated MTs.
  • the hyper-activate nature of Golgi-based MT nucleation during HCMV infection may make differences between the functions of EB1 and EB3 more readily detectable. Indeed, although structurally similar and often functionally interchangeable, examples of diversification amongst EB proteins have emerged.
  • EB1 regulates spindle orientation while EB3 is required for daughter cell reattachment during mitosis, when EBB stability is specifically increased (Ban et al., 2009; Ferreira et al., 2013).
  • HCMV's induction of a pseudo-mitotic state may underlie how it specifically exploits EB3.
  • EB3 also plays specific roles in Focal Adhesion and Adherens Junction formation, ciliogenesis, and myoblast and epithelial apico-basal elongation (Bazellieres et al., 2012; Geyer et al., 2015; Komarova et al., 2012; Schroder et al., 2011; Straube and Merdes, 2007). While in many systems these functional differences can be subtle, in HCMV-infected cells robust divergence is evident; EB1 and EB3 specifically regulate CLIP170 behavior and acetylated MTs, respectively, with each contributing differently to AC structure and the extent of HCMV replication.
  • Nuclear movement which involves elements of rotation and the distinct process of front-rear repositioning, helps generate cell polarity in migrating cells (Maninova et al., 2013; Zhu et al., 2017).
  • MTs contribute to this by directly pushing the nucleus or by enabling force exertion by motors associated with the nuclear envelope (Daga et al., 2006; Hui et al., 2016; Levy and Holzbaur, 2008; Maninova et al., 2013; Szikora et al., 2013; Wu et al., 2011; Zhao et al., 2012).
  • Golgi-derived MTs are rapidly acetylated and may be the primary source of acetylated MT subsets in many cell types (Chabin-Brion et al., 2001; Rivero et al., 2009; Sanders and Kaverina, 2015), the data suggest Golgi-derived MTs may be underappreciated regulators of nuclear rotation.
  • Rotation of nuclei is a fundamental part of intracellular reorganization during polarization and migration (Gundersen and Worman, 2013; Maninova et al., 2013). Nuclear rotation may be central to how HCMV remodels its host cell to replicate and spread, in part through polarizing the cell for migration. ACs also exhibited structural abnormalities in EB3- depleted or HEBTRON-treated cells, suggesting that in order to maintain their structure ACs may need acetylated MTs to tether to the nucleus. This tight coupling to a dynamic AC may in turn cause nuclei to rotate. Rotation has also been suggested to contribute to chromosome organization during meiosis (Christophorou et al., 2015).
  • HCMV forms discrete nuclear replication compartments that may be organized, similar to chromosomes, by rotating the nucleus, or be positioned relative to sites of AC tethering to the nucleus for efficient virus budding into the AC.
  • the findings described herein suggest this is a fundamental aspect of HCMV infection driven by EB3-regulated MTs. Targeting host proteins like EB3 with highly specialized functions is an attractive approach to avoid the emergence of drug-resistance commonly associated with therapeutics targeting evolutionarily adaptable viral proteins.
  • the subject may be a mammal, which may be a human. In certain embodiments, the subject is an adult human or child human.
  • the subject is a newborn human, including children less than one week old, less than two weeks old, less than 3 weeks old, less than 4 weeks old, less than 5 weeks old, less than 6 weeks old.
  • a "newborn” refers to a human that has an age between birth and about 2 months.
  • the subject is an infant human, including children that are about 2 months old, about 3 months old, about 4 months old, about 5 months old, about 6 months old, about 7 months old, about 8 months old, about 9 months old, about 10 months old, about 11 months old or about 12 months old.
  • An “infant” refers to a human that has an age between about 2 months and about 1 year.
  • the subject is infected with HMCV but may be asymptomatic.
  • the subject may be at risk of being infected or developing symptoms associated with HCMV infection or a cytomegalovirus (CMV) related-condition.
  • CMV cytomegalovirus
  • the subject infected with HCMV may be an organ transplant recipient, a pregnant woman, a subject infected with HIV, a subject that has been significantly burned, or a subject that is immune compromised.
  • HMCV related-conditions include CMV hepatitis, cytomegalovirus retinitis,
  • cytomegalovirus colitis CMV pneumonitis, CMV esophagitis, polyradiculopathy, transverse myelitis, subacute encephalitis, CMV mononucleosis, Gullain-Barre syndrome, type I diabetes and type 2 diabetes.
  • Treating each may mean to alleviate, suppress, repress, eliminate, prevent or slow the appearance of symptoms, clinical signs, or underlying pathology of a condition or disorder on a temporary or permanent basis.
  • Preventing a condition or disorder involves administering a peptide, agent or compound described herein to a subject prior to onset of the disease or prior to evidence of symptoms, for example in subjects infected with HCMV but are asymptomatic.
  • Suppressing a condition or disorder involves administering a peptide, agent or compound described herein to a subject after induction of the condition or disorder but before its clinical appearance, for example "suppression” includes inhibiting or preventing clinical symptoms after HCMV infection.
  • Repressing the condition or disorder involves administering a peptide, agent or compound described herein to a subject after clinical appearance of the disease, for example "repression” includes inhibiting or reducing the clinical symptoms after HCMV infection. Treating also includes reducing HCMV viral load and/or inhibiting the spread of the HCMV infection and/or inhibiting HCMV proliferation in the infected an infected subject.
  • the term "therapeutically effective” depends on the condition of a subject and the specific peptide, agent or compound administered. The term refers to an amount effective to achieve a desired clinical effect. A therapeutically effective amount varies with the nature of the condition being treated, the length of time that activity is desired, and the age and the condition of the subject, and ultimately is determined by the health care provider.
  • Suitable methods of administering a physiologically-acceptable composition such as a pharmaceutical composition comprising a peptide, agent or compounds described herein, are known in the art. Although more than one route can be used to administer a peptide, a particular route can provide a more immediate and more effective reaction than another route.
  • a pharmaceutical composition comprising the peptide is applied or instilled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled and/or introduced into circulation.
  • drug exposure can be optimized by maintaining constant drug plasma concentrations over time. Such a steady-state is generally accomplished in clinical settings by continuous drug infusion at doses depending on the drug clearance and the plasma concentration to be sustained.
  • the composition is administered regionally via intratumoral, administration, intrathecal administration, intracerebral (intra-parenchymal) administration, intracerebroventricular administration, or intraarterial or intravenous administration targeting the region of interest.
  • the peptide is administered locally via implantation of a matrix, membrane, sponge, or another appropriate material onto which the desired compound has been absorbed or encapsulated.
  • the device is, in one aspect, implanted into any suitable tissue or organ, and delivery of the desired compound is, for example, via diffusion, timed-release bolus, or continuous administration.
  • Ocular administration of the peptides may be carried using intraocular implants, intravitreal injections, systemic administration, topical application, nanoparticles,
  • the peptide may be administered by intravitreal injection or topically such as in the form of an eye drop.
  • the provided peptide, agent or compound may be administered as a monotherapy or simultaneously or metronomically with other treatments.
  • “simultaneously” as used herein means that the peptide and other treatment be administered within 48 hours, preferably 24 hours, more preferably 12 hours, yet more preferably 6 hours, and most preferably 3 hours or less, of each other.
  • the term “metronomically” as used herein means the administration of the peptide at times different from the other treatment and at a certain frequency relative to repeat administration.
  • the provided peptide is administered with one or more anti-viral agents, hyperimmune globulin enhanced for CMV or with an anti-HMCV vaccine.
  • the provided peptide, agent or compound may be administered simultaneously or metronomically with ganciclovir (Cytovene), valganciclovir (Valcyte), foscarnet (Foscavir), and cidofovir (Vistide), hexadecyloxypropyl-cidofovir, leflunomide (Avara), letermovir (Prevymis), and/or Maribavir.
  • ganciclovir Cytovene
  • valganciclovir Valcyte
  • foscarnet Foscavir
  • cidofovir Vistide
  • hexadecyloxypropyl-cidofovir leflunomide
  • leflunomide Avara
  • Prevymis letermovir
  • Maribavir Maribavir
  • the provided peptide, agent or compound may be administered to a subject that is infected with HCMV that is resistant to one or more of with ganciclovir (Cytovene), valganciclovir (Valcyte), foscarnet (Foscavir), and cidofovir (Vistide), hexadecyloxypropyl-cidofovir, leflunomide (Avara), letermovir (Prevymis), and/or Maribavir.
  • ganciclovir Cytovene
  • valganciclovir Valcyte
  • foscarnet Foscavir
  • cidofovir Vistide
  • hexadecyloxypropyl-cidofovir leflunomide
  • Avara letermovir
  • Prevymis letermovir
  • the peptide may be administered at any point prior to another treatment including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36
  • the peptide may be administered at any point prior to a second treatment of the peptide including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr, 106 hr, 104 hr,
  • the peptide may be administered at any point after another treatment including about 1 min, 2 mins., 3 mins., 4 mins., 5 mins., 6 mins., 7 mins., 8 mins., 9 mins., 10 mins., 15 mins., 20 mins., 25 mins., 30 mins., 35 mins., 40 mins., 45 mins., 50 mins., 55 mins., 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40 hr, 42 hr, 44 hr, 46 hr, 48 hr, 50 hr, 52 hr, 54 h
  • the peptide may be administered at any point prior after a second treatment of the peptide including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110 hr, 108 hr. 106 hr, 104 hr,
  • the method may comprise administering one or more of the peptides, agents or compounds disclosed herein.
  • the peptides, agents or compounds provided herein may be in the form of tablets or lozenges formulated in a conventional manner.
  • tablets and capsules for oral administration may contain conventional excipients may be binding agents, fillers, lubricants, disintegrants and wetting agents.
  • Binding agents include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone.
  • Fillers may be lactose, sugar, microcrystalline cellulose, maize starch, calcium phosphate, and sorbitol.
  • Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica.
  • Disintegrants may be potato starch and sodium starch glycollate.
  • Wetting agents may be sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.
  • the peptides, agents or compounds provided herein may also be liquid formulations such as aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs.
  • the peptides may also be formulated as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain additives such as suspending agents, emulsifying agents, nonaqueous vehicles and preservatives.
  • Suspending agent may be sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats.
  • Emulsifying agents may be lecithin, sorbitan monooleate, and acacia.
  • Nonaqueous vehicles may be edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol.
  • Preservatives may be methyl or propyl p-hydroxybenzoate and sorbic acid.
  • the provided peptides may be in aqueous formulations for topical administration such as in the form of an eye drop.
  • the peptides, agents or compounds provided herein may also be formulated as suppositories which may contain suppository bases such as cocoa butter or glycerides.
  • Peptides provided herein may also be formulated for inhalation, which may be in a form such as a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane.
  • Peptides provided herein may also be formulated as transdermal formulations comprising aqueous or nonaqueous vehicles such as creams, ointments, lotions, pastes, medicated plaster, patch, or membrane.
  • Peptides provided herein may also be formulated for parenteral administration such as by injection, intratumor injection or continuous infusion.
  • Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents.
  • the peptide may also be provided in a powder form for reconstitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water.
  • the peptides, agents or compounds provided herein may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection.
  • the peptides may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).
  • the method may comprise administering a therapeutically effective amount of the peptides to a patient in need thereof.
  • the therapeutically effective amount required for use in therapy varies with the nature of the condition being treated, the length of time desired to activate Toll-like receptors, and the age/condition of the patient. In general, however, doses employed for adult human treatment typically are in the range of 0.001 mg/kg to about 200 mg/kg per day. The dose may be about 0.05 mg to about 10 g per day.
  • the desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day. Multiple doses may be desired or required.
  • the dosage may be at any dosage such as about 0.05 pg/kg. 0.06 pg/kg, 0.07 pg/kg, 0.08 pg/kg, 0.09 pg/kg, 0.1 pg/kg, 0.2 pg/kg, 0.3 pg/kg, 0.4 pg/kg, 0.5 pg/kg, 0.6 pg/kg, 0.7 pg/kg, 0.8 pg/kg, 0.9 pg/kg, 1 pg/kg, 1.5 pg/kg, 2 pg/kg, 3 pg/kg, 4 pg/kg, 5 pg/kg, 10 pg/kg, 15 pg/kg, 20 pg/kg, 25 pg/kg, 50 pg/kg, 75 pg/kg, 100 pg/kg, 125 pg/kg, 150 pg/kg, 175 pg/kg, 200 pg/kg, 225 pg/kg,
  • the dosage may be at any dosage such as about 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 275 mg/kg, BOO mg/kg, 325 mg/kg,
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly contemplated.
  • Identical or “identity” as used herein in the context of two or more polypeptides or nucleotide sequences may mean that the sequences have a specified percentage of residues or nucleotides that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the
  • substantially identical may mean that a first and second protein or nucleotide sequence are at least 50%-99% identical over a region of 6-100 or more amino acids nucleotides.
  • NHDFs Primary Normal Human Dermal Fibroblasts isolated from human male neonatal foreskin were purchased from Lonza (CC-2509).
  • HEK-293-T, HEK- 293-A, VERO and BSC-40 cells were from Dr. Ian Mohr, NYU.
  • Phoenix-Ampho cells were purchased from ATCC. All cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Fisher Scientific) supplemented with 5% Fetal Bovine Serum (FBS), 2 mM L-Glutamine and penicillin-streptomycin and maintained at 37°C, 5% C0 2 .
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS Fetal Bovine Serum
  • FBS Fetal Bovine Serum
  • penicillin-streptomycin penicillin-streptomycin
  • confluent cultures of NHDFs were growth-arrested by washing three times in PBS before being maintained in DMEM supplemented with 0.2% Fetal Bovine Serum (FBS), 2 mM L-Glutamine and penicillin- streptomycin for 72 h.
  • FBS Fetal Bovine Serum
  • HCMV strain AD169 and bacterial artificial chromosome (BAC)-derived HCMV strains AD169, TB40/E and FIX expressing GFP reporters were grown on NHDFs until >90 cytopathic effect was observed.
  • Cells and medium were collected and freeze-thawed to release virus.
  • Cell debris was removed by centrifugation and virus was titrated by serial dilution on NHDFs and plaque counting.
  • Generation of HCMV TB40/E expressing UL99-eGFP or UL32-mCherry, as well as viral expression vectors is described in Method Details.
  • VacV-B5-GFP was grown on BSC-40 cells until >90% cypopathic effect was observed.
  • HCMV BAC-derived strain TB40/E (clone 4) was used to generate either TB40/E expressing UL99-eGFP or UL32-mCherry using galK BAC recombineering protocols and transformation in SW105 E. coli.
  • the galK ORF was inserted into UL99 or UL32 locus in BAC- TB40/E (clone 4) using the following primers:
  • ACTTGTACAGCTCGTCCATGCCGAGAGT-3' (SEQ ID NO: 10) using eGFP as a template, or UL32- Rev-5': 5'-CCG TGC AGA ACA TCC TCC AAA AGA TCG AGA AGA TTA AGA AAA CGG AGG AAA TGG TGA GCA AGG GCG AGG AG-3' (SEQ ID NO: 11) coupled with UL32 Rev-3': 5'-CGT CAC TAT CCG ATG ATT TCA TTA AAA AGT ACG TCT GCG TGT GTG TTT CTT TAC TTG TAC AGC TCG TCC ATG CCG-3' (SEQ ID NO: 12) using mCherry as a template.
  • Recombinant clones were selected for loss of GalK expression by screening on 2- Deoxy galactose/Glycerol containing plates. Clones were further validated by PCR analysis and then analyzed by sequencing. Virus stocks were grown and titrated on NHDFs as described above.
  • GFP-CLIP170, GFP, HCMV IE1 and HCMV IE2 retroviral expression vectors were produced by transfecting Phoenix-Ampho cells with pBABE-puro vectors described below. Cell culture medium was changed 24 h after transfection. Supernatant containing virus was then collected at 48 hours and 72 hours post-transfection and filtered through a 0.45 mM filter.
  • NHDFs were transduced with a pBABE-puro-AA-GFP-CLIP170-derived or pBABE-puro-AA-NLS-mCardinal- derived retroviral vector in the presence of polybrene.
  • NHDFs were washed with PBS and fresh cell culture media was added.
  • pools of stably expressing cells were generated by selecting with either 0.8 mg/ml puromycin (for eGFP-CLIP170 and NLS-mCardinal). Following selection, cells were maintained in growth medium containing either 160 ng/ml puromycin.
  • GFP or IE1/2 were transduced with retroviral vectors described above and processed at the indicated times.
  • RNA interference RNA interference (RNAi) and inhibitors
  • siRNAs were obtained from Life Technologies (Thermo Fisher Scientific); See Key Resources Table for details. siRNAs were transfected as described previously (Jovasevic et al., 2015). To avoid effects on early HCMV infection, cells were transfected with 150 pmol/ml siRNA using RNAiMax (Invitrogen) and 30 h later infected with HCMV at the indicated MOI. When examining late stages of infection, 3 d.p.i. cells were transfected with siRNA for a second time to counter HCMV-induced increases in EB expression. In the case of spreading assays, this was done without changing cell culture medium. Infected cultures were then processed for imaging, western blot analysis or titration of virus production as described below.
  • NHDFs were treated with 300 pmol/ml siRNA. 1 day later, cells were infected with HCMV AD169 at MOI 3. Lysates were collected at 3 d.p.i. and analyzed by
  • HEBTRON and EB3(200-281) were dialyzed into PBS. Titration experiments were performed using a VP-ITC Microcalorimeter (MicroCal LLC, Northampton, MA) at 25°C. For titration, 25-27 aliquots (5 m ⁇ each) of 60 mM EB3 (200-281) were injected into the ITC cell containing 1.4 mL of 4 mM HEBTRON. Each titration was preceded by a single 2pL injection to address diffusion artifacts. Two reference titrations were run. One of these titrations controlled for protein dilution effects and the other controlled for the peptide dilution effects.
  • the reference data were subtracted from the protein titration data points.
  • the integrated heat values were analyzed using the Origin 7.0'-software as well as the Engineer 3.0 software (Micromath Scientific Software). Data were fit using the 'One set of sites' model, to yield the dissociation constant (Kd), the stoichiometry, the enthalpic and entropic contributions to the Gibbs free energy of complex formation.
  • FRET measurements were carried out using a PHERAstar FS (BMG LABTECH Inc., Cary, NC) microplate reader equipped with a FRET module for CFP and YFP pair (Komarova et al., 2012).
  • (His6)-YFP-EB3 and (His6)-CFP-EB3 were mixed in equimolar concentration.
  • the protein and the peptide were mixed in 1:1 molar ratio.
  • NHDFs were grown on glass bottom dishes for 2 days at 37 °C and 5% C02. Cells were kept at 37 °C for the experiment.
  • the 5'6-FAM (Fluorescein)-conjugated Myr-HEBTRON was added at a concentration of 10 mM directly to the media during imaging. Images were acquired every 10 seconds for 10 minutes in both the FITC and DIC channels (to mark cell boundaries) using a Zeiss LSM 880 confocal microscope equipped with a 63x 1.4 NA oil objective.
  • the fluorescent intensity inside each cell for each time point was measured using ImageJ and normalized to the initial fluorescent background intensity inside the cell. The normalized fluorescent intensity was plotted over time. The half time to maximum uptake as well as the maximum uptake in the cells was found by fitting a Sigmoidal dose response curve to the data using GraphPad Prism 7.
  • NHDFs were treated and infected as outlined above before phase and fluorescent (GFP channel) images of plaques were acquired using a Leica DMI6000B-AFC microscope with a 370C InVivo environmental chamber, lOx objective, X-Cite XLED1 illumination, ORCA FLASH 4.0 cMOS camera and Metamorph software (Molecular devices) using the multi-dimensional acquisition function. Plaque areas were determined by using the threshold area function of the FIJI. To determine the effect of HEBTRON treatment on virus plaque sizes, NHDFs seeded in 12-well plates were pretreated with DMSO, 25 mM HEBTRON or 25 pM HEBTRON controls for 1 h.
  • HCMV AD169-GFP or TB40/E-GFP HCMV AD169-GFP or TB40/E-GFP
  • VACV-B5-GFP MOI 0.001.
  • cell culture media containing DMSO or 25 pM HEBTRON or controls was replaced every 48h. Images were acquired at the indicated times and analyzed as described above. 50 pixel rolling ball background subtraction was used to reduce autofluorescence from the cell culture vessel evident in VACV-B5-GFP plaque images using FIJI.
  • Resolved proteins were transferred to a nitrocellulose membrane (GE Healthcare Life Sciences) at 57 V for 60 min (Mini Trans-Blot system, Bio-Rad), washed in Tris-Buffered Saline (TBS) containing 0.1% Tween (TBS-T) and blocked (5% non-fat milk TBS-T) before incubating with primary antibodies diluted in 3% BSA TBS-T overnight at 40C.
  • TBS Tris-Buffered Saline
  • TBS-T 0.1% Tween
  • TBS-T blocked (5% non-fat milk TBS-T) before incubating with primary antibodies diluted in 3% BSA TBS-T overnight at 40C.
  • Membranes were washed with TBS-T and incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (GE Healthcare Life Sciences) diluted 1:3,000 in TBS-T containing 5% non-fat milk for 1 h at room temperature.
  • HRP horseradish peroxidas
  • NHDFs were seeded onto glass coverslips and grown to confluence. Cultures were then mock infected or infected with HCMV strain AD169 at MOI 3. At 3 d.p.i. culture medium was changed to DMEM supplemented with 10 pM nocodazole for 8 h to completely depolymerize MTs. Cells were then washed with PBS and incubated in normal culture medium for 20 min. Cells were then fixed in ice cold methanol, stained with the indicated antibodies for immunofluorescence and imaged as described above.
  • centrosomal tyrosinated tubulin staining after nocodazole washout were determined using the threshold area function on single confocal slices obtained at the centrosomal focal plain indicated by g-tubulin staining. Identical thresholds were used for all conditions. Non- centrosomal microtubules were quantified using the cell counter plugin for FIJI, centrosomal microtubules were excluded from analysis using pericentrin staining. CDK5RAP2 staining area was determined for infected cells with consistent gB staining using the threshold area function of the FIJI.
  • NHDFs stably expressing eGFP-CLIP170 were imaged at 500 ms intervals for 1 min using a lOOx objective (HC PL APO 100x/1.44NA OIL), X-Cite XLED1 illumination and an ORCA FLASH 4.0 CMOS camera with Metamorph software.
  • the second approach to tracking nuclear rotation involved labeling nuclei in NHDFs stably expressing mCardinal fused to a nuclear localization signal (NLS), which enabled the tracking of nuclear movement in both infected and uninfected cells. Nuclear movement was measured as described above, this time using far-red fluorescent nuclei. For uninfected cells, the two points used to track rotation were directly opposite each other at the center of the elongated side of the nuclear membrane.
  • NLS nuclear localization signal
  • IPTG isopropyl l-thio- -D-galactopyranoside
  • Ni-NTA beads (Thermo Scientific) as previously described (Geyer et al., 2015). Ni-NTA beads (1 ml) in a 20 ml column (Bio-Rad) were equilibrated with 50 bed-volumes of binding buffer (25 mM Tris, pH 7.4, 300 mM NaCI, 5 mM 2-mercaptoethanol, 2 mM PMSF). Bacterial lysate (50 ml) was then added to the column, followed by washing (150 bed-volumes of wash buffer, ⁇ 75 ml). The protein-bound beads were washed with phosphate-bq ered saline (PBS) supplemented with 2 mM CaCI2 and protease inhibitor cocktail (Sigma) and stored in the same buffer.
  • PBS phosphate-bq ered saline
  • recombinant His6-EB3 was eluted by addition of wash buffer containing 150 mM imidazole. Peak elution fractions were pooled, exchanged to imidazole-free buffer using PD-10 desalting columns (GE Life Sciences), and concentrated with an Amicon Ultra-15 with 10 kDa cut-off concentrator unit (Millipore, Inc.). The His6 tag was removed by addition of 1.5% (w/w) recombinant TEV protease and incubation at 0C for 16 hr.
  • EB3 proteins were then subjected to gel filtration chromatography over tandem Superdex 200 HR 10/30 columns connected in series and controlled by an AKTA FPLC (GE Life Sciences). Peak fractions containing EB3 proteins were then pooled and concentrated as described above.
  • Peptides were synthesized using the stepwise solid-phase method by 9- fluorenylmethoxycarbonyl (Fmoc) chemistry on Wang resin (AnaSpec, Fremont, CA, USA) with a 12-channel multiplex peptide synthesizer (Protein Technologies, Arlington, AZ, USA) according to the manufacturer's procedures. Detachment of peptide from the resin and removal of the side chain protection groups were done by incubating the resin with a mixture of trifloroacetic acid (TFA):Thioanisole:Water:Phenol:l,2-ethanedithio (82.5:5:5:5:2.5 v/v) for 2 hours.
  • Fmoc 9- fluorenylmethoxycarbonyl
  • the crude peptide was purified on a preparative Kinetex reversed-phase C18 column, 150 x 21.1 mm (Phenomenex, Torrance, CA, USA) using a BioCad Sprint (Applied Biosystems, Foster City, CA, USA). A flow rate of 30 mL/min with solvent A (0.1% TFA in deionized water) and solvent B (0.1% TFA in acetonitrile) was used. The column is equilibrated with 5% solvent B before sample injection. Elution is performed with a linear gradient from 5% solvent B to 100% solvent B in 60 min. The absorbance of the column effluent is monitored at 214 nm, and peak fractions are pooled and lyophilized.
  • the pure peptide fraction is identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) or electrospray ionization mass
  • the AC is a dynamic structure that controls host cell behavior
  • UL99 localizes to trans-Golgi vesicles and labels the AC, and also binds virus particles (Moorman et al., 2010; Sanchez et al., 2000a; Sanchez et al., 2000b).
  • UL32 localizes to the nucleus very early (l-2d.p.i.) but becomes predominantly cytoplasmic thereafter, associating with clathrin-containing compartments recruited to the AC.
  • HCMV clinical strain TB40/E was engineered to express either UL99-eGFP or UL32-mCherry.
  • NHDFs primary normal human dermal fibroblasts
  • Cultures were fixed at different days post-infection (d.p.i.) and stained for the trans-Golgi marker, TGN46.
  • the HCMV AC acts as a Golgi-derived MTOC
  • nocodazole washout assays were performed to identify sites of MT nucleation. Nocodazole disrupts the Golgi in uninfected cells and the AC in infected cells (Chabin-Brion et al., 2001; Sanchez et al., 2000a). While this prevented imaging of MT growth from an intact AC, this effect was advantageous as it spatially separated the centrosome from surrounding Golgi sites and avoided uncertainty over the origin of new MTs. Staining for TGN46, pericentrin and tyrosinated tubulin, which detects dynamic MTs, confirmed that nocodazole depolymerized MTs and dispersed Golgi and AC structures (Figure 2C).
  • Golgi-derived MTs A characteristic of Golgi-derived MTs is their rapid acetylation (Chabin-Brion et al., 2001; Efimov et al., 2007; Rios et al., 2004; Sanders and Kaverina, 2015). Uninfected or infected samples were stained for acetylated tubulin and TGN46. Imaging and quantification revealed that beyond singular asters, uninfected NHDFs contained very few acetylated MTs (Figure 8C- D). However, HCMV induced the formation of acetylated MTs that co-localized with TGN46- positive regions of the AC ( Figure 2G), suggesting Golgi domains nucleated these MT subsets.
  • qRT-PCR revealed that HCMV increased the transcript abundance of each EB relative to mock-infected cells (Figure 3C).
  • the relative increases in transcript and protein levels for EB1 and EB2 largely corresponded but increases in EB3 protein were greater than those of its transcript. This suggested that EB3 might be additionally regulated at post-transcriptional levels.
  • CDKl Cyclin Dependent Kinase 1
  • JNJ-7706621 demonstrated that CDK, or the combined action of IE2 and CDK were required to increase EB3.
  • both CDK substrate phosphorylation and increases in EB3 abundance became insensitive to JNJ-7706621, despite daily inhibitor replenishment.
  • HCMV encodes a CDK homolog, pUL97 that is resistant to host CDK inhibitors (Hertel et al., 2007).
  • CDK activity regulates EB3 abundance during infection, but that the viral CDK homolog likely takes over from host kinases once infection is established.
  • these findings established that HCMV uses multiple strategies to increase the abundance of EB proteins.
  • EBs were required for the formation of acetylated MTs by HCMV was also tested. Staining showed that EB1 depletion increased acetylated MTs, while EBB depletion suppressed their formation compared to controls ( Figure 5A). These effects were quantified scoring cells as 1) lacking acetylated MTs (none), 2) having acetylated MTs extending from the AC (medium), 3) having extensive acetylated arrays filling the cytoplasm (high). This quantification approach confirmed that EB1 depletion increased, while EB3 depletion decreased the extent of acetylated MT formation by HCMV ( Figure 5B).
  • EB3 formed longer and brighter comets upon EB1 depletion, while EB3 depletion resulted in brighter and longer EB1 tracks.
  • NHDFs were treated with control or EB-targeting siRNAs followed by infection with TB40/E- UL99-eGFP. Live cell imaging over 3-5 d.p.i together with nuclear displacement measurements revealed that in control or EB1 siRNA-treated samples -86% (37/43) nuclei rotated >180°
  • ITC isothermal titration calorimetry
  • NHDFs were treated with DMSO or 25mM HEBTRON before low MOI infection with an unrelated poxvirus, Vaccinia Virus (VacV).
  • CDK5RAP2 not only binds EB proteins but also stimulates the nucleating activity of g-TuRC (Choi et al., 2010; Fong et al., 2009; Wang et al., 2010; Wu et al., 2016).
  • CDK5RAP2 was present at centrosomes in uninfected NHDFs, but upon infection it localized throughout Golgi regions of the AC ( Figure 7A). Imaging and measurements threshold brightness revealed that HEBTRON, but not the MutN-MutC form of HEBTRON, significantly reduced CDK5RAP2 recruitment to the AC ( Figure 7B).
  • HEBTRON did not decrease CDK5RAP2 or EB3 abundance (Figure 7C), demonstrating that HEBTRON specifically affected CDK5RAP2 enrichment in Golgi regions. Cumulatively, these data supported the notion that HEBTRON suppressed infection by interfering with EB3-mediated enrichment of factors such as CDK5RAP2 at non-centrosomal sites within the AC. This, combined with recent reports that EB proteins function in MT minus-end organization and nucleation at the Golgi (Yang et al., 2017), suggested that EB3 likely regulated the nucleation of MTs at the AC.
  • DMSO-treated cells contained a dense AC core while the cytoplasm was filled with UL99-positive structures, many of which were uniform in size and exhibited bi-directional motility suggestive of HCMV particles (Figure 7E).
  • HEBTRON treatment resulted in aberrant ACs and large structures resembling MVBs in the cytoplasm, similar to effects of EB3 depletion.
  • HEBRON- ASLIP and HEBTRON-mutN-mutC which are set out in Table 1.
  • FIG. 12 demonstrates that treatment with 25 mM of HEBTRON and HEBTRON short interferes with HCMV replication.
  • the mutant harboring deletion of SLIP motif (RSMKRRWIGNKR) has similar inhibitory effect on HCMV replication whereas introduction of negatively changed amino acids in flanking region abolished the effect.
  • HEBTRON and the SLIP deletion mutant suppressed HCMV spreading.
  • the mutant harboring deletion of SLIP motif (RSMKRRWIGNKR)
  • Recipient mice are treated with 3 different doses of HEBTRON (5mM, 25mM and 50 mM per kg body weight, i.p.) daily for 5 days. All organs are frozen in liquid nitrogen immediately after removal. Viral replication is assessed in various organs by qPCR analysis of viral DNA copy number and by plaque forming assay.
  • Golgi complex is a microtubule-organizing organelle. Mol Biol Cell 12, 2047- 2060.
  • Three-dimensional structure of the human cytomegalovirus cytoplasmic virion assembly complex includes a reoriented secretory apparatus. J Virol 81, 11861-11869.
  • Microtubule-Associated Protein EB3 Regulates IP3 Receptor Clustering and Ca(2+) Signaling in Endothelial Cells. Cell Rep 12, 79-89.
  • p53 downstream target DDA3 is a novel microtubule-associated protein that interacts with end binding protein EB3 and activates beta-catenin pathway. Oncogene 26, 4928-4940.
  • the linear and rotational motions of the fission yeast nucleus are governed by the stochastic dynamics of spatially distributed microtubules. J Biomech 49, 1034-1041.
  • GTPgammaS microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs). Proc Natl Acad Sci U S A 108, 3988-3993.
  • EB1 a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle. Oncogene 17, 3471-3477.
  • Roubin R., Acquaviva, C., Chevrier, V., Sedjai, F., Zyss, D., Birnbaum, D., and Rosnet,
  • HIV-1 induces the formation of stable microtubules to enhance early infection.

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Abstract

La présente invention concerne des peptides permettant de traiter des infections par le cytomégalovirus humain. La présente invention concerne également des méthodes de traitement d'infections par le cytomégalovirus humain faisant appel aux peptides décrits dans la description.
PCT/US2019/020052 2018-02-28 2019-02-28 Compositions peptidiques et méthode de traitement d'une infection par le cytomégalovirus humain WO2019169131A1 (fr)

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WO2022099185A1 (fr) * 2020-11-09 2022-05-12 Thrive Bioscience, Inc. Procédé d'essai de comptage de plaque

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US20070059712A1 (en) * 2000-10-13 2007-03-15 Gish Kurt C Methods of diagnosis of prostate cancer, compositions and methods of screening for modulators of prostate cancer
US20110214199A1 (en) * 2007-06-06 2011-09-01 Monsanto Technology Llc Genes and uses for plant enhancement
US20110296543A1 (en) * 2006-06-01 2011-12-01 The University Of California Nucleic acids and proteins and methods for making and using them

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US20070059712A1 (en) * 2000-10-13 2007-03-15 Gish Kurt C Methods of diagnosis of prostate cancer, compositions and methods of screening for modulators of prostate cancer
US20110296543A1 (en) * 2006-06-01 2011-12-01 The University Of California Nucleic acids and proteins and methods for making and using them
US20110214199A1 (en) * 2007-06-06 2011-09-01 Monsanto Technology Llc Genes and uses for plant enhancement

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LAROSA, C ET AL.: "Preclinical development of an adjuvant-free peptide vaccine with activity against CMVpp65 in HLAtransgenic mice", IMMUNOBIOLOGY, vol. 100, no. 10, 12 July 2002 (2002-07-12), pages 3681 - 3689, XP002277230, doi:10.1182/blood-2002-03-0926 *
PROCTER, DJ ET AL.: "The HCMV Assembly Compartment Is a Dynamic Golgi-Derived MTOC that Controls Nuclear Rotation and Virus Spread", DEVELOPMENTAL CELL, vol. 45, no. 1, 9 April 2018 (2018-04-09), pages 83 - 100, XP085376814 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022099185A1 (fr) * 2020-11-09 2022-05-12 Thrive Bioscience, Inc. Procédé d'essai de comptage de plaque

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