WO2020186211A1 - Procédés et compositions pour le traitement d'une infection par le polyomavirus - Google Patents

Procédés et compositions pour le traitement d'une infection par le polyomavirus Download PDF

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WO2020186211A1
WO2020186211A1 PCT/US2020/022751 US2020022751W WO2020186211A1 WO 2020186211 A1 WO2020186211 A1 WO 2020186211A1 US 2020022751 W US2020022751 W US 2020022751W WO 2020186211 A1 WO2020186211 A1 WO 2020186211A1
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polyomavirus
subject
pyv
atmi
atri
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Sunnie THOMPSON
Joshua JUSTICE
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UAB Research Foundation
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UAB Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure is directed generally to treatment of viral infections and conditions related to a viral infection in a subject and pharmaceutical compositions useful in such treatments.
  • the present disclosure is more specifically dircted to treatment of a polyomavirus infection and conditions related to a polyomavirus infection in a subject and pharmaceutical compositions useful in such treatments.
  • Polyoma virus is a family of viruses that contains over 70 species, of which 13 are known to infect humans. Of the thirteen human PyVs that cause human infection, BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), and Merkel cell polyomavirus (MCPyV) cause the majority of PyV related maladies in humans. Most PyV infections appear to cause little or no symptoms and PyVs are probably persistent lifelong among most adults. Diseases caused by human polyomavirus infections are most common among immunocompromised subjects and subjects undergoing certain medical procedures or treatment.
  • BKPyV BK polyomavirus
  • JCPyV JC polyomavirus
  • MCPyV Merkel cell polyomavirus
  • PyV is an emerging pathogen that reactivates in immunosuppressed patients.
  • BKPyV latently infects the genitourinary tract of >90% of the adult population.
  • Immunosuppression increases the risk of viral activation leading to active PyV infection.
  • Immunosuppression can occur as a result of a disease or condition that suppresses the immune system (either as a result of the natural course of the disease or condition or a treatment directed to treating the disease or condition) as well as when a subject is given a treatment that is aimed at suppressing the immune system.
  • Organ and tissue transplant procedures often require that the subject (the transplant recipient) receive a treatment that suppresses the immune system.
  • a treatment is administered in order to decrease the possibility of a host-graft immune response, which can lead to damage to the transplanted organ or tissue (graft dysfunction) or cause failure of the organ or tissue (graft failure).
  • graft dysfunction graft dysfunction
  • graft failure graft failure
  • subjects with certain autoimmune conditions are administered drugs to suppress the immune system as a treatment for the autoimmune disease.
  • treatments may involve the administration of agents that modulates or inhibits the immune system.
  • JCPyV virus has been known to activate or reactivate in multiple sclerosis patients taking immunosuppressive drugs which can lead to progressive multifocal leukoencephalopathy (PML), which can cause dementia, blindness, paralysis and seizures.
  • PML progressive multifocal leukoencephalopathy
  • agents that can be used to treat PyV infections include activation of a latent PyV. Such agents would provide a significant benefit to subjects experiencing PyV infections. Such agents would provide a significant benefit to subjects experiencing active PyV infection caused (in whole or in part) by a treatment that suppresses the immune system as such agents would provide for treatment of the PyV infection without requiring that the treatment that suppresses the immune system be decreased.
  • FIG. 1 shows BKPyV activation of the DDR induced cell cycle arrest.
  • FIG. IB shows DNA damage assessed by nuclear morphology.
  • the left panel shows representative IFA images of nuclear morphologies (DAPI; blue) in BKPyV infected cells (TAg; red).
  • FIG. 1G shows the effects of DDR inhibition on cell cycle arrest, mitosis and premature mitosis events as measured by cell cycle analysis (FACS).
  • S phase EdU pulse labeling
  • M phase pH3 S10
  • DNA content FxCycle Violet
  • FIG. 1H shows quantification of mitotic cells (pH3 S10 ) from FIG. 1G.
  • FIG. 1 J shows a working model of activation of the DDR during a BKPyV.
  • BKPyV infection arrests the cell cycle and prevents entry into mitosis.
  • ATRi but not ATMi, causes premature mitosis.
  • FIG. 2 shows blocking mitosis prevented BKPyV-dependent host DNA damage when ATR was inactive.
  • ATRi 5mM VE-821
  • ATMi ImM KU-55933
  • DMSO vehicle control
  • FIG. 2A shows a diagram of the G2/M checkpoint.
  • DDR activation phosphorylates Cdkl (inactive; gray) and arrests the cell cycle.
  • Cdkl green
  • FIG. 2E shows data from FIG. IB for a direct comparison to siCdkl nuclear morphology, which were performed in the same manner as the experiments in FIG. IB.
  • FIG. 2E shows data from the experiment shown in Fig. IB are shown again here for a direct comparison to siCdkl nuclear morphology, which was determined as described for the experiments shown in FIG. IB.
  • FIG. 2G shows a working model illustrating that mitotic entry leads to DNA damage during BKPyV infection when ATR is inhibited.
  • FIG. 3 A shows Cdkl silencing was used to determine if mitotic entry due to DDRi reduced viral titers. The mean ⁇ SD are shown. Significant differences from the NTC (black asterisks) were determined by One-Way ANOVA with Dunnet’s post-test. Significant differences from the NTC within treatment conditions (red asterisks) were determined by Student’s t-test (*p ⁇ 0.05).
  • FIG. 3B shows representative Western blots of TAg (viral infection) and Cdkl knockdown.
  • FIG. 3C shows cell cycle analysis as performed by FACS of mock or BKPyV infected RPTE cells treated with ATRi or ATMi and shown as contour plots (5%) to determine how DDR activation influences the cell cycle profile of a BKPyV infection.
  • FIG. 3D shows the percent of cells in G1 (gray), S (green) and G2 + M (blue) phases from FIG. 3C as quantified and reported as the % of total.
  • FIG. 3E shows the average percent of cells in G1 phase (re-graphed from FIG. 3D to show the differences in the populations). Shown are mean ⁇ SD.
  • FIG. 3F shows the average percent of cells in S phase (re-graphed from FIG. 3D to show the differences in the populations). Shown are mean ⁇ SD.
  • FIG. 3G shows the average percent of cells in G2 + M phase (re-graphed from FIG. 3D to show the differences in the populations). Shown are mean ⁇ SD.
  • FIG. 3H shows G2 and M phase population of cells from FIG. 1C as further separated into non-mitotic (gray) and mitotic (orange) cells by pH3 S10 expression.
  • FIG. I shows average % of mitotic cells from FIG. 3H quantified as % of total G2 and M phase cells. Shown are mean ⁇ SD.
  • FIG. 3J shows a working model illustrating that if ATR is inhibited during infection, then mitotic entry is increased, which decreases BKPyV titers and reduces S phase levels.
  • FIG 4 show the effect of small molecule inhibitors of ATR and ATM prolong BKPyV induced S phase and increase viral titers.
  • FIG. 4A shows cell cycle analysis by flow cytometry.
  • FIG. 4B shows the percent of cells in G1 (gray), S (green) and G2 or M (blue) phases from FIG. 4A as quantified and reported as the % of the total.
  • FIG. 4C shows the average percent of cells in G1 phase (re-graphed separately from FIG. 4B to show the differences in the populations). Shown are the mean ⁇ SD.
  • FIG. 4D shows the average percent of cells in S phase (re-graphed separately from FIG. 4B to show the differences in the populations). Shown are the mean ⁇ SD.
  • FIG. 4E shows the average percent of cells in G2 or M phase (re-graphed separately from FIG. 4B to show the differences in the populations). Shown are the mean ⁇ SD.
  • FIG. 4F shows the average percent of cells in M phase determined based on the percent of pH3 S10 positive cells in the total cell population. Shown are the mean ⁇ SD.
  • FIG. 4H shows quantification of the average % of premature mitosis from FIG. 4G. Shown are the mean ⁇ SD.
  • FIG. 41 shows a comparison of the average proportion of cells in S phase caused by chemical inhibition with both sets of inhibitors to ATM and ATR.
  • FIG. 4J shows a comparison of the average proportion of cells in premature mitosis caused by chemical inhibition with both sets of inhibitors to ATM and ATR.
  • FIG. 4K shows inhibition of ATM reduced BKPyV titers.
  • ATM inhibitors were added: 10 mM KU-55933 (positive control), lOpM to InM AZD1390, or DMSO (vehicle only).
  • FIG. 4L shows inhibition of ATM increases mitosis during BKPyV infection.
  • ATM inhibitors were added: 10 pM KU-55933 (positive control), lOpM to InM AZD1390, or DMSO (vehicle only).
  • IFA immunofluorescence assay
  • FIG. 5 shows constitutive ATM and ATR activation was required to prolong BKPyV -induced S phase.
  • Full (24-72hpi) treatment data were re-graphed from FIG. 3C for direct comparison. Significant differences were determined by Two-Way ANOVA with Tukey post-hoc test.
  • FIG. 5A shows a DDRi treatment diagram to indicate the late (48-72 hpi; black) and full (24- 72 hpi; dark gray) treatment windows.
  • FIG. 5B shows cell cycle analysis performed at the late time point (top) or throughout full infection (bottom) by flow cytometry.
  • FIG. 5H shows the cell cycle protein expression profile during BKPyV infection.
  • the top diagram shows the expression levels of cyclins D (gray line), E (blue line), A (green line) and B (orange line) during the phases of a normal cell cycle (x-axis).
  • FIG. 51 shows how DDR activation affects the expression of cyclins during infection.
  • Proteins involved in cell cycle control, DDR activation, and viral infection following DDR inhibition late (48-72 hpi) during infection were analyzed by Western blot.
  • FIG. 6 shows ATM enhanced S phase entry while ATR induced cell cycle arrest during BKPyV infection.
  • FIG. 6A shows a model of the viral life cycle with DDR inhibition scheme.
  • 24hpi TAg yellow
  • capsid proteins capsid proteins
  • Viral progeny can be detected by 48hpi and peaks by 72hpi. Bars represent the drug treatment window for DDR inhibitors during viral infection: early (24 to 30 hpi), mid (24 to 48 hpi), and full (24 to 72 hpi).
  • FIG. 6B shows a time course study to reveal the proportion of cells in S phase and their DNA content during a normal BKPyV infection.
  • Cell cycle analyses were performed at 30, 48, and 72 hpi.
  • S phase (EdU + ) (green) cells were superimposed on the total population (grey). The average % of cells with > 4N DNA content were quantified and mean ⁇ SD for n 3 to 7 biological replicates are shown.
  • FIG. 6C shows how ATRi or ATMi affected the cell cycle in early or mid-infection.
  • FIG. 6F shows the effect of ATR or ATM inhibition on the incidence of premature mitosis (red). All S phase cells (grey) were plotted based on DNA content and mitosis (pH3 S10 ).
  • FIG. 6H shows how ATRi or ATMi affected the cell cycle over time during a BKPyV infection.
  • a ternary analysis was utilized to represent the proportional relationship of Gl, S, and G2 phases for each of 3-7 replicates from the early (circle), mid (square) and full (triangle) DDRi treatment windows quantified from FIG. 6C and FIG. 3C.
  • 95% COI is represented for each treatment population (line). Symbol color represents vehicle (black), ATMi (green), and ATRi (blue) treatment.
  • FIG. 61 shows a working model illustrating that ATM is required for efficient S phase entry early during infection while ATR activation prevents mitotic entry of actively replicating cells later during infection. Together, this results in a prolonged S phase during infection.
  • FIG. 7 shows premature mitosis was the source of DNA damage due to ATR inhibition during BKPyV infection.
  • Weel and/or Cdkl were silenced with siRNAs to induce or block premature mitosis, respectively.
  • At 72 hpi cells were harvested to assess the amount of premature mitosis and DNA damage during BKPyV infection. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P0.001; ****, PO.OOOl.
  • FIG. 7A shows a diagram for how ATM and ATR regulate of the G2/M checkpoint.
  • ATR and ATM activation stimulates Chkl and Chk2, respectively.
  • Activation of the Weel kinase inhibits Cdkl through phosphorylation leading to G2/M arrest.
  • DDR activation inhibits Cdc25C, the phosphatase that reactivates Cdkl to promote mitosis.
  • Cdc25C turnover requires protein PP2A.
  • FIG. 7D shows quantification of cells from FIG. 7C that are mitotic.
  • FIG. 7K shows the percentage of cells in S-phase.
  • RPTE cells were mock or BKPyV infected (multiplicity of infection of 0.5) and then at 24 hpi treated with Weeli (300 nM MK1775).
  • Cell cycle analysis to identify S phase (EdU) was performed by FACS at 72 hpi.
  • FIG. 7L shows the percentage of cells with premature mitosis based on pH3 S10 expression. The experiment was conducted as described in FIG. 7K.
  • FIG. 7M shows a working model illustrating that ATR prolongs S phase by activating Weel to inhibit mitotic entry by inhibiting Cdkl.
  • ATR activation prevents premature mitosis, which avoids DNA damage and reduced viral titers.
  • FIG. 8 shows activated Cdkl is required for S phase exit during BKPyV infection.
  • FIG. 8B shows a diagram of Chkli treatment during BKPyV infection.
  • FIG. 8C shows the percent of cells in S phase quantified from FIG. 8A. Symbols: mock (white) or BKPyV (black) infected, vehicle (square), Chkli (diamond).
  • FIG. 8D shows the percent of cells in premature mitosis quantified from FIG. 8A. Symbols are as described in FIG. 8C.
  • FIG. 8E shows Cdkli blocks ATRi-associated mitotic entry.
  • Mock or infected RPTE cells were treated at 48 hpi with ATRi (5mM, VE-821), Cdkli (IOmM, RO-3306) or vehicle control (DMSO).
  • FIG. 8H shows small molecule inhibitor treatment and EdU labeling schema for the ATRi treatment conditions (Control, Cdkli, and Cdkli wash). The control, no ATRi treatment schema is not shown.
  • Cdkli was used to synchronize BKPyV RPTE cells treated with ATRi, then Cdkl was washed out to determine if cells entered mitosis immediately (premature mitosis) or completed S phase prior to entering mitosis.
  • FIG. 8K shows a working model wherein ATR prolongs S phase by activating Wee 1 to inhibit mitotic entry by inhibiting Cdkl.
  • ATR activation prevents premature mitosis, which in turn maintains S phase, prevents DNA damage, and enhances viral titers.
  • Weel is no longer activated to inhibit Cdkl.
  • Activated Cdkl in S phase induces premature mitosis, leading to DNA damage and reducing viral titers.
  • the present disclosure provides for methods of treating a PyV infection in a subject as well as methods for treating conditions related to a PyV infection in a subject.
  • the present disclosure provides for a method of treating a subject having a PyV infection (including activation of a latent PyV).
  • Such methods comprise administering to the subject an amount of an ATM inhibitor (ATMi), an ATR inhibitor (ATRi), or both an ATMi and an ATRi.
  • ATMi ATM inhibitor
  • ATRi ATR inhibitor
  • Such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the present disclosure provides for a method of reducing a viral titer of a PyV in a subject having a PyV infection or a latent PyV.
  • Such methods comprise administering to the subject an amount of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the present disclosure provides for a method of treating a subject suffering from a disease or condition that suppresses the immune system.
  • Such methods comprise administering to the subject an amount of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the present disclosure provides for a method for treating an adverse effect of a treatment that suppresses the immune system in a subject administered the treatment that suppresses the immune system.
  • Such methods comprise administering to the subject an amount of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the present disclosure provides a method for treating a side effect of immune suppression in a subject who has received or is being prepared to receive an organ or tissue transplant, wherein the subject is administered a treatment that suppresses the immune system and the subject has a PyV infection or a latent PyV.
  • Such methods comprise administering to the subject an amount of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • Such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the present disclosure provides for a method for inhibiting the DDR in a subject having a PyV infection.
  • Such methods comprise administering to the subject an amount of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the present disclosure provides for a method for treating a disease or condition caused by or resulting from a PyV infection in a subject (including activation of a latent PyV).
  • Such methods comprise administering to the subject an amount of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the present disclosure provides a pharmaceutical composition for use in the methods described herein, including, but not limited to the methods of the first to seventh aspects, wherein the composition comprises an ATMi, an ATRi, and an optional pharmadeutically acceptable carrier or excipient.
  • the present disclosure provides a pharmaceutical composition for use in the methods described herein, including, but not limited to the methods of the first to seventh aspects, wherein the composition comprises an ATMi and an optional pharmadeutically acceptable carrier or excipient.
  • the present disclosure provides a pharmaceutical composition for use in the methods described herein, including, but not limited to the methods of the first to seventh aspects, wherein the composition comprises an ATRi and an optional pharmadeutically acceptable carrier or excipient.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in a method of treatment or therapy.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in treating a PyV infection.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in reducing a viral titer of a polyomavirus PyV having a PyV infection or a latent PyV.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in treating a disease or condition that suppresses the immune system.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in treating an adverse effect of a treatment that suppresses the immune system.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in treating a side effect of immune suppression in a subject who has receiveeid or is being prepared to receive an organ or tissue transplant, wherein the subject has a PyV infection or a latent PyV.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in decreasing the DDR.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in the manufacture of a medicament for treating a disease or condition caused by or resulting from a PyV infection.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in the manufacture of a medicament for reducing a viral titer of a polyoma virus PyV having a PyV infection or a latent PyV.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in the manufacture of a medicament for treating a disease or condition that suppresses the immune system.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in the manufacture of a medicament for decreasing an adverse effect of a treatment that suppresses the immune system.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in the manufacture of a medicament for treating a side effect of immune suppression in a subject who has received or is being prepared to receive an organ or tissue transplant, wherein the subject has a PyV infection or a latent PyV.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use the manufacture of a medicament for decreasing the DDR.
  • the present disclosure provides an ATMi, an ATRi, both an ATMi and an ATRi, or a pharmacetuical composition of the eighth to tenth aspects, for use in the manufacture of a medicament for treating a disease or condition caused by or resulting from a PyV infection.
  • a disease or condition that suppresses the immune system refers to a disease or condition in which the immune system is suppressed, either as a consequence of the pathology of the agent causing the disease or condition or as a result of a treatment of the disease or condition (for example, treating a subject having an autoimmune disease with a compound that modulates or inhibits the immune system).
  • Exemplary diseases or conditions that suppress the immune system include, but are not limited to, acquired immunodeficiency syndrome (AIDS), cancers of the immune system, immune-complex diseases (for example, viral hepatitis and complications of monoclonal antibody treatment), combined immunodeficiency disease, complement deficiencies, DiGeorge syndrome, hypogammaglobulinemia, Job syndrome, leukocyte adhesion defects, panhypogammaglobulinemia, Bruton’s disease, congenital agammaglobulinemia, selective deficiency of IgA, Wiskott-Aldrich syndrome, ataxia-telangiectasia, cartilage-hair hypoplasia, PyV nephropathy, nephropathy non-renal solid organ transplants, interstitial nephritis and progressive multifocal leukoencephalopathy (PML), dementia, blindness, paralysis, and seizures associated with PML, Merkel cell cancer, hemorrhagic cystitis, diseases or conditions in which in which the immune system is
  • the term “suppressed” in describing the immune system means a change in the function of the immune system that decreases a subject's ability to respond to self and/or non-self-antigens (such as, but not limited to, bacteria, viruses, fungi, and transplanted organs or tissues).
  • the change may be the result of a treatment that decreases the effectiveness of the immune system or the result of a disease or condition that decreases the effectiveness of the immune system.
  • the normal immune system involves a complex interaction of certain types of cells that can recognize and attack non-self-antigens and in certain cases (i.e., autoimmune diseases) attack self-antigens.
  • the immune system has both innate and adaptive components. Innate immunity is made up of immune protections people are bom with. Adaptive immunity develops throughout life. Adaptive immunity is divided into two components: humoral immunity and cellular immunity. The change in the immune system can occur in any component of the immune system or in more than one component of the immune system.
  • organ or tissue transplant means a procedure in which an organ or tissue is removed from the body of a donor and placed in the body of a recipient, generally to replace a damaged or dysfunctional organ or tissue, wherein the recipient is administered a treatment in which the immune system is suppressed.
  • organs and tissues that may be the subject of an organ or tissue transplant include, but are not limited to, kindey, heart, intestine (small bowel), pancreatic islet cell, liver, lung, thymus, pancrease, bone marrow, bone, tendon (musculoskeletal graft), cornea, skin, heart valve, nerve, and vein.
  • the donor and recipient are the same subject (for example, in the case of a bone marrow transplant).
  • the donor and recipient are not the same subject (for example, in the case of a kidney transplant).
  • the donor is an animal (for example, a heart valve).
  • polyomavirus or“PyV” is meant to include any virus of the family Polyomaviridae that infects or is capable of infecting a human subject.
  • PyV include, but are not limited to, Merkel cell polyomavirus (MCPyV), Trichodysplasia spinulosa polyomavirus, human polyomavirus 9, human polyomavirus 12, New Jersey polyomavirus, BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), KI, polyomavirus (KIPyV), WU polyomavirus (WUPyV), human polyomavirus 6, human polyomavirus 7, MW polyomavirus, STL polyomavirus, and Lyon IARC polyomavirus.
  • the polyomavirus is BKPyV.
  • the polyomavirus is JCPyV.
  • phosphatidylinositol 3-kinase-related kinase family means the proteins ATM, ATR, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), mammalian target of rapamycin (mTOR), suppressor of morphogenesis in genitalia (SMG1), and transformation/transcription domain-associated protein (TRRAP).
  • ATM DNA-dependent protein kinase catalytic subunit
  • mTOR mammalian target of rapamycin
  • SMG1 suppressor of morphogenesis in genitalia
  • TRRAP transformation/transcription domain-associated protein
  • treatment refers a course of action (such as administering an ATMi or ATRi of the present disclosure) taken to reduce a symptom, aspect, or characteristics of a disease or condition. Such treating need not be absolute to be useful.
  • the term“in need of treatment” refers to a judgment made by a caregiver that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a disease or condition that is treatable by a method or compound of the disclosure.
  • the term“individual”,“subject” or“patient” refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans.
  • mammals such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans.
  • the term may specify male or female or both, or exclude male or female.
  • a subject is a human.
  • the term“therapeutically effective amount” refers to an amount of a compound (such as an ATMi or ATRi of the present disclosure), either alone or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease or condition. Such effect need not be absolute to be beneficial.
  • PyVs are the group of DNA tumor viruses with the smallest genome of DNA viruses, encoding only five to eight proteins (1).
  • the BKPyV early genes dramatically alter the host cell cycle and are expressed by alternative splicing of a single transcript to generate large (TAg), small (tAg), and truncated (truncTAg) tumor antigens.
  • the late genes encode for agnoprotein and three structural proteins (VP1, VP2, and VP3).
  • the recently solved structure of the BKPyV virion revealed an outer shell that consists of 72 pentamers of the major capsid protein, VP1, and an inner shell that is not solvent exposed consisting of VP2 and VP3 monomers.
  • the minor capsid proteins, VP2 and VP3 are made from the same transcript, but VP2 has an N-terminal extension.
  • VP1 is required for viral entry through endocytosis and then carried by microtubules to the endoplasmic reticulum at which point the capsid is uncoated and VP2 and/or VP3 are required for nuclear import by the importin pathway where the genome remains episomal.
  • Expression of the early gene TAg drives the cells into S phase by sequestering retinoblastoma protein (pRb) and blocking apoptosis by inactivating the p53 tumor suppressor protein.
  • pRb retinoblastoma protein
  • Viral DNA replication requires only one viral protein, TAg, and several cellular proteins, such as: replication protein A (RPA), DNA polymerase d (Pol d), and topoisomerase I. PyV infection has also been shown to require the mismatch repair pathway for cell cycle arrest (BK polyomavirus infection activates the DNA damage response to prolong S Phase, Justice, JL, Verhalen, Brandy, and Thompson, SR, J Virol 93:e00130- 19).
  • RPA replication protein A
  • Poly d DNA polymerase d
  • Topoisomerase I topoisomerase I.
  • PyV infection has also been shown to require the mismatch repair pathway for cell cycle arrest (BK polyomavirus infection activates the DNA damage response to prolong S Phase, Justice, JL, Verhalen, Brandy, and Thompson, SR, J Virol 93:e00130- 19).
  • PyVs rely on host DNA replication machinery to amplify the viral genome.
  • PyVs drive the host into S phase by expressing TAg and tAg.
  • TAg inactivates the S phase-suppressor, retinoblastoma protein (pRb) (3, 4) while tAg inactivates protein phosphatase 2A (PP2A), a master phosphatase that is essential for cell cycle progression (5, 6).
  • P2A protein phosphatase 2A
  • the DDR is a cellular response to genotoxic stress that governs three processes: DNA repair, cell cycle arrest, or cell death (10).
  • the DDRs that impact PyV infection are regulated by ataxia telangiectasia mutated (ATM) and ATM-Rad 3 related (ATR) protein.
  • ATM coordinates homologous recombination to repair double-stranded breaks.
  • RPA association with ssDNA activates the ATR pathway causing replication to slow and mediates recovery from replication fork collapse (11).
  • Both ATR and ATM mediate cell cycle arrest by activating the downstream checkpoint kinases (Chk), Chkl (ATR) and Chk2 (ATM), respectively.
  • ATM and ATR phosphorylate potentially hundreds of proteins in response to DNA damage (12).
  • the present disclosure identifies the source of host DNA damage that occurs during PyV infection when the DDR is inhibited.
  • the present disclosure shows that the DDR is required to prevent host DNA damage rather than to repair existing damage caused intrinsically by PyV induced replication stress.
  • the present disclosure shows that ATR prevented premature mitosis during PyV infection by blocking activation of cyclin dependent kinase 1 (Cdkl). Consistent with this finding, blocking premature mitosis by Cdkl- depletion prevented DNA damage and rescued viral titers that were attenuated by both ATM and ATR inhibition.
  • the present disclosure shows that the DDR is required late during infection when the majority of viral replication and assembly occurs to prolong S phase and prevent mitotic entry, thereby extending the window for viral production.
  • the present disclosure also reveals differences between the role of ATM and ATR during a Py V infection.
  • ATM was required for efficient S phase entry as well as prolonging S phase as inhibiting ATM drove cells into regulated mitosis following S phase termination.
  • inhibition of ATR during BKPyV infection resulted in a dramatic shift of the population of cells that entered mitosis while actively synthesizing DNA, resulting in severe DNA damage.
  • the present disclosure shows PyV activates the DDR in order to keep PyV infected cells in S phase.
  • This extended S phase provides a greater time to replicate the viral DNA. Absent activation of the DDR in PyV infected cells (i.e., when the DDR was inhibited in PyV infected cells), significant DNA damage in PyV infected cells occurs. The DNA damage was primarily due to actively replicating cells with uncondensed chromosomes entering directly into mitosis. The DNA damage decreased PyV viral titer due to death of PyV infected cells.
  • the present disclosure also provides insights into therapeutic targets that may be used to inhibit PyV replication and to treat PyV infection in a subject.
  • the present disclosure demonstrates that inhibition of ATM and/or ATR represent viable methods of treatment as described herein. Therefore, inhibiting activation of the DDR through the use of the methods and compositions of the present disclosure provides a novel and inventive approach to treating PyV infection and diseases and conditions related to PyV infection.
  • the present disclosure provides methods for various methods of treatment based on inhibiting ATM, ATR, or both ATM and ATR.
  • inhibiting ATM results in decreased damage to host cell DNA making inhibition of ATM a particularly preferred option.
  • the methods rely on inhibiting the DDR that is induced in PyV infected cells.
  • the present disclosure provides a method for treating a subj ect having a PyV infection.
  • Such methods comprise administering to the subject an amount, including a therapeutically effective amount, of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the Py V infection result from the activation of a latent Py V in the subject.
  • the PyV infection may result from any virus of the family Polyomaviridae that infects or is capable of infecting a human subject.
  • PyVs include, but are not limited to, MCPyV, Trichodysplasia spinulosa polyomavirus, human polyomavirus 9, human polyomavirus 12, New Jersey polyomavirus, BKPyV, JCPyV, KIPyV, WUPyV, human polyomavirus 6, human polyomavirus 7, MW polyomavirus, STL polyomavirus, Lyon IARC polyomavirus, or a combination of the foregoing.
  • the PyV infection is caused by JCPyV, MCPyV, or BKPyV. In another particular embodiment, the PyV infection is caused by BKPyV.
  • the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • an ATMi and an ATRi such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits the DDR.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits PyV replication in the subject and/or reduces PyV viral titers in the subject.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits activation of a latent PyV.
  • the subject has a disease or condition that suppresses the immune system.
  • the subject is a recipient of an organ or tissue transplant (including, but not limited to, a kidney transplant) and the subject is administered a treatment that suppresses the immune system.
  • the subject is administered a treatment that suppresses the immune system in preparation for an organ or tissue transplant (including, but not limited to, a kidney transplant).
  • the subject has an autoimmune disease.
  • the subject has AIDS.
  • any ATMi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATMi compounds specifically disclosed herein. Suitable ATMi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATMi is AZD1390, AZD0156, or KU-55933.
  • ATRi any ATRi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATRi compounds specifically disclosed herein. Suitable ATRi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATRi is AZD6738 or VE-821.
  • the present disclosure also provides methods for reducing a viral titer of a PyV in a subjectv having a PyV infection or a latent PyV.
  • Such methods comprise administering to the subject an amount, including a therapeutically effective amount, of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • Such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • PyV infection results from the activation of a latent PyV in the subject.
  • the viral titer is reduced by over 90%, 75%, 50%, 40%, 30%, or 20% as compared to a subject not administered an ATMi, ATRi, or combination of ATMi and ATRi of the present disclosure.
  • the PyV infection may result from any virus of the family Polyomaviridae that infects or is capable of infecting a human subject.
  • PyVs include, but are not limited to, MCPyV, Trichodysplasia spinulosa polyomavirus, human polyomavirus 9, human polyomavirus 12, New Jersey polyomavirus, BKPyV, JCPyV, KIPyV, WUPyV, human polyomavirus 6, human polyomavirus 7, MW polyomavirus, STL polyomavirus, Lyon IARC polyomavirus, or a combination of the foregoing.
  • the PyV infection is caused by JCPyV, MCPyV, or BKPyV. In another particular embodiment, the PyV infection is caused by BKPyV.
  • the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • an ATMi and an ATRi such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits the DDR.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits PyV replication in the subject and/or reduces PyV viral titers in the subject.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits activation of a latent PyV.
  • the subject has a disease or condition that suppresses the immune system.
  • the subject is a recipient of an organ or tissue transplant (including, but not limited to, a kidney transplant) and the subject is administered a treatment that suppresses the immune system.
  • the subject is administered a treatment that suppresses the immune system in preparation for an organ or tissue transplant (including, but not limited to, a kidney transplant).
  • the subject has an autoimmune disease.
  • the subject has AIDS.
  • any ATMi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATMi compounds specifically disclosed herein. Suitable ATMi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATMi is AZD1390, AZD0156, KU-55933, or a combination of the foregoing.
  • ATRi any ATRi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATRi compounds specifically disclosed herein. Suitable ATRi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATRi is AZD6738, VE-821 or a combination of the foregoing.
  • the present disclosure further provides for methods for treating a subject suffering from a disease or condition that suppresses the immune system.
  • Such methods comprise administering to the subject an amount, including a therapeutically effective amount, of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • Such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the disease or condition that suppresses the immune system includes, but is not limited to, AIDS, cancers of the immune system, immune-complex diseases (for example, viral hepatitis and complications of monoclonal antibody treatment), combined immunodeficiency disease, complement deficiencies, DiGeorge syndrome, hypogammaglobulinemia, Job syndrome, leukocyte adhesion defects, panhypogammaglobulinemia, Bruton’s disease, congenital agammaglobulinemia, selective deficiency of IgA, Wiskott-Aldrich syndrome, ataxia-telangiectasia, cartilage-hair hypoplasia, PyV nephropathy, nephropathy non-renal solid organ transplants, interstitial nephritis and PML, dementia, blindness, paralysis, and seizures associated with PML, Merkel cell cancer, hemorrhagic cystitis, diseases or conditions in which in which the immune system is suppressed during treatment of the disease or condition (such, as,
  • such a disease or condition is an autoimmune disease, AIDS, cancers of the immune system, or cartilage-hair hypoplasia.
  • the immune system is suppressed in the subject either as a consequence of the pathology of the agent causing the disease or condition or as a result of a treatment of the disease or condition.
  • the subject subject suffering from a disease or condition that suppresses the immune system also has a PyV infection.
  • the PyV infection result from the activation of a latent PyV in the subject.
  • the PyV infection may result from any virus of the family Polyomaviridae that infects or is capable of infecting a human subject.
  • PyVs include, but are not limited to, MCPyV, Trichodysplasia spinulosa polyomavirus, human polyomavirus 9, human polyomavirus 12, New Jersey polyomavirus, BKPyV, JCPyV, KIPyV, WUPyV, human polyomavirus 6, human polyomavirus 7, MW polyomavirus, STL polyomavirus, Lyon IARC polyomavirus, or a combination of the foregoing.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV.
  • the PyV infection is caused by BKPyV.
  • the disease or condition that suppresses the immune system is a disease or condition listed above and the subject is treated with an ATMi only, such as but not limited to, AZD1390, AZD0156 and/or KU-55933.
  • the disease or condition that suppresses the immune system is an autoimmune disease, AIDS, or a cancer of the immune system and the subject is treated with an ATMi only, such as but not limited to, AZD1390, AZD0156 and/or KU-55933.
  • the PyV infection may be caused by at least one of JCPyV, MCPyV, or BKPyV. In any of the foregoing, the PyV infection may be caused by BKPyV.
  • the disease or condition that suppresses the immune system is a disease or condition listed above and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the disease or condition that suppresses the immune system is an autoimmune disease, AIDS, or a cancer of the immune system, or cartilage-hair hypoplasia and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the PyV infection may be caused by at least one of JCPyV, MCPyV, or BKPyV. In any of the foregoing, the PyV infection may be caused by BKPyV.
  • the disease or condition that suppresses the immune system is a disease or condition listed above and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, and KU-55933, and one or more of AZD6738 and VE-821.
  • the disease or condition that suppresses the immune system is an autoimmune disease, AIDS, or a cancer of the immune system, and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, and KU-55933, and one or more of AZD6738 and VE-821.
  • the PyV infection may be caused by at least one of JCPyV, MCPyV, or BKPyV. In any of the foregoing, the PyV infection may be caused by BKPyV.
  • the subject is being administered a treatment that suppresses the immune system.
  • the subject is being administered a treatment that suppresses the immune system in preparation for an organ or tissue transplant (including, but not limited to, a kidney transplant).
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits the DDR.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits PyV replication in the subject and/or reduces PyV viral titers in the subject.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits activation of a latent PyV.
  • any ATMi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATMi compounds specifically disclosed herein. Suitable ATMi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATMi is AZD1390, AZD0156, KU-55933, or a combination of the foregoing.
  • ATRi any ATRi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATRi compounds specifically disclosed herein. Suitable ATRi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATRi is AZD6738, VE-821 or a combination of the foregoing.
  • the present disclosure further provides methods for treating an adverse effect of a treatment that suppresses the immune system in a subject.
  • Such methods comprise administering to the subject an amount, including a therapeutically effective amount, of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • Such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the subject administered the treatment that suppresses the immune system also has a PyV infection.
  • a PyV infection may result from the activation of a latent PyV in the subject.
  • the PyV infection may result from any virus of the family Polyomaviridae that infects or is capable of infecting a human subject.
  • PyVs include, but are not limited to, MCPyV, Trichodysplasia spinulosa polyomavirus, human polyomavirus 9, human polyomavirus 12, New Jersey polyomavirus, BKPyV, JCPyV, KIPyV, WUPyV, human polyomavirus 6, human polyomavirus 7, MW polyomavirus, STL polyomavirus, Lyon IARC polyomavirus, or a combination of the foregoing.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV.
  • the PyV infection is caused by BKPyV.
  • the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • an ATMi and an ATRi such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits the DDR.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits PyV replication in the subject and/or reduces PyV viral titers in the subject.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits activation of a latent PyV.
  • the subject has a disease or condition that suppresses the immune system.
  • the subject is a recipient of an organ or tissue transplant (including, but not limited to, a kidney transplant).
  • the subject is administered a treatment that suppresses the immune system in preparation for an organ or tissue transplant (including, but not limited to, a kidney transplant).
  • the organ or tissue transplant is a heart, intestine (small bowel), pancreatic islet cell, kidney, liver, lung, thymus, pancrease, bone marrow, bone, tendon (musculoskeletal graft), cornea, skin, heart valve, nerve, or vein transplant.
  • the tissue or organ transplant is a kidney transplant.
  • the subject has an autoimmune disease.
  • the subject has AIDS.
  • the adverse effect an activation of a latent PyV, including, but not limited to, a PyV infection caused by BKPyV, JCPyV, and/or MCPyV.
  • the adverse effect is rejection of a transplanted tissue or organ, dysfunction of a transplanted tissue or organ, or failure of a transplanted tissue or organ.
  • the adverse effect the occurrence of PML or dementia, blindness, paralysis, and seizures associated with PML.
  • any ATMi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATMi compounds specifically disclosed herein. Suitable ATMi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATMi is AZD1390, AZD0156, KU-55933, or a combination of the foregoing.
  • ATRi any ATRi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATRi compounds specifically disclosed herein. Suitable ATRi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATRi is AZD6738, VE-821 or a combination of the foregoing.
  • the present disclosure further provides a method for treating a side effect of immune suppression in a subject who has received or is being prepared to receive an organ or tissue transplant, wherein the subject is administered a treatment that suppresses the immune system and the subject has a PyV infection or a latent PyV.
  • Such methods comprise administering to the subject an amount, including a therapeutically effective amount, of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • Such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the subject administered the treatment that suppresses the immune system also has a PyV infection.
  • a PyV infection may result from the activation of a latent PyV in the subject.
  • the PyV infection may result from any virus of the family Polyomaviridae that infects or is capable of infecting a human subject.
  • PyVs include, but are not limited to, MCPyV, Trichodysplasia spinulosa polyomavirus, human polyomavirus 9, human polyomavirus 12, New Jersey polyomavirus, BKPyV, JCPyV, KIPyV, WUPyV, human polyomavirus 6, human polyomavirus 7, MW polyomavirus, STL polyomavirus, Lyon IARC polyomavirus, or a combination of the foregoing.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV. In another particular embodiment, the PyV infection is caused by BKPyV.
  • the side effect of immune suppression is rejection of the tissue or organ, dysfunction of the tissue or organ, and/or failure of the tissue or organ. In another embodiment, the side effect of immune suppression is nephropathy.
  • the side effect of immune suppression is a side effect listed above and the subject is treated with an ATMi only, such as but not limited to, AZD1390, AZD0156 and/or KU-55933.
  • the side effect of immune suppression is rejection of the tissue or organ, dysfunction of the tissue or organ, and/or failure of the tissue or organ and the subject is treated with an ATMi only, such as but not limited to, AZD1390, AZD0156 and/or KU-55933.
  • the side effect of immune suppression is nephropathy and the subject is treated with an ATMi only, such as but not limited to, AZD1390, AZD0156 and/or KU-55933.
  • the PyV infection may be caused by at least one of JCPyV, MCPyV, or BKPyV. In any of the foregoing, the PyV infection may be caused by BKPyV.
  • the side effect of immune suppression is a side effect listed above and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the side effect of immune suppression is rejection of the tissue or organ, dysfunction of the tissue or organ, and/or failure of the tissue or organ and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the side effect of immune suppression is nephropathy and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the PyV infection may be caused by at least one of JCPyV, MCPyV, or BKPyV. In any of the foregoing, the PyV infection may be caused by BKPyV.
  • the side effect of immune suppression is a side effect listed above and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, and KU-55933, and one or more of AZD6738 and VE-821.
  • the side effect of immune suppression is rejection of the tissue or organ, dysfunction of the tissue or organ, and/or failure of the tissue or organ and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, and KU-55933, and one or more of AZD6738 and VE-821.
  • the side effect of immune suppression is nephropathy and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, and KU-55933, and one or more of AZD6738 and VE-821.
  • the PyV infection may be caused by at least one of JCPyV, MCPyV, or BKPyV. In any of the foregoing, the PyV infection may be caused by BKPyV.
  • the organ or tissue transplant is a heart, intestine (small bowel), pancreatic islet cell, kidney, liver, lung, thymus, pancrease, bone marrow, bone, tendon (musculoskeletal graft), cornea, skin, heart valve, nerve, or vein transplant.
  • the tissue or organ transplant is a kidney transplant.
  • the organ or tissue transplant is a kidney transplant and the subject has a BKPyV infection, including a BKPyV infection caused by activation of a latent BKPyV virus by the treatment that suppresses the immune system.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits the DDR.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits PyV replication in the subject and/or reduces PyV viral titers in the subject.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits activation of a latent PyV.
  • the subject is administered a treatment that suppresses the immune system in preparation for an organ or tissue transplant (including, but not limited to, a kidney transplant).
  • a treatment that suppresses the immune system in preparation for an organ or tissue transplant including, but not limited to, a kidney transplant.
  • the subject has an autoimmune disease.
  • the subject has AIDS.
  • any ATMi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATMi compounds specifically disclosed herein. Suitable ATMi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATMi is AZD1390, AZD0156, KU-55933, or a combination of the foregoing.
  • ATRi any ATRi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATRi compounds specifically disclosed herein. Suitable ATRi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATRi is AZD6738, VE-821 or a combination of the foregoing.
  • the present disclosure further provides methods for inhibiting the DDR in a subject having a PyV infection.
  • Such methods comprise administering to the subject an amounttt, including a therapeutically effective amount, of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • Such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the subject has a PyV infection.
  • the PyV infection results from the activation of a latent PyV in the subject. Such activation may result from suppression of the immune system (such as by a treatment that suppresses the immune system or a disease or condition that suppresses the immune system).
  • the PyV infection may result from any virus of the family Polyomaviridae that infects or is capable of infecting a human subject.
  • PyVs include, but are not limited to, MCPyV, Trichodysplasia spinulosa polyomavirus, human polyomavirus 9, human polyomavirus 12, New Jersey polyomavirus, BKPyV, JCPyV, KIPyV, WUPyV, human polyomavirus 6, human polyomavirus 7, MW polyomavirus, STL polyomavirus, Lyon IARC polyomavirus, or a combination of the foregoing.
  • the subject has a PyV infection caused by at least one of JCPyV, MCPyV, or BKPyV. In another particular embodiment, the subject has a PyV infection caused by BKPyV. In one embodiment, the PyV infection is caused by activation of a latent PyV.
  • the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the subject has a PyV infection caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the subject has a PyV infection caused by BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the subject has a PyV infection caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the subject has a PyV infection caused by BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the subject has a PyV infection caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the subject has a PyV infection caused by BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • an ATMi and an ATRi such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits PyV replication in the subject and/or reduces PyV viral titers in the subject. In certain embodiments, of the methods for inhibiting a DDR, treatment with the ATMi, ATRi, or ATMi and ATRi inhibits activation of a latent PyV.
  • the subject has a disease or condition that suppresses the immune system.
  • the subject is a recipient of an organ or tissue transplant (including, but not limited to, a kidney transplant) and the subject is administered a treatment that suppresses the immune system.
  • the subject is administered a treatment that suppresses the immune system in preparation for an organ or tissue transplant (including, but not limited to, a kidney transplant).
  • the subject has an autoimmune disease.
  • the subject has AIDS.
  • any ATMi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATMi compounds specifically disclosed herein. Suitable ATMi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATMi is AZD1390, AZD0156, KU-55933, or a combination of the foregoing.
  • ATRi any ATRi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATRi compounds specifically disclosed herein. Suitable ATRi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATRi is AZD6738, VE-821 or a combination of the foregoing.
  • the present disclosure further provides methods for treating a disease or condition caused by or resulting from a PyV infection in a subject.
  • Such methods comprise administering to the subject an amount, including a therapeutically effective amount, of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • Such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the subject administered a treatment that suppresses the immune system and the PyV infection that results from the treatment that suppresses the immune system.
  • the PyV infection results from the activation of a latent PyV in the subject. Such activation may result from suppression of the immune system (such as by a treatment that suppresses the immune system or a disease or condition that suppresses the immune system).
  • the disease or condition resulting from a PyV infection is nephropathy rejection of a tissue or organ transplant, dysfunction of a tissue or organ transplant, or failure of a tissue or organ transplant, PML (generally associated with JCPy V), and Merkel cell cancer (generally associated with MCPyV).
  • the PyV infection may result from any virus of the family Polyomaviridae that infects or is capable of infecting a human subject.
  • PyVs include, but are not limited to, Merkel cell polyomavirus (MCPyV), Trichodysplasia spinulosa polyomavirus, human polyomavirus 9, human polyomavirus 12, New Jersey polyomavirus, BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), KI polyomavirus (KIPyV), WU polyomavirus (WUPy V), human polyomavirus 6, human polyomavirus 7, MW polyomavirus, STL polyomavirus, and Lyon IARC polyomavirus.
  • MCPyV Merkel cell polyomavirus
  • Trichodysplasia spinulosa polyomavirus human polyomavirus 9, human polyomavirus 12, New Jersey polyomavirus
  • BKPyV
  • the subject has a PyV infection caused by at least one of JCPyV, MCPyV, or BKPyV. In another particular embodiment, the subject has a PyV infection caused by BKPyV. In one embodiment, the PyV infection is caused by activation of a latent PyV.
  • the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the subject has a PyV infection caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the subject has a PyV infection caused by BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the subject has a PyV infection caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the subject has a PyV infection caused by BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the subject has a PyV infection caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the subject has a PyV infection caused by BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • an ATMi and an ATRi such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits the DDR.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits PyV replication in the subject and/or reduces PyV viral titers in the subject.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits activation of a latent PyV.
  • the subject has a disease or condition that suppresses the immune system.
  • the subject is a recipient of an organ or tissue transplant (including, but not limited to, a kidney transplant) and the subject is administered a treatment that suppresses the immune system.
  • the subject is administered a treatment that suppresses the immune system in preparation for an organ or tissue transplant (including, but not limited to, a kidney transplant).
  • the subject has an autoimmune disease. In certain embodiments of the methods for treating a disease or condition caused by or resulting from a PyV infection, the subject has from AIDS.
  • Any ATMi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATMi compounds specifically disclosed herein. Suitable ATMi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATMi is AZD1390, AZD0156, KU-55933, or a combination of the foregoing.
  • ATRi any ATRi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATRi compounds specifically disclosed herein. Suitable ATRi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATRi is AZD6738, VE-821 or a combination of the foregoing.
  • the present disclosure further provides a method for treating a subject at risk for activation of a latent PyV.
  • Such methods comprise administering to the subject an amount, including a therapeutically effective amount, of an ATMi, an ATRi, or both an ATMi and an ATRi.
  • Such methods comprise administering to the subject a therapeutically effective amount of an ATMi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATRi.
  • such methods comprise administering to the subject a therapeutically effective amount of an ATMi and an ATRi.
  • the subject is determined to have a latent PyV before such treatment is administered.
  • the subject is not determined to have a latent PyV but is undergoing a procedure or treatment that suppresses the immune system (which would result in activation of any latent PyV if such were present)
  • the subject is one who is receiving an organ or tissue transplant.
  • the subject is one who is donating an organ or tissue for transplantation (including both autologous and allogeneic transplantation).
  • the subject is undergoing a treatment that suppresses the immune system and a PyV infection results from the activation of the latent PyV in the subject.
  • the treatment prevents the activation of a latent PyV in a subject.
  • the PyV infection may result from any virus of the family Polyomaviridae that infects or is capable of infecting a human subject.
  • PyVs include, but are not limited to, MCPyV, Trichodysplasia spinulosa polyomavirus, human polyomavirus 9, human polyomavirus 12, New Jersey polyomavirus, BKPyV, JCPyV, KIPyV, WUPyV, human polyomavirus 6, human polyomavirus 7, MW polyomavirus, STL polyomavirus, Lyon IARC polyomavirus, or a combination of the foregoing.
  • the latent PyV is at least one of JCPyV, MCPyV, or BKPyV.
  • the latent PyV is BKPyV.
  • the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATMi only, such as, but not limited to, AZD1390, AZD0156, KU-55933 or a combination thereof.
  • the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATRi only, such as but not limited to, AZD6738 and/or VE-821.
  • the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the PyV infection is caused by at least one of JCPyV, MCPyV, or BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • the PyV infection is caused by BKPyV and the subject is treated with an ATMi and an ATRi, such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • an ATMi and an ATRi such as but not limited to, one or more of AZD1390, AZD0156, or KU-55933 and one or more of AZD6738 and VE-821.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits the DDR when the latent PyV is activated.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits PyV replication in the subject and/or reduces PyV viral titers in the subject when the latent PyV is activated.
  • treatment with the ATMi, ATRi, or ATMi and ATRi inhibits activation of the latent PyV.
  • the subject has a disease or condition that suppresses the immune system.
  • the subject is a recipient of an organ or tissue transplant (including, but not limited to, a kidney transplant) and the subject is administered a treatment that suppresses the immune system.
  • the subject is administered a treatment that suppresses the immune system in preparation for an organ or tissue transplant (including, but not limited to, a kidney transplant).
  • the subject has an autoimmune disease. In certain embodiments of the methods for treating a subject at risk for activation of a latent PyV, the subject has AIDS.
  • any ATMi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATMi compounds specifically disclosed herein. Suitable ATMi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATMi is AZD1390, AZD0156, KU-55933, or a combination of the foregoing.
  • ATRi any ATRi known in the art may be used in the methods for treating a subject having a PyV infection, including the ATRi compounds specifically disclosed herein. Suitable ATRi compounds are discussed herein and the discussion is hereby incorporated by reference. In certain embodiments, the ATRi is AZD6738, VE-821 or a combination of the foregoing.
  • a disease or condition that suppresses the immune system includes, but is not limited to, AIDS, cancers of the immune system, immune-complex diseases (for example, viral hepatitis and complications of monoclonal antibody treatment), combined immunodeficiency disease, complement deficiencies, DiGeorge syndrome, hypogammaglobulinemia, Job syndrome, leukocyte adhesion defects, panhypogammaglobulinemia, Bruton’s disease, congenital agammaglobulinemia, selective deficiency of IgA, Wiskott-Aldrich syndrome, ataxia- telangiectasia, cartilage-hair hypoplasia, PyV nephropathy, nephropathy non-renal solid organ transplants, interstitial nephritis and PML, dementia, blindness, paralysis, and seizures associated with PML, Merkel cell cancer, hemorrhagic cystitis, diseases or conditions in which in which the immune system
  • an autoimmune disease or condition such a disease or condition includes, but is not limited to, Achalasia, Addison’s disease, Adult Still's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti- GBM/Anti-TBM nephritis, Antiphospholipid syndrome, Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy, Balo disease, Behcet’s disease, Benign mucosal pemphigoid, Bullous pemphigoid,
  • the autoimmune disease or condition is Multiple sclerosis, vasculitis, myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, Rheumatoid arthritis, systemic lupus erythematosus (lupus), inflammatory bowel disease, type 1 diabetes mellitus, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, or psoriasis.
  • compositions that comprise an amount of a compound of the present disclosure (such as an ATMi and/or an ATRi).
  • such pharmaceutical compositions contain a therapeutically effective amount of a compound of the present disclosure.
  • the compound is an ATMi, an ATRi, or both an ATMi and an ATRi.
  • other active agents may be included in such pharmaceutical compositions. Additional active agents to be included may be selected based on the disease or condition to be treated.
  • compositions disclosed may comprise one or more compound of the present disclosure, alone or in combination with additional active agents, in combination with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier examples of such carriers and methods of formulation may be found in Remington: The Science and Practice of Pharmacy (20 th Ed., Lippincott, Williams & Wilkins, Daniel Limmer, editor).
  • Such pharmaceutical compositions may be used in the manufacture of a medicament for use in the methods of treatment and prevention described herein.
  • the compounds of the disclosure are useful in both free form and in the form of pharmaceutically acceptable salts.
  • the pharmaceutically acceptable carriers described herein, including, but not limited to, vehicles, adjuvants, excipients, or diluents, are well-known to those who are skilled in the art.
  • Suitable excipients are also well-known to those who are skilled in the art. The choice of excipient will be determined in part by the particular compound(s), as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following methods and excipients are merely exemplary and are in no way limiting.
  • Suitable carriers and excipients include solvents such as water, alcohol, and propylene glycol, solid absorbants and diluents, surface active agents, suspending agent, tableting binders, lubricants, flavors, and coloring agents.
  • the pharmaceutically acceptable carriers can include polymers and polymer matrices.
  • acceptable pharmaceutical carriers include carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc and water, among others.
  • the pharmaceutically acceptable carrier is chemically inert to the active agents in the composition and has no detrimental side effects or toxicity under the conditions of use.
  • the term“pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • Surfactants such as, for example, detergents, are also suitable for use in the formulations.
  • Specific examples of surfactants include polyvinylpyrrolidone, polyvinyl alcohols, copolymers of vinyl acetate and of vinylpyrrolidone, polyethylene glycols, benzyl alcohol, mannitol, glycerol, sorbitol or polyoxyethylenated esters of sorbitan; lecithin or sodium carboxymethylcellulose; or acrylic derivatives, such as methacrylates and others, anionic surfactants, such as alkaline stearates, in particular sodium, potassium or ammonium stearate; calcium stearate or triethanolamine stearate; alkyl sulfates, in particular sodium lauryl sufate and sodium cetyl sulfate; sodium dodecylbenzenesulphonate or sodium dioctyl sulphosuccinate; or fatty acids, in particular those derived from coconut oil,
  • the compound(s) of the present disclosure will ordinarily be present in an amount of about 0.5-95% weight based on the total weight of the composition.
  • Multiple dosage forms may be administered as part of a single treatment.
  • the active agent can be administered internally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as milk, elixirs, syrups and suspensions. It can also be administered parenterally, in sterile liquid dosage forms.
  • the compound(s) of the present disclosure can also be administered intranasally (nose drops) or by inhalation via the pulmonary system, such as by propellant based metered dose inhalers or dry powders inhalation devices.
  • Other dosage forms include topical administration, such as administration transdermally, via patch mechanism or ointment.
  • the compounds of the disclosure are administered to the subject (or are contacted with cells of the subject) in a therapeutically effective amount.
  • a therapeutically effective amount decreases the level of PyV in the subject and/or limits or prevents an increase in the level of viral particles in the subject.
  • a therapeutically effective amount decreases the viral titer of PyV in the subject.
  • the therapeutically effective amount of a compound of the disclosure ranges from about 0.01 mg/kg/day to about 500 mg/kg/day. In certain embodiments, the therapeutically effective amount ranges from about 0.01 mg/kg/day to about 400 mg/kg/day, from about 0.01 mg/kg/day to about 300 mg/kg/day, from about 0.01 mg/kg/day to about 200 mg/kg/day, from about 0.01 mg/kg/day to about 100 mg/kg/day, from about 0.01 mg/kg/day to about 50 mg/kg/day, from about 0.01 mg/kg/day to about 25 mg/kg/day, from about 0.01 mg/kg/day to about 15 mg/kg/day, from about 0.01 mg/kg/day to about 10 mg/kg/day, from about 0.01 mg/kg/day to about 5 mg/kg/day, or from about 0.01 mg/kg/day to about 2.5 mg/kg/day.
  • the therapeutically effective amount ranges from about 5 mg/kg/day to about 100 mg/kg/day, from about 5 mg/kg/day to about 50 mg/kg/day, from about 2 mg/kg/day to about 30 mg/kg/day, or from about 1 mg/kg/day to about 10 mg/kg/day.
  • the therapeutically effective amount is administered in one or more doses according to a course of treatment (where a dose refers to an amount of a compound administered in a single day).
  • the dose is administered q.d. (1 time/administration per day), b.i.d. (2 times/administrations per day; for example, one- half of the therapeutically effective amount in two administrations a day), or t.i.d. (three times/administrations per day; for example, one-third of the therapeutically effective amount in two administrations a day).
  • the dose may be divided equally or the dose may be divided unequally at each administration. Any given dose may be delivered in a single dosage form or more than one dosage form (for example, a tablet).
  • the methods may comprise the administration of a single dose of a therapeutically effective amount of a compound of the disclosure during the entire course of treatment.
  • the dose is delivered by parenteral administration.
  • the dose is delivered by oral administration.
  • the dose may be delivered in a single dosage form or more than one dosage form (for example, a tablet).
  • the methods may comprise the administration of multiple doses of a therapeutically effective amount of a compound of the disclosure during the course of treatment.
  • the course of treatment may range from 2 days to years, from 2 days to months, or from 2 days to 4 weeks.
  • the course of treatment for a patient who is receiving an immunosuppressive drug the course of treatment is for the complete duration of the time he subject is receiving the immunosuppressive drug; the compounds of the disclosure may be administered before the subject receives the immunosuppressive drug and/or extend for a period of time after the subject receives the last dose of the immunosuppressive drug.
  • a therapeutically effective amount of a compound of the disclosure may be delivered every day during the course of treatment.
  • the therapeutically effective amount need not be the same for every dose during a course of treatment.
  • a course of treatment may comprise administering at least one dose as a loading dose and at least one dose as a maintenance dose, wherein the loading dose contains a greater amount of a compound of the invention as compared to the maintenance dose (such as, but not limited to, 2 to 10 times higher).
  • the dose may comprise a compound of the disclosure alone or a compound of the disclosure in a pharmaceutical composition.
  • the precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the infection or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • ATMi and ATRi for use in the methods and pharmaceutical compositions described herein. Any known ATMi or ATRi may be used in the methods and pharmaceutical compositions described herein.
  • the ATMi is a compound of the formula I:
  • R 1 is methyl
  • R 2 is hydro or methyl; or R 1 and R 2 together with the nitrogen atom to which they are bonded form an azetidinyl, pyrrolidinyl or piperidinyl ring;
  • R 3 is hydro or fluoro
  • R 4 is hydro or methyl
  • R 5 is hydro or fluoro.
  • the ATMi is AZD1390, AZD0156, KU-55933, KU-60014, or KU-59403.
  • the ATMi is a compound described in one or more of US Patent Nos. 9,428,503, 9822,111, 7,049,313, or 8,802,680, US Patent Publication 2018- 134699, Patent Cooperation Treaty application PCT/EP2016/071782, PCT/EP/2017/076416, or PCT/EP2016/076412.
  • the ATMi has a selectivity for ATM as compared to other members of the phosphatidylinositol 3-kinase-related kinase family of 50 or more, 100 or more, 250 or more, or 500 or more.
  • 1 pM of the ATMi does not inhibit another serine/threonine or tyrosine kinase at a level greater than 50% when tested in vitro using the Thermo Fisher Scientific Kinase panel.
  • 1 pM of the ATMi does not inhibit ATR at a level greater than 50% when tested in vitro using the Thermo Fisher Scientific Kinase panel.
  • 1 pM of the ATMi does not inhibit ATM, PI-3K, or mTOR when tested in vitro using the Thermo Fisher Scientific Kinase panel.
  • the ATMi is AZD1390. In another particularly preferred embodiment, the ATMi is AZD0156. In another particularly preferred embodiment, the ATMi is KU-55933.
  • the ATRi is AZD6738, VE-821, VE-822 (VX-970), MAY1895344, CGK-733, M6620, BAY 1895344, or BEZ-235.
  • the ATRi is a compound described in one or more of US Patent Nos. 9,334,244, 8,841,308, 9,701,674, 9,365,557, 8,552,004, 8,252,802, 8,999,997, 9,155,742, or 9,421,213, US Patent Publication Nos. 2018-170,922, 2016-311,809, or 2015-164,908, or Patent Cooperation Treaty Publication WO 2016/020320, WO 2010/071837, or WO 2011/154737.
  • the ATRi has a selectivity for ATR as compared to other members of the phosphatidylinositol 3-kinase-related kinase family of 50 or more, 100 or more, 250 or more, or 500 or more.
  • 1 mM of the ATMi does not inhibit another serine/threonine or tyrosine kinase at a level greater than 50% when tested in vitro.
  • 1 pM of the ATRi does not inhibit another ATM at a level greater than 50% when tested in vitro using the Thermo Fisher Scientific Kinase panel.
  • the ATRi is AZD6738.
  • the ATMi is VE-821.
  • Kits are provided for use in the methods disclosed herein, comprising any of the active agents disclosed herein.
  • the active agent is an ATMi only.
  • the active agent is an ATRi only.
  • the active agent is an ATMi and an ATRi. Any of the foregoing kits may further comprise compositions useful in the administration of the active agents.
  • kits comprise any of the pharmaceutical compositions disclosed herein.
  • instructions for administration may also be provided.
  • devices for administration of the active agents or pharmaceutical compositions may also be provided.
  • RPTE cells were used to study an in vivo lytic BKPyV infection to study BKPyV host- pathogen interactions in cells with intact cell cycle control pathways (15) (16), avoiding complications with using immortalized cells that commonly harbor mutations and epigenetic modifications in the DDR and cell cycle control pathways.
  • ATM and ATR inhibition (ATMi and ATRi) reduced the BKPyV- induced DDR activation as measured by reduced Chkl phosphorylation at Ser317 (pChkl S317 ) and ATM autophosphorylation at Serl981 (pATM S1981 ) (FIG. 1A).
  • the decreased Chkl phosphorylation observed in the ATMi treatment group is likely due to cross talk between the ATM and ATR pathways, (19).
  • abnormal mitosis herein
  • Cells treated with ATMi showed an increased percentage of mitotic cells and a decreased percentage of cells with abnormal mitosis compared to the control group and ATRi groups.
  • Activation of the ATR pathway prevents metaphase shattering, which is similar to what is observed when an S phase cell goes into mitosis (hereafter referred to as“premature mitosis”) (7, 21). Therefore, ATR appears to be important during infection to prevent premature mitotic entry.
  • FIG. 1G To determine if BKPyV requires ATR or ATM activation for cell cycle arrest and to prevent premature mitosis, the cell cycle distribution of BKPyV infected cells was examined when ATM or ATR was inhibited (FIG. 1G). Both ATR and ATM inhibition significantly increased mitotic entry of BKPyV infected cells, but had no effect on uninfected cells (FIG. 1H). ATRi increased premature mitosis 30-fold in BKPyV infected cells (FIG. II), while premature mitosis was rarely observed for uninfected cells under any condition ( ⁇ 0.5% of S phase cells).
  • Example 2- Blocking mitosis prevented BKPyV-denendent host DNA damage when ATR was inactive
  • This example shows blocking mitosis prevented BKPyV-dependent host DNA damage when ATR was inactive.
  • siCdkl or control siRNA siNTC
  • At 24 hpi cells were treated with ATRi, ATMi or vehicle control and harvested at 72 hpi.
  • the ATRi was VE-821 (5mM)
  • the ATMi was KU-55933 (IOmM)
  • vehicle control was DMSO.
  • This example shows DDR activation enhanced BKPyV titers by arresting the cell cycle.
  • MOI 0.5
  • At 24 hpi cells were treated with ATRi, ATMi or vehicle control and harvested at 72 hpi for analysis.
  • the ATRi was VE-821 (5mM)
  • the ATMi was KU-55933 (IOmM)
  • vehicle control was DMSO.
  • BKPyV infection significantly increased the fraction of cells in S phase (FIG. 3F) and correspondingly decreased the number of cells that were in G1 phase compared to the uninfected control (FIG. 3E and 3F).
  • a portion of BKPyV -infected, S-phase cells were marked by >4N DNA content reflecting the reduplication of host chromatin (FIG. 3C).
  • BKPyV infection induces cell cycle arrest, BKPyV infection alone did not significantly increase the portion of cells in G2 or M phase compared to mock infected cells (FIG. 3G).
  • Example 4- The effect of inhibiting DDR activation is observed with multiple ATRis and ATMis
  • FIG. 4A shows cell cycle analysis after treatment with the ATRi AZD6738.
  • FIGS. 4B-4F show the percentage of cells in S phase, Gl phase, G2 + M phase, and M phase.
  • BKPyV infection significantly increased the fraction of cells in S phase (FIG. 4D) and correspondingly decreased the number of cells that were in Gl phase compared to the uninfected control (FIG. 4C).
  • a portion of BKPyV -infected, S-phase cells were marked by >4N DNA content reflecting the reduplication of host chromatin (FIG. 4A).
  • FIG. 4G shows the total fraction of cells in S phase analyzed to determine if the cells were undergoing premature mitosis with the results quantified as in FIG 4H.
  • treatment with the ATRi AZD6738 induced a higher percentage of cell in premature mitosis than did treatment with the ATMi AZD0156 or vehicle control.
  • FIGS. 4I-K Comparison of the average proportion of cells in S phase and premature mitosis caused by chemical inhibition with structurally different inhibitors of ATM (KU-55933 and AZD0156) and ATR (VE-821 and AZD0156) is shown in FIGS. 4I-K for percentage of cells in S phase and percentage of cells in premature mitosis, respectively, to show a direct comparison between the various inhibitor compounds.
  • AZD1390 is an ATMi that shows less inhibition of other phosphatidylinositol-3-kinase-related kinases, such as, but not limited to, ATR, allowing a more specific inhibition of ATM (Durant, et al, Sci. Adv., 4(6) 2018; DOI: 10.1126/sciadv.aatl719).
  • FIG. 4K shows the ATMi AZD1390 reduced BKPyV viral titers to the same extent as the ATMi KU-55933.
  • FIG. 4L shows the ATMi AZD1390 increased mitosis in BKPyV infected cells to the same extent as the ATMi KU-55933.
  • FIGS 4K-L show that the decrease in viral titers correlates with an increase in mitosis.
  • BKPyV viral titers are decreased when ATM is inhibited as the host replication machinery, which is required for BKPyV replication, is destroyed during the mitotic process. When ATM is inhibited the cells complete S phase and then enter mitosis.
  • FIGS 4A-L show the effect of inhibiting the DDR is not specific to a particular ATRi or ATMi.
  • Example 5- Increased cell populations in BKPyV infected cells is due to increased mitosis
  • This example shows constitutive ATM and ATR activation is required to prolong BKPyV -induced S phase.
  • the ATRi was VE-821 (5mM)
  • the ATMi was KU-55933 (IOmM)
  • the vehicle control was DMSO.
  • An elevated G1 population may reflect cells that failed to enter S phase or cells that failed to proceed through mitosis due to loss of BKPyV -mediated cell cycle arrest. Since the DDR inhibition treatments were applied after the virus reached the nucleus and TAg was expressed driving the cells into S phase, it is more likely that this cell cycle distribution was due to increased mitosis rather than failure to enter S phase. However, similar results were obtained when the DDR was inhibited at 48 hpi (hours post infection), well after S phase induction (FIGS. 5A-G). These results suggested that DDR is required late in infection to block cell cycle progression.
  • Examples 3-5 show that ATR and ATM prevent cell cycle progression from S phase into mitosis during a BKPyV infection (see model shown in FIG. 3J).
  • Example 6- ATM enhanced S phase entry while ATR induced cell cycle arrest during BKPyV infection
  • S phase is a tightly regulated process during which the 2N DNA content of the cell is replicated only once to 4N before entering G2 phase.
  • TAg expression drives the host cell into S phase and when coupled to cell cycle arrest leads to host polyploidization (>4N DNA content) (7).
  • FOG. 6A Early during infection (30 hpi) most cells in S phase had ⁇ 4N DNA content suggesting that host DNA re-replication had not begun for most of the cells (FIG. 6B).
  • By 48 hpi there was a prominent EdU + accumulation of cells at 4N and some cells had >4N DNA content.
  • ATR inhibition did not affect S-phase levels at the early time point (30 hpi) (FIGS. 6C-D), but failed to keep pace with the percent of S phase cells at later time points. This suggests that ATR may be required later during infection to prevent mitotic entry but is not required for S-phase entry early during infection. In fact, mitosis levels at 30 hpi were not affected, but by 48 hpi both ATRi and ATMi increased overall mitotic entry (FIG. 6E). Interestingly, at 48 hpi cells that were undergoing premature mitosis had >4N DNA content when ATR was inhibited, but ATMi did not induce premature mitosis over the control at any time point (FIGS. 5G and 6F-G).
  • the mitosis and S phase analysis suggested that ATM and ATR were required synergistically but at different time points to maintain S-phase levels during infection (FIGS. 6C-E).
  • a ternary analysis was employed to show significant shifts in the populations (FIG. 6H) (28).
  • Ternary analysis characterizes each cell cycle replicate as a single point with three coordinates (% G1 phase, % S phase and % G2+M phase). From this data a transformation confidence interval can be calculated to determine if populations of points are statistically dissimilar to each other.
  • the ternary analysis demonstrates that inhibiting ATM early in infection significantly reduced the population of cells in S-phase compared to that in untreated cells (FIG. 6H, left), whereas early ATRi and vehicle control levels were completely overlapping with no statistical difference (FIG. 6H right).
  • This data show that ATM, but not ATR, is required early during infection.
  • Late during BKPyV infection both ATM and ATR inhibition significantly reduced S-phase and increased mitosis compared to the vehicle control.
  • ATR and ATM are required synergistically, but for different roles in reprogramming the cell cycle during BKPyV infection. Specifically, ATM is required for efficient S phase entry and blocking mitosis after DNA replication was completed, while ATR is required later in infection for cell cycle arrest to prevent entry into mitosis in actively replicating cells (FIG. 61).
  • Example 7- Premature mitosis was the source of DNA damage due to ATR inhibition during BKPvV infection
  • This example shows premature mitosis was the source of DNA damage due to ATR inhibition during BKPyV infection.
  • Weel and/or Cdkl were silenced with siRNAs to induce or block premature mitosis, respectively.
  • At 72 hpi cells were harvested to assess the amount of premature mitosis and DNA damage during BKPyV infection.
  • the DDR activates the G2/M checkpoint and temporarily blocks cell cycle progression, which allows DNA repair to occur in G2 phase (FIG. 7A) (29).
  • ATM and ATR activities were both required to block overall mitotic entry, but only ATR inhibition significantly increased premature mitosis (FIGS. 1H-I).
  • ATR inhibition reduced Chkl (pChkl) and Weel kinase (pWeel) activation, but ATM inhibition did not (FIG. 7B) (30, 31).
  • BKPyV replication activates the DDR (14) and protects the host from DNA damage.
  • the present disclosure shows that the DNA damage resulted not from a lack of DNA repair, but rather from a failure to arrest the cell cycle and allowing cells in S phase to enter mitosis prematurely.
  • the present disclosure shows separate and synergistic roles for ATR and ATM during a BKPyV infection. Inhibiting ATR resulted in cells entering mitosis when S phase DNA synthesis was ongoing. This premature mitosis correlated with severe host DNA damage. In addition, if the block to mitosis was removed in ATR inhibited BKPyV infected cells there was a dramatic and immediate induction of premature mitosis.
  • the present disclosure shows that activation of both ATR and ATM was required non- redundantly to prevent BKPyV infected cells from entering mitosis. This suggests at least two distinct mechanisms by which the DDR promotes cell cycle arrest during infection. Inhibiting the function of ATR and ATM during defined windows of the BKPyV replication cycle revealed Weel was required to arrest the cell cycle and prolong S phase. Recently, ATR has been found to mediate the S/G2 phase transition by monitoring DNA replication in an unperturbed cell cycle and blocking Cdkl activation (34). Thus, DNA replication itself is an essential signal to restrict premature Cdkl activation (35).
  • ATR inhibition resulted in premature mitosis of S phase cells with a >4N DNA content.
  • the present disclosure revealed that for cells with ⁇ 4N DNA content during infection, ATR was not necessary to block premature mitosis through Cdkl inactivation. This may suggest that in certain primary cells there are additional layers of regulation in S phase that prevent progression into G2 phase in the absence of ATR.
  • ATR mutations are lethal in utero and the Chkl response is required to prevent mitotic entry in trophoblast giant cells, therefore it is likely that the ATR pathway regulates genomic reduplication events such as those involved in embryogenesis, cellular differentiation, and tissue repair (36-38).
  • ATM activation was required to block mitotic entry, but premature mitosis was not linked to ATM inhibition during BKPyV infection, which is likely due to intact ATR activity in the ATMi cells.
  • ATM and ATR contribute to PyV replication are directly required to replicate the circular PyV genome and prevent viral DNA damage.
  • ATM has been suggested to be required to prevent rolling circle replication of the viral DNA, which must undergo theta replication to produce genomes that are packaged (9, 40).
  • ATR has been suggested to prevent replication fork collision of the two forks that fire from the Py V origin of replication on the circular genome (9, 40).
  • ATM or ATR inhibition sharply decreased overall viral DNA replication 60% to 80%, yet only 10-20% of the viral genomes had replication defects (9, 40). The relatively small fraction of viral genomes damaged does not account for the substantial decrease in viral DNA levels.
  • PyV-associated cancers are not concomitant with a productive infection, but are driven by clonal integration of TAg with mutations in the helicase domain of TAg or in the viral origin of replication (2, 44). In these tumors, episomal viral DNA is depleted and viral replication is limited suggesting that oncogenic transformation does not occur during a normal productive PyV infection (45). In fact, both SV40 and BKPyV cause cancer in a non- permissive host when viral replication is attenuated. (46, 47). Since viral DNA amplification is the trigger for PyV-mediated DDR activation and non-permissive tissue culture models similarly limit DDR activation (14, 48).
  • results of the present disclosure in combination with those in the field, provide a framework to understand the cause of oncogenic transformation by PyVs and how DDR activation may be a countermeasure to oncogenesis.
  • the results of the present disclosure support a model by which viral replication activates the DDR to inhibit cellular proliferation and prevent DNA damage by blocking entry into mitosis. This model underscores the role of the DDR during PyV infection not only to increase viral production, but also as a mechanism to counteract the oncogenic potential of TAg.
  • the present disclosure therefore provides mechanisms to inhibit PyV replication in a human subject, including inhibition of ATM using an ATMi, inhibition of ATR using an ATRi, and a combination of the foregoing.
  • RPTE cells were purchased from LONZA (Basel, Switzerland) and maintained according to the supplier’s recommendation in renal epithelial growth medium (REGM) (CC- 3190) Cells were expanded in T75 flasks twice from the original vial of cells and approximately 2.25 x 10 6 cells were frozen REGM + 10% DMSO and stored in liquid nitrogen (49). All studies utilize these expanded RTPE cell stocks, which can be passaged one additional time.
  • REGM renal epithelial growth medium
  • BKPyV (Dunlop strain) is maintained on the pBR322 vector (Addgene #25466, a gifr from Peter Howley) ligated into the BamHI site (50).
  • Original stocks of infectious BKPyV were prepared from the pBR322:Dunlop genomic clone which was digested and purified prior to transfection into a T-75 flask of RPTE cells at 60% confluency with Lipofectamine 2000 (Thermo) (7). The cells were scrapped in media after 21 days and subjected to three freeze- thaw cycles and the viral titer was determined by focus forming assay.
  • RPTE cells were chilled at 4°C for 15 min, and then incubated with BKPyV in REGM at 1/4* of the volume of the dish at 4°C for lh with intermittent shaking before replacing the inoculum with REGM at 37°C (7).
  • Viral infections were at 0.5 FFU (focus forming units )/cell unless otherwise specified. For example, such as when performing comet assay where it is impossible to distinguish an infected cell from an uninfected cell.
  • Silencer select siRNA for Weel (s21), Cdkl (s464), and non-targeting siRNA were purchased from Ambion (Thermo). Reverse transfected into RPTE cells was performed using 10.8 m ⁇ /ml Lipofectamine RNAiMax (Thermo) and lOnM siRNA using a protocol adapted from (7). Briefly, siRNA and lipofectamine were pre-incubated for 20 min in 1/2 of the final volume of the tissue culture plate with REGM in the dish prior to the addition of cells. Subsequently, RPTE cells were suspended at 1.5 x 10 5 cells/mL and added to the lipid:siRNA mixture in 1/2 of the final volume of the well. In the case of double knockdowns, single knockdown and controls conditions were supplemental with additional non-targeting control siRNA such that all conditions were transfected with equal concentrations of siRNA. Inhibitor Treatment
  • ATRi and ATMi were by VE-821 (5 mM in DMSO) and KU-55933 (10 mM in DMSO) except for in FIG. 7 in which case AZD6738 (ATRi, 5 mM in DMSO) and AZD0156 (ATMi, 5 mM in DMSO) were utilized.
  • Cdkl was inhibited by the potent and selective inhibitor RO-3306 (Cdkli, 10 mM in DMSO) (51).
  • Chkli and Weeli were MK8766 (2 mM in DMSO) and MK1775 (0.3 mM in DMSO), respectively. All inhibitors were purchased from Selleck Chemicals (Houston, Tx) and final concentrations of the vehicle (DMSO) were kept constant between conditions.
  • Coverslips were mounted onto glass microscopy slides with ProLong Gold anti-fade reagent (Thermo) with 4', 6- diamidino-2-phenylindole (DAPI) and stored overnight prior to quantification. Analysis was performed on a Nikon Eclipse Ti-S inverted microscope using the Nikon 40X/0.60 S Plan Fluor ELWD AMD objective.
  • Viral titers were determined using a focus forming unit assay (50). Briefly, RPTE cells were infected as above in duplicate with undiluted, 10 _1 , and 10 2 dilutions of viral stocks in REGM. Cells were fixed at 48hpi and stained for TAg as above. The average number of TAg positive nuclei in 7 fields of view (FOV) at 10X magnification per technical replicate were counted for dilutions that had TAg stained between 20 and 200 cells per field of view (FOV).
  • FOV focus forming unit assay
  • This number was multiplied by the dilution factor and number of FOV per surface area of a 12 well plate (determined by dividing the area of the well by the area of one FOV) then technical duplicates were averaged to determine viral titer. All titers were performed in biological triplicate of technical duplicates.
  • DNA damage was visualized by IFA for the fragmented nuclei assay to assess nuclear fragmentation of BKPyV infected RPTE cells at 72 hpi (7). At least 100 TAg + nuclei per biological replicate were scored on the basis of nuclear morphology. Nuclear classifications were either: normal (rounded), fragmented (multiple smaller nuclei in which DAPI and TAg staining overlap), or diffuse (TAg staining is dispersed throughout the cytoplasm).
  • the diffuse TAg staining pattern was then subdivided into two categories, normal mitosis and abnormal mitosis, where abnormal mitosis was marked by aberrant DAPI staining such as: misaligned chromatin in reference to the metaphase plate, lagging chromatin in anaphase, and anaphase bridges connecting separating chromatids.
  • Protein lysates were collected in E1A lysis buffer (HEPES pH 7, 50mM; NaCl 250mM; NP40 0.1%) with protease inhibitors (50). An equal amount of protein lysates (minimum 20pg) were loaded on either an 8% or 10% polyacrylamide gel for SDS-PAGE. Gel electrophoresis was performed at either 45V overnight or 120V for 6 h then transferred using a wet transfer method at 60V overnight to a PVDF membrane. Membranes were cut on the basis of molecular weight based on the protein marker. Membranes were blocked in either 2% fat free dry milk or 2% FBS in IX PBS with 0.1% Tiroton X-100 and probed with primary antibodies in blocking buffer (see table SI).
  • FACS Fluorescence Activated Cell Sorting
  • a 10cm 2 dish of RPTE cells was labeled by adding a thymidine analogue, 10 mM 5- ethynyl-2’-deoxyuridine (EdU) (Click Chemistry Tools; Scottdale, AZ) to the media for 3 h to detect newly synthesized DNA prior to fixation in 4% PFA for 20 min at room temperature (22°C) (52).
  • Samples were washed in wash buffer (IX PBS with 2% FBS) 2x from with centrifugation at 1200 x g for 8 min then permeabilized in 0.3% Triton X-100 in wash buffer for 15 min on ice.
  • samples that were prepared for flow cytometry as above were fixed onto microscopy slides by addition of Prolong Gold Anti-Fade reagent with DAPI 1: 1 (v/v) with the sample and covered with a glass coverslip. After an overnight incubation samples were analyzed using a Nikon Eclipse Ti-S inverted microscope using the Nikon 100X/1.45 oil S Plan APO l objective. At least 10 images of cells in each of G1/G2 phase, S phase, and premature mitosis were collected per biological replicate. Images were analyzed on the basis of nuclear morphology.
  • the comet assay detects DNA damage with single cell resolution by detecting the comet-like appearance of broken DNA migrating away from the unbroken genomic pellet of cells embedded in agarose and pulsed in an electric field.
  • test of significance herein are One-way ANOVA with Dunnet’s post-test for multiple comparisons. The p-values are always: *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001. In the event of a significant Bartlett’s test a Kruskal-Wallace was utilized instead. Significant difference was determined against the BKPyV infected DMSO control unless otherwise noted by brackets on the graph. The GraphPad PRISM analysis suite was utilized for all statistical analysis.
  • Ternary plots allow visualization of proportional data from three conditions or states allowing to visually and mathematically distinguish distinct populations from the data (28).
  • Ternary analyses were performed using the ggtem package within the R software environment version 3.4.3 using Rstudio version 1.1.447. Briefly, the percent of cells in Gl, S, and G2 (or M) phase under various conditions were determined by flow cytometry and plotted. The 95% confidence interval was determined by the Mahalnobis Distance and log-ratio transformation. Ternary plot visualization was performed using the ggplot2 package version 3.1.0.
  • PP2A targeting by viral proteins a widespread biological strategy from DNA/RNA tumor viruses to HIV-1. Biochim Biophys Acta 1812: 1498-1507.
  • Vendetti FP Lau A, Schamus S, Conrads TP, O'Connor MJ, Bakkenist CJ. 2015.
  • the orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti tumor effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo. Oncotarget 6:44289-44305.
  • ATM protein physically and functionally interacts with proliferating cell nuclear antigen to regulate DNA synthesis. J Biol Chem 287: 12445-12454.
  • SV40 utilizes ATM kinase activity to prevent non-homologous end joining of broken viral DNA replication products.
  • Cidofovir inhibits polyomavirus BK replication in human renal tubular cells downstream of viral early gene expression. Am J Transplant 8: 1413-1422.
  • Gyori BM Venkatachalam G, Thiagarajan PS, Hsu D, Clement MV. 2014. OpenComet: an automated tool for comet assay image analysis. Redox Biol 2:457- 465.

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Abstract

L'invention concerne des procédés pour le traitement d'une infection par le polyomavirus, destinés à diminuer le titre viral d'un PyV chez un sujet atteint d'une infection par le PyV, à traiter un effet indésirable d'un traitement qui supprime le système immunitaire, à inhiber la voie de réponse à l'endommagement de l'ADN, et des procédés associés. L'invention concerne également des composés, des kits et des compositions utiles dans les procédés décrits. Les composés appropriés utiles dans les procédés décrits comprennent des inhibiteurs de protéines mutées de l'ataxie télangiectasie et de protéines associées à l'ATM-Rad 3.
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CN115531382A (zh) * 2021-06-29 2022-12-30 中国科学院生物物理研究所 喹啉噻唑啉酮衍生物Ro-3306在治疗β-冠状病毒感染中的应用
CN115531382B (zh) * 2021-06-29 2024-01-30 中国科学院生物物理研究所 喹啉噻唑啉酮衍生物Ro-3306在治疗β-冠状病毒感染中的应用

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