First-in-class phospho-BRD4 inhibitors

Abstract

The present invention relates to novel small molecule of Formula I, and the preparation and use thereof: Formula (I).

Description

FIRST-IN-CLASS PH0SPH0-BRD4 INHIBITORSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 584,118, filed on September 20, 2023, the contents of which are hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0001] The field of the invention relates generally to target- selective inhibitors for the treatment of human diseases, and the method of preparation and use thereof. More specifically, the invention relates to molecules targeting an intrinsically disordered region (IDR) of human bromodomain-containing protein 4 (BRD4).BACKGROUND

[0002] Small molecule inhibitors targeting epigenetic regulators, particularly histone deacetylases, protein methyltransferases, and bromodomain and extra-terminal (BET) family members, have emerged as promising anti-inflammatory, anticancer and antiviral agents. Nevertheless, side effects and drug resistance following prolonged treatment remain concerning with these epigenetic compounds. More potent target-selective inhibitors that do not impair normal cellular function and organismal health are urgently needed for therapeutic development.

[0003] BRD4 is a ubiquitously expressed epigenetic ‘reader’ protein that binds preferentially to acetyl-lysine (Kac) residues in histone and nonhistone proteins via its N-terminal tandem bromodomains (BD1 and BD2), which are found in all BET family proteins that also include BRD2, BRD3, and testis-specific BRDT. Over 30 BET bromodomain inhibitors (BBDz) are currently under clinical evaluation for inflammatory, cardiovascular and cancer treatments, although the outcomes are generally limited due to the broad suppression of BET chromatin binding and targeted therapy-associated drug resistance. While shared functions among BET proteins in disease progression including diabetes and neurodegeneration have been reported, the existence of multiple protein isoforms for BRD3 and BRD4 with distinct and even opposingfunctions, as observed during breast cancer initiation and progression, demands more careful target selection in clinical treatment.

[0004] Particularly, among breast cancer (BC) cases, triple-negative breast cancer (TNBC) is one of the most aggressive BC subtypes and does not respond to hormonal or receptor-targeted therapies due to lack of ER / PR expression and HER2 amplification. Although progress has recently been made in treating TNBC, patients frequently develop loco-regional and systemic metastases with recurrence typically occurring < 3 years following diagnosis, thus resulting in much poorer overall survival (OS) compared to other BC subtypes. TNBC is also prevalent in young women of underrepresented populations, often initially presenting with rapidly growing, larger metastatic tumors. African- American women have a 2.21 -fold higher risk of developing TNBC compared to others. This disparity is further compounded by a higher prevalence in underserved and lower socioeconomic groups, rendering TNBC a global health / social / economic issue.

[0005] Besides the cancer link, BRD4 also plays an important role in viral latency and reactivation in which the long isoform (BRD4-L) has been shown to regulate Tat-independent human immunodeficiency virus 1 (HIV-1) gene activation whereas the short isoform (BRD4-S) is implicated in provirus inhibition during HIV-1 latency. The involvement of BET proteins has likewise been found in other RNA / DNA viruses, including SARS-CoV-2 and human papillomavirus (HPV). In a study of HPV gene regulation, BRD4 was identified as the long- sought cellular factor needed for HPV-encoded early protein 2 (E2)-mediated inhibition of the HPV early promoter that drives E6 and E7 oncogene expression, which was subsequently confirmed by genome-wide knockdown library screening. BRD4 also modulates HPV replication via enhancing E2 binding to its cognate binding sequences (E2BS) flanking the HPV origin of replication (orz), with E2 binding in turn facilitating loading of viral El helicase essential for ori- dependent HPV DNA replication. All other replication enzymes, proteins, and substrates are furnished by the host cells. Whether BRD4-L and BRD4-S are both involved in HPV transcription and replication or only unique isoforms are implicated in specific spatiotemporal regulation of HPV gene expression and genome replication, as found in BRD4-L-mediated non-homologous end joining (NHEJ) during immunoglobulin class switch recombination and oncogenic genomic rearrangement and BRD4-S-promoted breast cancer progression, remains to be elucidated.

[0006] The diverse functions of BRD4 are in part regulated by post-translational modification including phosphorylation and protein isoform switching. A phosphorylation cluster at the N- terminal phosphorylation sites (NPS, aa 484-503) of BRD4, situated in an intrinsically disorderedregion (IDR) and counter-regulated by CK2 and protein phosphatase 2A (PP2A), plays a key role in conferring phospho-BRD4 (pBRD4) binding to acetylated chromatin and recruitment of sequence-specific transcription factors, such as p53, AP-1, NF-KB and cancer-associated high- risk (HR) HPV E2. The extent of NPS phosphorylation correlates with triple-negative breast cancer progression and drug resistance, cerebellar neurogenesis, mouse learning / memory, cocaine addiction, and HPV El / E2-dependent ori DNA replication. Clearly, phosphorylation at distinct serine / threonine residues within the eight CK2 phosphorylation sites in the NPS creates a combinatorial phosphorylation code, analogous to RNA polymerase II (Pol II) CTD phosphorylation, for recruitment of different phospho-NPS (pNPS) interactors to regulate diverse functions of BRD4.

[0007] Given the importance of NPS phosphorylation, two pNPS-binding peptoids (DC-1 : Nlys- Nlys-Nmea-Nlys-Ntyr-Nleu; and DC-2: Nlys-Nlys-Nffa-Nlys-Nleu-Napp) were previously identified by screening a combinatorial library of -38,500 peptoids. These amino acid-like peptoids exhibited impressive target selectivity specifically blocking phosphorylation-dependent BRD4 interaction with NF-KB and with HR HPV E2 without perturbing pBRD4 association with other partner proteins, such as AP-1 and noncancer-associated low-risk (LR) HPV E2 or bovine papillomavirus type 1 (BPV1) E2, and with minimal disruption of BRD4 binding to its target chromatin. With the successful discovery of peptoids binding to a unique phospho-IDR, a chemical library of -200,000 compounds was further screened to identify pNPS-binding small molecule inhibitors with more diverse scaffolds amenable to subsequent structural design and optimization. Two pNPS-binding compounds thus identified (14 and 90) showed strong inhibition of HPV genome replication by disrupting pBRD4 interaction with HR HPV E2 and DNA damage response (DDR) factors 53BP1 and BARD1, highlighting the development of specific BRD4 IDR-targeting compounds as the first-in-class PPI inhibitors (PPIz) for HR HPV genome replication. Further, these pBRD4 inhibitors, 14 and 90, effectively suppress TNBC PDX tumor growth and brain metastasis in TNBC mouse models significantly better than pan-BBDz (+)-JQl and with no overt toxicity.

[0008] This background information is provided for the purpose of making information believed by the applicants to be of possible relevance to the present invention. No admission is necessarily intended, nor should it be construed, that any of the information disclosed herein constitutes prior art against the present invention.DESCRIPTION

[0009] Recent discovery of the phospho switch mechanism underlying BRD4 recruitment of cellular factors provides an innovative strategy for targeted therapy focusing on disrupting unique PPI contact surfaces between pBRD4 and transcription / replication / DDR factors using non-peptoid small molecule inhibitors disclosed herein. Exemplary embodiments of the compounds disclosed herein demonstrates selectivity and low cytotoxicity. Of note, compounds disclosed herein (PPIz) was shown to selectively disrupt upstream protein-protein interactions (PPI) without disruption of BET protein binding to global chromatin as seen with BET bromodomain inhibitors (BBDz). This selectivity makes these PPIz compounds useful across a broad range of diseases and conditions including various cancers (such as breast cancer, e.g., TNBC, prostate cancer, cervical cancer, lung cancer, GI cancers, neuroblastoma, acute myeloid leukemia, mantle cell lymphoma, etc.), inflammation (such as Crohn’s disease, ulcerative colitis, airway inflammation and fibrosis, etc.), viral infection (such as HPV, HIV, SARS-CoV-2 infection, etc.), and neurodegeneration (such as Alzheimer's disease, Parkinson’s disease, Huntington’s disease, etc.).

[0010] One aspect of the invention pertains to a compound of Formula (I) or a pharmaceutically acceptable salt, tautomer, or isotopologue thereof:Formula (I)

[0011] Another aspect of the invention pertains to a compound of claim 1, wherein said compound of Formula I is chosen from:Formula laFormula IcFormula Ic’Formular Id

[0012] In some embodiments, the invention encompasses a compound chosen from:

[0013] A compound or a pharmaceutically acceptable salt, tautomer, or isotopologue thereof:22.

[0014] In some aspects, the invention pertains to a method for inhibiting phospho-BRD4(pBRD4)-dependent human papillomavirus (HPV) genome replication, said method comprising contacting one or more cells with a compound disclosed herein.

[0015] In some aspects, the invention pertains to a method for inhibiting phospho-BRD4 (pBRD4)-dependent cancer (such as triple negative breast cancer (TNBC)), said method comprising contacting one or more cells with a compound disclosed herein.

[0016] In some aspects, the invention pertains to a method for treating a disease or condition, said method comprising administering an effective amount of a compound disclosed herein to a subject.

[0017] A further aspect of the invention pertains to a method for treating a disease or condition, said method comprising administering an effective amount of a compound disclosed herein (e.g. a compound of Formulas la, la’, lb. Ic, Ic’ and / or Id) to a subject. In some embodiments, said method involves targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that effectively inhibits phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication, and / or disturbing the interaction between phospho-BRD4 and its partners including HPV E2, p53, and DNA damage response factors, such as 53BP1 and BARD1.

[0018] All publications mentioned herein are incorporated by reference to the extent they support the present invention.1.0 Definitions

[0019] For the purposes of promoting an understanding of the principles of the invention, references will now be made to certain embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, and alterations and modifications in the illustrated invention, and further applications of the principles of the invention as illustrated therein are herein contemplated as would normally occur to one skilled in the art to which the invention relates.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used).

[0022] The use of “or” means “and / or” unless stated otherwise.

[0023] The use of “a” herein means “one or more” unless stated otherwise or where the use of “one or more” is clearly inappropriate.

[0024] The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of’ and / or “consisting of.”

[0025] As used herein, the term “about” refers to a ±10% variation from the nominal value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0026] Any ranges given either in absolute terms or in approximate terms are intended to encompass both, and any definitions used herein are intended to be clarifying and not limiting. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges (including all fractional and whole values) subsumed therein.

[0027] The term "pharmaceutically acceptable salt" refers to those salts of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, and the like. As used herein, the term "pharmaceutically acceptable salt" may include acetate, hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate and laurylsulphonate salts, and the like. (See S. M. Barge et al., "Pharmaceutical Salts," J. Pharm. Sci., 66: 1-19 (1977), which is incorporated herein by reference in its entirety, for further examples of pharmaceutically acceptable salt).

[0028] The term “HATU” refers to l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate.

[0029] The term “HBTU” refers to 3-[Bis(dimethylamino)methyliumyl]-3H-benzotriazol-l- oxide hexafluorophosphate (also known as 2-(lH-benzotriazol-l-yl)-l, 1,3,3- tetramethyluronium hexafluorophosphate).

[0030] The term “HOBt” refers the following structure, known as 1 -hydroxybenzotriazole, (including hydrates and polymorphs, thereof):

[0031] The term “DIEA” refers to N,N-Diisopropylethylamine (also known as Hiinig’s base, DIPEA, and ethyldiisopropylamine).

[0032] The term “DCM” refers to dichloromethane (also known as methylene chloride).

[0033] The term “TFA” refers to trifluoroacetic acid.

[0034] The term “rt” refers to room temperature.

[0035] The term “alkyl” as used herein by itself or as part of another group refers to both straight and branched chain radicals, and cyclic alkyl groups. In one embodiment, the alkyl group has 1-12 carbons. In another embodiment, the alkyl group has 1-7 carbons. In another embodiment, the alkyl group has 1-6 carbons. In another embodiment, the alkyl group has 1-4 carbons. The term “alkyl” may include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, and dodecyl.

[0036] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a linear or branched chain having at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, S, P, and Si. In certain embodiments, the heteroatoms are selected from the group consisting of O, and N. The heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Up to two heteroatoms may be consecutive.

[0037] The term “alkylene” as used herein refers to straight and branched chain alkyl linking groups, i.e., an alkyl group that links one group to another group in a molecule. In some embodiments, the term “alkylene” may include -(CH2)n— where n is 2-8.

[0038] The term “aryl” means a polyunsaturated hydrocarbon substituent. Aryl groups can be monocyclic or polycyclic (e.g., 2 to 3 rings that are fused together or linked covalently). Nonlimiting examples of aryl and heteroaryl rings are phenyl, naphthyl, pyranyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyrazolyl, pyridinyl, furanyl, thiophenyl, thiazolyl, imidazolyl, isoxazolyl, and the like.

[0039] The term “heteroaryl” as used herein refers to groups having 5 to 14 ring atoms; 6, 10 or 14 77t-electrons shared in a cyclic array; and containing carbon atoms and 1, 2 or 3 oxygen, nitrogen or sulfur heteroatoms. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Especially preferred heteroaryl groups include 1,2,3-triazole, 1,2,4-triazole, 5-amino 1,2,4-triazole, imidazole, oxazole, isoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 3-amino-l,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, 2- aminopyridine, 4-aminopyridine, 2-aminoimidazoline, and 4-aminoimidazoline.

[0040] An “amino” group refers to an -NH2 group.

[0041] An “amido” group refers to an -CONH2 group. An alkylamido group refers to an - CONHR group wherein R is as defined above. A dialkylamido group refers to an -CONRR' group wherein R and R' are as defined above.

[0042] The term “halogen” or “halo” as used herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine.

[0043] The term “hydroxy” or “hydroxyl” as used herein by itself or as part of another group refers to an — OH group.

[0044] An “alkoxy” group refers to an -O-alkyl group wherein “alkyl” is as defined above. In one embodiment, the alkyl group has 1-12 carbons. In another embodiment, the alkyl group has 1-7 carbons. In a further embodiment, the alkyl group has 1-6 carbons. In another embodiment, the alkyl group has 1-4 carbons.

[0045] A “thio” group refers to an -SH group.

[0046] An “alkylthio” group refers to an -SR group wherein R is alkyl as defined above.

[0047] The term “heterocycle” or “heterocyclic ring”, as used herein except where noted, represents a stable 5- to 7-membered monocyclic-, or stable 7- to 11-membered bicyclic heterocyclic ring system, any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. Rings may contain one oxygen or sulfur, one to three nitrogen atoms, or one oxygen or sulfur combined with one or two nitrogen atoms. The heterocyclic ring may be attached at any heteroatom or carbon atom that results in the creation of a stable structure.

[0048] The term “alkylamino” as used herein by itself or as part of another group refers to an amino group which is substituted with one alkyl group having from 1 to 6 carbon atoms. The term “dialkylamino” as used herein by itself or as part of another group refers to an amino group which is substituted with two alkyl groups, each having from 1 to 6 carbon atoms.

[0049] The term “arylamine” or “arylamino” as used herein by itself or as part of another group refers to an amino group which is substituted with an aryl group, as defined above.

[0050] As used herein, the term “arylalkyl” denotes an alkyl group substituted with an aryl group, for example, PI1-CH2- etc.

[0051] In certain embodiments, the term “cycloalkyl” includes bicyclic ring systems. The bicyclic ring system may be in the form of a bridged, fused, or spiro form.

[0052] As used herein, the term “(R3)m” refers to multiple substituents that are either same or different independently on the single ring or fused ring selected from R3.

[0053] Various groups are described herein as substituted or unsubstituted (i.e., optionally substituted). Optionally substituted groups may include one or more substituents independently selected from: halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, oxo, carbamoyl, alkyl, heteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In certain aspects the optional substituents may be further substituted with one or more substituents independently selected from: halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl ( — C(O)NR2), unsubstituted alkyl, unsubstituted heteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl, unsubstituted cycloalkyl, unsubstituted heterocyclyl, unsubstituted aryl, or unsubstituted heteroaryl. Exemplary optional substituents include, but are not limited to: — OH, oxo (=0), — Cl, — F, Br, Ci-4alkyl, phenyl, benzyl, — NH2, — NH(Ci-4alkyl), — N(Cl-4alkyl)2, — NO2, — S(Cn 4alkyl), — SO2(Ci-4alkyl), — CO2(Ci-4alkyl), and — O(Ci-4alkyl).

[0054] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0055] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof, such as gastric cancer, and / or other cancers and / or tumors. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition.

[0056] The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or thosein which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0057] BRIEF DESCRIPTION OF THE FIGURES

[0058] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0059] FIGS. 1A-1D. Figure 1 displays high-throughput Screening (HTS) of pBRD4-targeting compounds. Fig. 1 A displays domain features of BRD4 long (L) and short (S) isoforms as well as cancer-associated high-risk (HR) HPV16-encoded E2. Numbers correspond to amino acid residues. BD1, first bromodomain; BD2, second bromodomain; PDID, phosphorylationdependent interaction domain; NPS, N-terminal cluster of CK2 phosphorylation sites; ET, extraterminal domain; CTM, C-terminal motif; GPA, the last 3 amino acids unique to BRD4-S; AD, activation domain; Hinge, hinge domain; DBD, DNA-binding domain. FIG. IB displays rationale for compound screening and predicted outcomes, (top) Cell-based luciferase (Luc) assay measuring compound (Cpd)-derepressed HPV18 long control region (LCR, containing enhancer / promoter)-driven Luc activity suppressed by the E2-BRD4 repressor complex mainly through binding to the promoter-proximal #3 and #4 E2 -binding sites (E2BS). Preferred compounds are those able to disrupt pNPS contact with HR-16E2 DBD. This contact is HR-E2- specific and different from the BRD4 CTM-E2 AD interaction commonly found in every type of HPV E2. Numbers in green correspond to the HPV18 genomic sequence, with +1 and +1707 in black indicating the boundaries of the Luc-coding sequence, (bottom) Model for compound- derepressed Luc activity unique to HR-16E2 but not LR-BE2. FIG. 1C displays the outline of cellbased HTS compound screening. Top hit compounds (1,021) selected from the primary screen (14,218) that increased Luc activity in 16E2-expressing C-33A cells were further confirmed and counter- screened with three Luc-reporter lines expressing 16E2, BE2, or no E2. FIG. ID displays compound IC50 in AlphaScreen calculated from a 5-dose titration, each in five replicates.AlphaScreen measures binary interaction between GST-tagged 16E2 DBD and FLAG-tagged pPDID expressed in and purified from insect Sf9 cells (Sf9-pPDID). Also shown is the dose response curve of cpd 14.

[0060] FIGS. 2A-2F. Figure 2 displays 14 inhibiting HPV ori replication and genome amplification but not genome maintenance. FIG. 2A displays a model for three stages of HPV replication. Abbreviations: pBRD4, phospho-BRD4; BM, basal membrane; ECM, extracellular matrix; trx, transcription; rep, replication, ec, extrachromosomal; DDR, DNA-damage response. FIG. 2B displays 14-reduced HPV18 genome amplification in three-dimensional (3D) raft cultures. HPV18 DNA was detected by fluorescence in situ hybridization (FISH, red) in two nonadj acent tissue sections of HPV18 rafts without (DMSO, top) or with 14 treatment (bottom) from day 6 to day 14 after lifting rafts to the air-medium interface. Bromodeoxyuridine (BrdU) was added 6 hours prior to harvesting on day 14 to detect host DNA synthesis (green) by indirect immunofluorescence (IF). DAPI (blue) is for nuclear staining. Basal and differentiated epithelial layers are indicated. Images were captured with 20X objective magnification. FIG. 2C displays hematoxylin-and-eosin (H&E) staining of HPV18 raft tissue with or without 14 treatment. FIG. 2D displays ElZE2-dependent HPV16 ori replication (black bars, Luc assay) inhibited by 14 but not JQ1, with cell viability measured by CTG reagent (gray bars). FIG. 2E displays reduced El / E2-dependent HPV16 ori replication (black bars, Hirt-qPCR) in C-33A cells following knockdown of BRD4-L or BRD4-S, each by two different siRNAs, with cell viability measured by CTG reagent (gray bars). siRNA was delivered 24 hours prior to transfection by 16orz-driven Luc reporter and 16E1 / E2 expression plasmids. Fold change is the ratio of cpd treatment vs. DMSO-only (-). Knockdown levels of BRD4-L and BRD4-S proteins were confirmed by immunoblotting (IB) with isoform-specific antibodies. FIG. 2F displays 14-suppressed vs. JQ1- stimulated ecHPV16 genome amplification. Maintenance (left) and amplification (right) assays were performed respectively in proliferating and methylcellulose-induced differentiated W12E human cervical keratinocytes (HCK), with replicating DNA quantified by Hirt-qPCR (black bars) and cell viability measured in parallel by Calcein AM (grey bars).

[0061] FIGS. 3A-3J. Figure 3 displays target validation of 14 and its analog 90. FIG. 3 A displays a table summarizing ATm of 14 and JQ1 binding to wild-type (WT) FLAG (f: )-tagged pPDID purified from Sf9 insect cells or unphosphorylated WT PDID and NPS-phosphomimetic 8E purified from bacteria (Bac) measured by thermal shift assay (TSA). Tm, the temperature with 50% of protein denatured and bound by SYBRO Orange fluorescent dye; ATm, difference in Tmresulting from compound binding (vs. DMSO-only). FIG. 3B displays a TSA assay showing that 14 does not bind GST-tagged 16E2 DBD. FIG. 3C displays the kd of 14 binding to Sf9-pPDID, with or without ZPPase treatment, or Bac-PDID measured by microscale thermophoresis (MST) in > 3 independent experiments. FIG. 3D displays an immobilized peptide binding assay, which shows that 14 disrupts GST-16E2 DBD binding to diphosphorylated but not unphosphorylated or monophosphorylated NPS peptides. FIG. 3E displays the mean IC50 of 14 and 90 averaged from multiple AlphaScreen assays, with each dot representing an individual experiment measuring interaction between FLAG-tagged Sf9-pPDID and GST-tagged 16E2 DBD. The central line in the violin plot shows the median value (50%) of compound IC50, with the top and bottom quartiles also indicated. FIG. 3F displays violin plots of ATm values showing 14, 90 and JQ1 binding to untagged Sf9-pPDID (left), Bac-PDID (middle), or Bac-PDID 8E (right) measured by TSA following normalization to the lowest Tm in the DMSO-only (-) group. FIG. 3G displays biotinylated 14 and 90 immobilized on beads bind to GST-tagged wild-type (WT) but not NPS- deleted (ANPS) Sf9-pPDID and to phosphomimetic Bac-PDID 8E rather than its WT counterpart. FIG. 3H displays D\the determination of the lowest temperature (49.2 °C) capable of denaturing endogenous BRD4-L / S but not P-actin by cellular TSA (CETSA) performed in HeLa cells with a heat gradient from 40 °C to 55 °C. FIG. 31 and FIG. 3J displays CETSA analysis showing that 14, 90, and JQ1 bind endogenous BRD4-L and BRD4-S in differentiated W12E (lanes 1-8) (FIG. 31) and proliferating HeLa cells (lanes 9-16) (FIG. 3J).

[0062] FIGS. 4A-4H. Figure 4 displays pBRD4 PPIz HPV DNA replication without perturbing global cellular and viral transcriptomes. FIG. 4A displays compounds 14 and 90 suppressing HPV18 genome amplification in 3D organotypic raft cultures. FIG. 4B displays 10-fold enhanced potency than 14 in inhibiting HPV16 ori replication (top) with little effect on cell viability. FIG. 4C displays 90 showing stronger suppression of HPV genome maintenance than 14 in proliferating W12E cells (top) with increased cytotoxicity. FIG. 4D displays 90 inhibiting HPV genome amplification 7.5-fold better than 14 while JQ1 enhances HPV genome amplification in differentiated W12E cells (top) with no overt cytotoxicity. FIG. 4E displays Venn diagrams of RNA-seq DEGs (differentially expressed genes) from proliferating or differentiated W12E cells with or without 14, 90, or JQ1 treatment. DEGs were obtained by using edgeR with cutoff p < 0.05 and fold change (FC) > 1.5 or < -1.5. Differentiation upregulated (Diff-Up) or downregulated (Diff-Dn) DEGs were obtained by comparing RNA levels between differentiation and proliferation datasets and were used to overlap cpd-altered DEGs in differentiation. FIG. 4Fdisplays a heatmap of HPV16 RNA abundance reflecting the levels of each virus-encoded transcript was shown using mean FPKM (Fragments Per Kilobase of transcript per Million mapped reads) from W12E RNA-seq datasets. Sequencing reads were mapped to HP VI 6 DNA K02718.1. Compounds 14 and 90, unlike JQ1, do not alter viral transcriptomes in proliferating and differentiated W12E cells. FIG. 4G displays RNA-seq MA plots. FIG. 4H displays PRO-seq MA plots. The read counts were normalized against the library size and total spike-in reads, then used for edgeR DEG analysis.

[0063] FIGS. 5A-5F. Figure 5 displays unique BRD4 isoform-associated DDR factors identified by proteomics in differentiated vs. proliferated W12E cells. FIG. 5 A displays the outline of BRD4- L / S IP -Proteomics. W12E cells in proliferation (Prol, 0.05 mM Ca2+) or differentiation (Diff, 1.4 mM Ca2+) were treated with compound (Cpd) 14, 90, or DMSO (-) before nuclear extracts were prepared for subsequent IP with BRD4 isoform-specific antibodies or IgG, each in triplicate (x3). BRD4-L / S-interacting proteins (2,352) with peptide spectrum match (PSM) > 2 and 1% FDR confidence were evaluated by principal component analysis (PCA). Significant interactors, based on FC and p values, with nuclear localization were further analyzed by Venn diagram overlap, Volcano plot, STRING networks, Gene Ontology (GO) / pathway, and heatmap clustering. FIG. 5B displays the key biological processes identified in BRD4-L / S interactomes. Venn diagrams show overlap of BRD4-L vs. -S significant interactors (FC > 2 and p < 0.05, BRD4 vs. IgG IP) in respective Prol and Diff datasets without cpd treatment. GO analysis identifies biological processes enriched in BRD4-L / S interactomes from Venn diagrams. Number of hits vs. input is listed in parenthesis. FIG. 5C displays DDR pathways enriched in BRD4-L-Unique interactomes. Venn diagrams show overlaps of all significant BRD4-L / S interactors in Prol and Diff with numbers indicating common or unique interactors identified in each group. 52 L-Prol-Unique and 308 L-Diff-Unique interactors were further analyzed by STRING to identify proteins involved in DNA damage response (DDR) with global-genome nucleotide excision repair (GG-NER) found in both cell states and transcription-coupled nucleotide excision repair (TC-NER) and doublestrand break (DSB) repair additionally enriched in differentiated W12E cells. FIG. 5D displays volcano plot showing a comparison of all BRD4 interactors enriched in BRD4-L (L > S) or BRD4- S (L < S) interactomes in differentiated W12E cells with FC in log2 scale on the x-axis and p values in -logic scale on the y-axis. Highlighted are components in Mediator (dark blue), DSB (red), GG-NER (green), and TC-NER (light blue). 53BP1 and BARD1, among others, are identified in BRD4-L but not BRD4-S interactomes in differentiated W12E cells. FIG. 5E displays a STRING network of 41 BRD4-L-Diff-Unique DDR interactors in DSB, GG-NER, and TC-NERpathways. Thickness of solid lines indicates the degree of confidence. Dashed lines generated by K-means clustering connect proteins between the pathways. Highlighted is BRD4 interaction with 53BP1 and BARD1. FIG. 5F displays a STRING network of 14 S-Diff-Unique interactors. Highlighted are proteins involved in proteosome (purple), RNA binding (pink), or chromatin remodeling (green).

[0064] FIGS. 6A-6D. Figure 6 displays 14 / 90-disrupted BRD4 association with 53BP1 and BARD1 impairing differentiation-dependent HPV genome amplification. FIG. 6A displays a heatmap showing 14 / 90-disrupted BRD4 isoform interaction with differentiation-associated pS / T- binding reader proteins identified in IP -Proteomics. Heatmap ranking is based on fold change (FC) of BRD4-L vs. IgG IP from differentiated W12E cells treated with DMSO-only (-) (i.e., 9th column). Listed are BRD4 interactors containing specific pS / T-binding domains - BRCT, FHA, PBD, FF - or belonging to the 14-3-3 family proteins. FIG. 6B displays IP -IB validation of endogenous protein-protein interaction between BRD4-L / S and 53BP1 or BARD1 in differentiated W12E cells. IP was performed with BRD4 isoform-specific antibodies with IB detection using anti-BRD4 N, 53BP1, or BARD1 antibodies. FIG. 6C displays a heatmap showing fold change (FC) of pS / T-reader RNA levels vs. that of untreated (-) Prol (1st column) analyzed by edgeR, with FDR indicating significant change in Diff vs. Prol DMSO-only samples (columns 1 vs. 5). 14 and 90 do not perturb gene expression of BRD4-L / S-associated pS / T readers. FIG. 6D displays an analysis of the replication of ecHPV16 DNA and cell viability, respectively, by Hirt- qPCR (black bars) and Calcein-AM labeling (grey bars). The efficiency of knockdown in W12E cells by two independent siRNAs targeting 53BP1, BARD1, BRD4-L, or BRD4-S was analyzed by IB with respective anti-protein antibodies. Knockdown of 53BP1 and BARD1 significantly reduces HPV genome amplification but not genome maintenance in W12E cells. FIG. 6E displays an analysis of HPV replication and cell viability by Hirt-qPCR (black bars) and Calcein-AM labeling (gray bars), respectively. Knockdown of both (Pan siRNA) or individual isoforms (L or S siRNA) was performed in differentiated (top) and proliferating (bottom) W12E cells. BRD4-L and BRD4-S are both important for HPV genome amplification and genome maintenance.

[0065] FIGS. 7A-7D. Figure 7 displays PPIz preventing BRD4 isoform-dependent recruitment of 53BP1 and BARD1 without globally inhibiting BRD4 binding to cellular and viral chromatin. FIG. 7A displays CUT&RUN BRD4 binding analysis showing that 90 slightly reduces BRD4-L without affecting BRD4-S genome-wide binding to W12E enhancers unlike JQ1 that globally inhibits both isoform occupancy. Heatmaps show BRD4-L and BRD4-S binding signals in W12E enhancers and flanking 1 kb regions with the average signal plot shown below. FIG. 7B displaysfactor-bound DNA from CUT&RUN which was analyzed by qPCR using orz-proximal primer pair #3 that shows highest binding signals compared to other sites analyzed. 14 / 90 selectively disrupt 53BP1 and BARD1 binding to HPV ori without reducing corresponding BRD4-L / S occupancy in differentiated W12E cells. FIG. 7C displays CUT&RUN-qPCR in differentiated W12E cells following respective siRNA knockdown of BRD4-L, BRD4-S, 53BP1, and BARD1, revealing that BRD4-L-dependent recruitment of 53BP1 and BARD1 to the HPV ori with BRD4- S selectively required for BARD 1 but not 53BP1 recruitment. Also shown is a schematic of factor entry to the ori.

[0066] FIGS. 8A-8E. Figure 8 displays anti-tumor and anti-metastasis effects of pBRD4 IDR- targeting compounds 14 and 90. FIG. 8A displays graphs showing that 14 suppresses TNBC mammary tumor growth in syngeneic mouse Py8119 and xenograft human PDX BCM-3887 models with no overt toxicity. Mouse Py8119 cells (IxlO5) and a 2-mm3BCM-3887 PDX tumor fragment were implanted at the fourth mammary fat pads in female C57BL / 6J and SCID / NSG mice, respectively (n = number of mice). When tumors reached -100 mm3, 14 (50 mg / kg) or DMSO vehicle (-) was administered via daily IP injection. Tumor volume and mouse weight were monitored twice per week for 27 days for Py8119 and 42 days for BCM-3887 PDX. Compound efficacy was determined by differences in tumor weight between 14- vs. vehicle- treated mice. Body weight was plotted against the treatment time. FIG. 8B displays graphs showing that 90 significantly inhibits primary tumor growth in the JQ1 -resistant SUM159R xenograft mouse model. IxlO6of SUM159R cells were injected as in (A) with compound treatment (daily IP, 50 mg / kg) and tumor / body weight measurement similarly conducted. FIG. 8C displays graphs showing that 14 and 90 suppress breast cancer brain metastasis better than JQ1. TNBC cells harboring a luciferase reporter from MDA-MB-231-BrM (IxlO4, left), MDA- MB-468-BrM (IxlO5, middle), or mouse 4Tl-BrM (IxlO5, right) were injected via intracardiac route into ten female 6- to 8-week-old athymic or BALB / c female mice. Bioluminescence (BLI) signals from metastatic brains harvested from mice treated with 14, 90, JQ1, or vehicle control (-) as shown in the scheme were plotted with representative images of dissected mouse brains shown below. Mouse body weight at termination is shown to the right of each BLI graph. FIGs. 8D and 8E display mouse overall survival (FIG. 8D) and Brain metastasis (BrM)-free percentage (FIG. 8E) was monitored for 100 days post MDA-MB-231-BrM cell injection with compound treatment scheme as in C (left). The period of compound administration is indicated by color shading. Compound treatment was stopped when mouse death occurred in 20% of the cohort.

[0067] FIGS. 9A-9D. Figure 9 displays on-target validation of 14 / 90 in MDA-MB-468-BrM cells. FIG. 9A displays a cell viability analysis using CellTiter-Glo (CTG) reagent. Each compound (3 pM of 14, and 1 pM of 90 and JQ1) was added to the cells at 72 hours post siRNA transfection for 4 days before cell viability was analyzed. The efficiency of knockdown was analyzed by immunoblotting (IB). FIG. 9B displays an IB graph showing that 90 and JQ1 reduced c-Myc oncoprotein level following individual compound treatment (10 pM of 14, and 1 pM of 90 and JQ1) for 24 hours. Total cell lysates were used for IB with the indicated antibodies. FIG. 9C displays an IB / IP graph showing that 14 / 90 but not JQ1 disrupt BRD4 interaction with p53-R273H. Nuclear extracts from compound-treated BrM cells (107), as shown in (B), were used for immunoprecipitation (IP) using BRD4 isoform-specific antibody followed by IB with BRD4-L, -S, and p53 DO-1 antibodies, respectively. FIG. 9D displays a CETSA assay showing that 14 / 90 bind endogenous BRD4-L and BRD4-S. A pre-determined temperature 49.2 °C (top) able to denature free BRD4 with no compound binding1was used to assess protein thermostability induced by 14 / 90 association. The permissible temperature 40.0 °C (bottom) was used as control.

[0068] FIG. 10. Figure 10 displays the mode of action by phosphor-BRD4 PPI inhibitors. Phospho-BRD4 (pBRD4) binds acetylated (Ac) lysine residues in chromatin and transcription factors (TFs) via its tandem bromodomains. This chromatin-binding activity of pBRD4 is jointly controlled by its interaction with different viral and cellular proteins, such as human papillomavirus (HPV)-encoded El and E2 proteins and cellular DNA damage response (DDR) factors, replication factors, TFs, and chromatin regulators. The current BET bromodomain inhibitors (BBDz) widely used by worldwide investigators not only block intended oncogenic gene transcription (e.g., c-Myc) but also unintentionally suppress homeotic gene transcription critical for normal cellular functions, thus eliciting many unwanted side effects during clinical trials. In contrast, the newly developed protein-protein interaction inhibitors (PPIz) only block pBRD4 interaction with select viral / cellular proteins without perturbing global gene expression and normal cellular activity, highlighting development of the first-in-class pBRD4 PPIz that possess effective antiviral, anticancer, and anti-inflammatory activity for future targeted therapy.

[0069] It is to be understood that both the foregoing descriptions are exemplary, and thus do not restrict the scope of the invention.

[0070] Table 1. Chemical structures of exemplified compoundsCompound R9R7Rl°

[0071] 2.2 Synthesis of Compounds of Formula I

[0072] The description of preparation of certain compounds of the invention is meant to be exemplary of certain embodiments of the invention. The reagents and reactant used for synthetic conversions outlined herein and below is merely exemplary. The invention contemplates using the same or different reagents discussed herein to achieve preparation of the compounds of the invention.

[0073] Certain compounds of Formula I can be prepared as exemplified by the following synthetic scheme (Scheme 1):Scheme 1. Synthetic route 1Reagents and conditions: (a) Chloroacetyl chloride, 1,4-Dioxane, Pyridine, 40 °C, 5 h, 82.1%; (b) (NH4)2S, H2O, Et3N, Pyridine, 80 °C, 12 h, 80.7%; (c) EtOH, 80 °C, overnight, 75.6%.

[0074] Certain compounds of Formula I can be prepared as exemplified by the following synthetic scheme (Scheme 2):Scheme 2. Synthetic route 2Reagents and conditions: (a) BBr3, DCM, 0 °C-rt, 10 min, 88.2%; (b) 2-Chloro-l-(2-methyl-177- indol-3-yl)ethan-l-one, EtOH, 80 °C, 12 h, 73.4%; (c) Biotin, DCC, DMAP, DMF, rt, 12 h, 69.2%.Certain compounds of Formula I can be prepared as exemplified by the following synthetic scheme (Scheme 3):Scheme 3. Synthetic route 3Reagents and conditions: (a) Chloroacetyl chloride, 1,4-Dioxane, Pyridine, 40 °C, 5 h, 81.4%; (b) Chlorosulfonyl isocyanate, DMF, MeCN, 0 °C to rt, 79.4%; (c) (NH4)2S, H2O, Et3N, Pyridine, 80 °C, 12 h, 75.1 %; (d) EtOH, 80 °C, 12 h, 67.1%; (e) NaH, DMF, RX (X= Cl, Br, I), 0 °C-r.t., 1 h or NaH, DMF, RX-HX (X= Cl, Br, I), KI, r.t.-60 °C, 12 h; (f) BBr3, CH2CI2, 0 °C- r.t., 2 h, 47%.

[0075] Certain compounds of Formula I can be prepared as exemplified by the following synthetic scheme (Scheme 4): Scheme 4. Synthetic route 4Reagents and conditions: (a) Chlorosulfonyl Isocyanate, CH3CN, DMF, 0 °C, 5 h, 83.3 %; (b) BBr3, CH2Q2, 0 °C, 10 min, 79.9 %; (c) Ammonium sulfide (20% in water), Pyridine, Et3N, 80 °C, 12 h, 73.2%; (d) EtOH, 80 °C, overnight, 12 h, 63.6%; (e) Biotin, DCC, DMAP, DMF, rt, 12 h, 67.3 %.

[0076] Certain compounds of Formula I can be prepared as exemplified by the following synthetic scheme (Scheme 5):Scheme 5. Synthetic route 5Reagents and conditions: (a) TFA, DCM, r.t. 2.0 h; (b) HATU, different amines, DIEA, DMF, r.t., 12 h.

[0077] Certain compounds of Formula I can be prepared as exemplified by the following synthetic scheme (Scheme 6):Scheme 6. Synthetic route 6Reagents and conditions: (a) Arylboronic acid or alkylpinacolyl boronate ester, Pd(dppf)C12, K2CO3, Dioxane / H2O (5: 1), 80 °C, 12 h, 60%-70%; (b) i. TFA, CH2CI2, r.t., 12 h; ii. NHrMeOH, MeOH, 30 min, 50-90%.Method of Use

[0078] In some embodiments, one or more of the compounds of Formulas 1, la, la’ and lb may be used for treating a disease or condition, said method comprising administering an effective amount of a compound of claim 1 to a subject, wherein said method involves targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that effectively inhibits phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication, and / or disturbing the interaction between phospho-BRD4 and its partners including HPV E2, p53, and DNA damage response factors, such as 53BP1 and BARD1.

[0079] LIST OF EMBODIMENTS

[0080] The following is a non-limiting list of embodiments:1. A compound of Formula (I) or a pharmaceutically acceptable salt, tautomer, or isotopologue thereof:Formula (I)R1is selected from hydrogen, alkyl (e.g., methyl, ethyl, isopropyl), cycloalkyl (e.g., cyclopropyl, cyclobutyl), alkoxy (e.g., methoxy, ethoxy), -OCD3, -CD3, CD2CD3, haloalkyl (e.g., trifluoromethyl, difluoromethyl), haloalkoxy (e.g., trifluoromethoxy, difluoromethoxy), halo, cyano, nitro, amino, aryl, heteroaryl, etc.R2is selected from hydrogen, alkyl, cycloalkyl, alkoxy, -OCD3, -CD3, CD2CD3, haloalkyl, haloalkoxy, halo, cyano, nitro, amino, carboxy, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, alkoxyalkyl, aminoalkyl, aminoalkoxy, hydroxy, hydroxyalkyl, wherein alkoxycarbonyl, alkylcarbonyl, alkylaminocarbonyl are unsubstituted or substituted with halo, hydroxy, cycloalkyl, alkoxy, alkylsulfonyl; etc.Ring A is a unsubstituted or substituted aryl (e.g., phenyl, naphthyl), heteroaryl (e.g., pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, furanyl, thiophenyl, imidazolyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl), fused heteroaryl (e.g., quinolinyl, indolyl, benzofuranyl, benzothiophenyl, benzothiazolyl, benzoimidazolyl, indazolyl), heterocyclicyl (e.g., morpholinyl, piperazinyl, homopiperazinyl, piperdinyl, thiomorpholinyl, pyrrolidinyl), spirocyclicyl, bridged bicyclicyl, etc. m is 1, 2, 3 or 4;R3is independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, haloalkyl, haloalkoxy, halo, -OCD3, -CD3, CD2CD3, amide, cyano, nitro, carboxy, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, alkoxyalkyl, aminoalkyl, aminoalkoxy, hydroxy, hydroxyalkyl, wherein alkoxycarbonyl, alkylcarbonyl, alkylaminocarbonyl, amide, or alkyl is optionally substituted with halo, hydroxy, cycloalkyl, alkoxy, alkylsulfonyl, ester, alkynyl, or substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroaryl, or Boc.X1is CH, O, S or NR4, wherein R4is hydrogen, alkyl, cycloalkyl, -OCD3, -CD3, CD2CD3, alkoxyalkyl, alkylcarbonyl, alkyl sulfonyl; etc.X2is CH, O, S or NR5, wherein R5is hydrogen, alkyl, cycloalkyl, -OCD3, -CD3, CD2CD3, alkoxyalkyl, alkylcarbonyl, alkyl sulfonyl; etc.X3is CH, O, S or NR6, wherein R6is hydrogen, alkyl, cycloalkyl, -OCD3, -CD3, CD2CD3, alkoxyalkyl, alkylcarbonyl, alkyl sulfonyl; etc. Yis O, S or NR7, wherein R7is hydrogen, alkyl, cycloalkyl, -OCD3, -CD3, CD2CD3, alkylcarbonyl, alkylsulfonyl, aminoalkyl, etc., wherein, alkyl, cycloalkyl, alkylcarbonyl, alkylsulfonyl, aminoalkyl are unsubstituted or substituted with halo, hydroxy, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, alkyloxy, carboxy, aminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, cycloaminecarbonyl, etc. 2. The compound of embodiment 1, wherein said compound of Formula I is chosen from:Formular IdFormula Ic’wherein R8, R9, and R9are independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, haloalkyl, haloalkoxy, halo, -OCD3, -CD3, CD2CD3, amide, cyano, nitro, carboxy, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, alkoxyalkyl, aminoalkyl, aminoalkoxy, hydroxy, hydroxyalkyl, wherein alkoxycarbonyl, alkylcarbonyl, alkylaminocarbonyl, amide, or alkyl is optionally substituted with halo, hydroxy, cycloalkyl, alkoxy, alkylsulfonyl, ester, alkynyl, or substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroaryl, or Boc.3. The compound of embodiment 1, wherein said compound is any one of:4. The compound of embodiment 1, wherein said compound is any one of the following:5. The compound of any of the preceding embodiments, wherein said compound is a phospho-5 BRD4 inhibitor.6. The compound of any of the preceding embodiments, wherein said compound is chosen from:7. A compound of Formula (II) or a pharmaceutically acceptable salt, tautomer, or isotopologue thereof:Formula (II) wherein R11is amino or alkylamino; and wherein R12is halo.8. The compound of embodiment 7, wherein R11is -NH2.9. The compound of embodiment 7, wherein R12is bromo.10. The compound of embodiment 7, wherein said compound of Formula (II) is:11. A method for inhibiting phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication, said method comprising contacting one or more cells with of a subject with a compound of any of the preceding claims.12. A method for treating a disease or condition, said method comprising administering an effective amount of a compound of any of the preceding claims to a subject.13. The method of embodiment 12, wherein said method involves targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that effectively inhibits phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication, and / or disturbing the interaction between phospho-BRD4 and its partners including HPV E2, p53, and DNA damage response factors, such as 53BP1 and BARD1.14. The method of embodiment 12, wherein said disease or condition is chosen from various cancers (such as breast cancer, e.g., TNBC, prostate cancer, cervical cancer, lung cancer, GI cancer, neuroblastoma, acute myeloid leukemia, mantle cell lymphoma, etc.), inflammation (such as Crohn’s disease, ulcerative colitis, airway inflammation and fibrosis, etc.), viral infection (such as HPV, HIV, SARS-CoV-2 infection, etc.), and neurodegeneration (such as Alzheimer's disease, Parkinson’s disease, Huntington’s disease, etc.)15. The method of embodiment 13, wherein said inflammation involves repressing LPS-induced proinflammatory cytokine / chemokine production.16. The method of embodiment 12, involves inhibition of bromodomain-containing protein 4 (BRD4) via upstream inhibition of protein-protein interaction inhibitor (PPIi).17. The method of embodiment 12, involves selectively inhibiting bromodomain-containing protein 4 (BRD4) via upstream inhibition of protein-protein interaction inhibitor (PPIi) without disrupting BBDi.EXAMPLES

[0081] The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, described herein. In addition to the examples below, more technical details relevant to the present disclosure can be found at: Wu et al., IDR- Targeting Compounds Suppress HPV Genome Replication via Disruption of Phospho-BRD4 Association with DNA Damage Response Factors, Molecular Cell, 2024, 84, 202-220.

[0082] GENERAL INFORMATION

[0083] (a) Star*Methods

[0084] Table 2. Key Resources Table.

[0085] Data and Code Availability

[0086] All RNA-seq, CUT&RUN and proteomics datasets, along with processed files used for data analysis, have been deposited in the Gene Expression Omnibus under accession number GSExxx or MassIVE under accession number MSV000091570.

[0087] Quantification And Statistical Analysis

[0088] Biological and biochemical assays are typically presented as mean + standard error of the mean (SEM) and performed with at least three independent biological samples, except standard deviation (SD) was used in RT-qPCR analysis and two independent biological samples were used for high-throughput CUT&RUN sequencing. Significance is defined as *p < 0.05, **p < 0.01,***p < 0.001, and ****p < 0.0001 using unpaired two-tailed Student’s / -test for pairwise comparison or two-way ANOVA followed by Tukey’s multiple comparison test for > 2 samples.

[0089] Example 1. Identification of pBRD4-Targeting Compounds by High-Throughput Screening (HTS)

[0090] The finding that ectopically expressed wild-type BRD4, but not its NPS phosphorylationdefective mutant protein 7A, could rescue the loss ofHRHPV type 16 (HPV16) ElZE2-dependent ori replication in endogenous BRD4-knockdown C-33A cervical carcinoma cells and that bothpNPS-binding DC-1 and DC-2 peptoids could reduce El / E2-dependent HPV16 ori replication prompted us to conduct a large-scale chemical library screen to identify pharmacologically expandable small compounds targeting pNPS. From mapping of BRD4-E2 interacting regions, pNPS, residing in a phosphorylation-dependent interaction domain (PDID, aa 287-530) common to both BRD4-L and BRD4-S, was found to uniquely contact the C-terminal DNA-binding domain (DBD) of E2 proteins encoded by HR HP Vs but not LR HP Vs or BPV-1 (Figure 1A). Since the E2-BRD4 complex typically inhibits HPV transcription in its native genomic configuration, a cellbased assay was developed for primary screening to identify pNPS-targeting compounds able to disrupt HR HPV E2-BRD4 interaction, leading to depression of HPV 18 long control region (LCR, containing HPV enhancer / promoter)-driven luciferase (Luc) reporter activity in a C-33A-derived cell line stably expressing HR HP VI 6 E2 (16E2). C-33A cell lines with vector alone or expressing LR BPV-1 E2 (BE2) that does not interact with pNPS served as negative controls and were additionally included in the secondary and counter screen (Figure IB).

[0091] Using a diversity -based 200K chemical library for high-throughput screening (HTS) at a single concentration of 5 pM, 14,218 hits were obtained from the primary screen showing increased (i.e., derepressed) Luc signals in the 16E2-expressing line (Figure 1C). Then 1,021 compounds representing top hits of all compound clusters with distinct chemical scaffolds were selected for a secondary (i.e., confirmatory and counter) screen using HPV18 LCR-Luc-harb oring C-33A lines stably expressing 16E2, BE2, or no E2, each in triplicate. Eighty-three compounds were obtained (Figure 1C), of which 16 compounds (19%) alleviated 16E2 but not BE2 repression (HR-unique), 54 compounds (65%) derepressed preferentially 16E2 rather than BE2 activity (HR- preferred), and 13 compounds diminished 16E2 and BE2 inhibition to a comparable level. AlphaScreen that measures binary interaction between GST-tagged 16E2 DBD and phosphorylated FLAG-tagged PDID (containing BD2 + pNPS) purified from insect Sf9 cells (Sf9- pPDID) was then performed with 37 commercially available compounds (Figures 1C). Compound (cpd) 14 showed strongest inhibition (Figure ID, left) with half-maximal inhibitory concentration (IC50) of 3.39 pM (Figure ID, right graph), ~30-fold better than the IC50 of DC-1 / 2 previously determined by GST pulldown.

[0092] Compound 14 Inhibits HPV Genome Amplification and ori Replication But Not Genome Maintenance

[0093] Since E2 and pBRD4 are both important for HPV DNA replication, it was first examined whether cpd 14 might inhibit HPV replication in infected cells. HPV replication typically occursat three distinct stages (establishment, maintenance, and amplification) in which HPV establishes infection in the basal stratum of a squamous epithelium and maintains a low viral genome copy in the lower strata, while productive genome amplification takes place in the mid and upper differentiating strata (Figure 2A). A three-dimensional (3D) organotypic raft culture of primary human keratinocytes (PHKs) that faithfully generates, in vitro, a stratified and differentiated squamous epithelium in which the productive phase of the HR HPV18 infection is recapitulated was employed for this compound analysis.

[0094] Remarkably, 14 effectively suppressed extrachromosomal (ec) HR HPV18 genome amplification in the upper strata of raft cultures harvested on days 14, 16, and 18 after culturing HPV18-containing PHKs at the air-medium interface to promote epithelial differentiation (Figure 2B, red DNA-FISH signals). Real-time quantitative PCR (qPCR) revealed dose-dependent and temporal inhibition of HPV genome replication, with 10 pM of 14 reducing HPV18 DNA levels to 1% or less relative to DMSO at all three monitored time points. In contrast, host DNA replication was not affected in the mid strata (BrdU in green; incorporated into cellular DNA over 6 h prior to tissue harvest) and had little or no colocalization with viral replication signals (Figure 2B), consistent with distinct cell cycle profiles for G2-associated HPV replication vs. S-dependent host DNA replication and highlighting 14 selectivity towards inhibition of HPV but not host DNA replication. Histology showed slightly thinner epithelium in 14-treated cultures without discernable abnormalities or apoptotic cells. Relative to the DMSO-treated control tissues, all morphologically discernable cell strata (basal, spinous, granular, and cornified) were retained (Figure 2C).

[0095] To examine if early stages (establishment and maintenance) of HPV replication were also inhibited by 14, a transient HPV replication assay was conducted which requires viral El helicase and E2 recognition of the HPV ori to initiate DNA replication (Figure 2D, left). 16E2 and 16E1 expression plasmids along with an HP VI 6 orz-containing Luc reporter were co-transfected into C-33A cells. After 24 hours, cells were treated with compound or vehicle for 24 hours. PPIz 14, but not BBDz JQ1, was found to significantly reduce HPV ori replication without affecting cell viability (Figure 2D, right), consistent with the requirement of both BRD4-L and BRD4-S for ori replication analyzed by Hirt-qPCR (Figure 2E) that quantifies the amount of replicating ecHPV DNA in Hirt lysatmda-e.

[0096] Using proliferating human cervical keratinocyte (HCK) W12E cells containing -300 copies of ecHPV16 for maintenance assay and methylcellulose (or high calcium)-induced W12E differentiation for amplification assay, JQ1, but not 14, was found to inhibit ecHPV16maintenance while both cpds reduced cell viability (Figure 2F, left). In contrast, only 14 suppressed HPV genome amplification in differentiated W12E cells with no effect on live cell viability (Figure 2F, right), consistent with the 3D raft culture results (Figures 2B). Unexpectedly, JQ1 enhanced differentiation-associated HPV genome amplification (Figure 2F, right), likely due to enhanced pools of free DDR factors available for amplification-dependent HPV replication following JQ1 -triggered dissociation of these factors from host chromatin. As HPV genome amplification coincides with increased DDR, ATR signaling was indeed found to activate in differentiated W12E cells as reflected by enhanced pATR, pChkl and pRPA, correlating with increased involucrin (a differentiation marker) but not BRD4-L / S levels and NPS phosphorylation.

[0097] Example 2. Target Validation of Cpd 14 and Its Modified Analog Cpd 90

[0098] To demonstrate direct compound binding to pNPS, thermal shift assay (TSA) was performed that measures increased protein thermostability upon compound binding by comparing the difference in melting temperatures (ATm) that denature 50% of the test protein in the absence vs. presence of a compound. 14 was found to enhance the thermostability of Sf9-f: pPDID but its effect on unphosphorylated PDID purified from bacteria (Bac-fPDID) was much reduced, contrasting to JQ1 that conferred comparable and higher thermostability through phosphorylationindependent BD2 binding to both Sf9-f: pPDID and Bac-fPDID (Figures 3 A). 14 binding to Bac- fPDID 8E, a phosphomimetic containing glutamic acid substitutions at all eight CK2 phosphorylation sites in the NPS, induced strong thermal shift comparable to that seen with JQ1 (Figures 3A), illustrating the importance of NPS phosphorylation for 14 binding. Notably, 14 did not bind 16E2 DBD in the TSA assay (Figure 3B), indicating that 14-inhibited protein-protein interaction (PPI) in AlphaScreen (Figure ID) is attributed to 14 binding to Sf9-pPDID but not 16E2 DBD.

[0099] To validate 14 binding to pNPS, microscale thermophoresis (MST) was used to measure 14 binding-altered movement of fluorescent Cy5-labeled PDID (untagged) and determine its binding affinity. Results showed the equilibrium dissociation constant (Kd) of 14 was 12.9 pM for Sf9-pPDID, 157.9 pM for ZPPase-dephosphorylated Sf9-pPDID, and ~1.0 mM for Bac-PDID (Figure 3C), validating the requirement of NPS phosphorylation for 14 binding. Furthermore, an NPS peptide with dual phosphorylation at S492 and S494, but not the unphosphorylated or monophosphorylated peptides, bound 16E2 DBD. This binding was specifically disrupted by 14 (Figure 3D), highlighting the importance of NPS diphosphorylation for 16E2 DBD contact and the mechanistic action of pNPS-targeting compounds.

[0100] Through chemical modification by adding an extra methoxy group to each indole moiety flanking the 1,3-thiazole ring of 14, cpd 90 was obtained, which exhibits improved IC50 from 5.1 pM (14) to 1.6 pM (90) in AlphaScreen measuring the efficiency of compound-disrupted Sf9- pPDID and 16E2 DBD interaction (Figure 3E). TSA analysis showed 90 induced a higher ATm (12.5 °C) than 14 (9.5 °C), comparable to that of JQ1 (11.7 °C), when bound to untagged Sf9- pPDID (Figure 3F, left) or phosphomimetic Bac-PDID 8E (Figure 3F, right). Moreover, 90 had slightly reduced background ATm (1.3 °C) vs. 14 (1.8 °C) when incubated with Bac-PDID (Figure 3F, middle). It is worth mentioning that background ATm seen with DMSO (1.5 °C) and 14 / 90 is not caused by compound binding but, rather, experimental variation. The lower thermostability of unphosphorylated PDID (Tm = 45 °C) vs. that of phosphorylated PDID (Tm = 50 °C) is consistent with earlier studies showing that phosphorylation-enhanced NPS interaction with BD2 helps stabilize the pPDID conformation to be more resistant to Glu-C protease cleavage than its unphosphorylated counterpart. pNPS-dependent compound action was further illustrated by showing that immobilized biotinylated 14 and 90 bind only wild-type (WT) Sf9-pPDID and phosphomimetic Bac-PDID 8E, but not NPS-deleted Sf9-pPDID nor unphosphorylated Bac- PDID (Figure 3G). Collectively, these analyses demonstrate 14 and 90 indeed target the pNPS of BRD4.

[0101] To confirm 14 and 90 bind endogenous BRD4 in cells, cellular TSA (CETSA) was conducted by pre-incubating differentiated W 12E or proliferating HeLa cells with 14, 90, or JQ1 and subjected the cells to heat treatment using a pre-determined temperature of 49.2°C (Figure 3H), wherein unbound BRD4 proteins form insoluble aggregates while cpd-bound BRD4 remains soluble in the supernatant detectable by immunoblotting after removing insoluble aggregates by centrifugation. CETSA showed 14 / 90 and JQ1 stabilized BRD4-L and BRD4-S in W 12E (Figure 31, lanes 1 vs. 2-4) and HeLa cells (lanes 9 vs. 10-12), while the levels of P-actin remain unaffected at 49.2°C with similar BRD4 levels present at the permissive temperature of 40°C (lanes 5-8 and 13-16), indicating that 14 / 90 and JQ1 indeed target both BRD4-L and BRD4-S isoforms in cells.

[0102] Example 3. pBRD4-Targeting Compounds Inhibit All Three Stages of HPVReplication with Minimal Cytotoxicity

[0103] Using 3D organotypic raft cultures of HPV18-containing PHKs, cpd 90 at 3 pM was found to effectively inhibit viral genome amplification in differentiated cells, comparable to that observed with 10 pM of cpd 14 (Figure 4A). Among the three stages of HPV replication, 90 showed 10-fold improved potency (IC50 = 0.1 pM) over 14 (IC50 = 1.0 pM) in inhibiting HPVestablishment replication with minimal cytotoxicity as analyzed by transient HPV16 ori replication assay performed in C-33A cells (Figure 4B). In HPV genome maintenance analysis, 90 had better inhibition than 14 but also exhibited higher cytotoxicity in proliferating W12E cells (Figure 4C). In HPV genome amplification assay performed in differentiated W12E cells cultured in high calcium medium, 90 is 7.5-fold better than 14 in suppressing ecHPV amplification with essentially no cytotoxicity similar to 14, while JQ1 again significantly enhanced HPV genome amplification (Figure 4D), consistent with that seen in methylcellulose-induced W12E differentiation (Figure 2F, right). Since JQ1 could inhibit viral genome maintenance in proliferating W12E cells (Figure 2F, left), it is likely that JQ1 may suppress HPV DNA replication in raft cultures when added prior to viral genome amplification which usually initiates on day 9 or 10 post induced differentiation. It seems cytotoxicity is in part determined by cellular context, as 14 and 90 only trigger cellular toxicity in the maintenance (proliferating) but not amplification (differentiated) or establishment (transiently transfected) stages of HPV replication. Interestingly, despite effective inhibition of HPV18 genome amplification in PHK raft cultures, neither 14 nor 90 at the concentrations used had morphologically discernable adverse effects on the epithelium over an extended duration (from day 6 to 14, 16, or 18).

[0104] As cytotoxicity remains a general concern in the development of anti -HPV inhibitors particularly during cell proliferation, 90 was further modified and ZM0145 was obtained by replacing the methoxy with a hydroxy group at each of the two indole moieties. ZM0145 showed significantly lower cytotoxicity than 90 in proliferating cells, while maintaining comparable potency in inhibiting pPDID interaction with 16E2 DBD and suppressing both ecHPV16 genome maintenance and amplification. These results highlight the potential for developing pBRD4 PPIz capable of inhibiting all three stages of HPV replication with minimal cytotoxicity.

[0105] Example 4. Compound Effects on Viral and Cellular Transcriptomes

[0106] To define whether 14 and 90 may reprogram transcriptomes to affect HPV DNA replication and genome amplification, RNA-seq was performed, with rRNA-depleted total RNA isolated from proliferating and differentiated W 12E cells with or without compound treatment for 4 hours, to identify primarily direct early response genes. The concentration chosen (5 pM for 14, and 1 pM for 90 and JQ1) was 2-3-fold lower than proliferation viability IC50 to avoid secondary effects triggered by viability inhibition. Cpd 90 was selected over ZM0145 due to its proliferation inhibition comparable to that of JQ1 (Figures 2F and 4C). Differentially expressed genes (DEGs) following RNA comparison between these two cell states were identified by edgeR and dividedinto differential on-upregulated (Diff-Up, 3,284 genes) and differential on-downregulated (Diff- Dn, 3,094 genes) gene sets using cutoff p < 0.05 and fold change (FC) > 1.5 or < -1.5 (Figure 4E). Compound-perturbed DEGs in differentiated W12E cells were then identified and, upon comparison with Diff-Up and Diff-Dn gene sets, were separated respectively into Cpd-Dn and Cpd-Up groups for identification of differentiation-related DEGs whose expression is deregulated by 14, 90, or JQ1. JQl-repressed Diff-Up DEGs (763 genes) was found to be > 14-fold more than those repressed by 14 (47 genes) and 90 (52 genes), and JQl-upregulated Diff-Dn DEGs (516 genes) are > 5-fold more of those upregulated by 14 (105 genes) and 90 (37 genes) (Figure 4E), indicating 14 / 90 do not globally perturb cellular gene expression to the same extent as JQ1 treatment.

[0107] Gene set enrichment analysis (GSEA) revealed that Cornified Envelope, the top Gene Ontology (GO) gene set with enrichment score (ES) = 0.85 and false discovery rate (FDR), and other differentiation-related datasets (bottom table) are significantly activated in differentiated W12E cells. Among the 36 Cornified Envelope genes strongly induced by differentiation 31 (except rows 2-4, 35 and 36) were severely repressed by JQ1, contrasting with generally minor and insignificant effects seen with 14 and 90 treatments (compare listed FDR values). Unlike JQ1 that significantly inhibited all early viral transcripts except El in both proliferation (Prol) and differentiation (Diff) states, 14 / 90 did not alter viral gene expression (Figure 4F). The RNA levels of select viral (E6 / E2) and host differentiation marker involucrin (IVE) transcripts were further validated by RT-qPCR.

[0108] It seems counterintuitive that JQ1 inhibition of viral transcription (Figure 4F) coincides with JQ1 -enhanced HPV replication (Figure 2F, right, and Figure 4D). Perhaps, transcriptionreplication conflicts (TRCs) occurring at actively replicating HPV genomes in differentiated W12E cells are resolved by JQ1 -dissociated transcription complexes that help clear roadblocks in the progression of stalled replication forks. It is also likely that BRD4 association with acetylated chromatin varies between proliferating and differentiated states, particularly in the TRC regions, leading to different sensitivities to JQ1 treatment. Taken together, these viral and cellular transcriptome analyses indicate 14 and 90 directly target pBRD4-mediated HPV genome replication with limited, if any, effects on transcriptional perturbance - a conclusion in agreement with minimal disturbance of normal stratification and differentiation of PHK raft cultures as judged by histology and the presence of BrdU-positive cells in the differentiated strata of 14- treated HPV- 18 raft cultures (Figures 2B and 4A).

[0109] Example 5. Proteomics Identification of BRD4 Isoform Interaction Networks

[0110] HPV replication relies heavily on host factors due to limited coding capacity of this small DNA tumor virus. Thus, it is important to identify cellular factors whose association with pBRD4 is specifically disrupted by 14 / 90. An unbiased proteomic analysis was carried out in proliferating and high Ca2+-induced differentiated W12E cells, with or without 14 / 90 treatment, each in triplicate. Immunoprecipitation (IP) with IgG or anti-BRD4 isoform-specific antibody was performed in the isolated nuclear extracts. BRD4 complexes pulled down were subjected to trypsin digestion and LC-MS / MS analysis (Figure 5 A). A total of 2,352 BRD4-L / S interactors were identified, each with peptide spectrum matches (PSMs) > 2 and confidence FDR cutoff 1%. Significant interactors were further defined by FC > 2 and p < 0.05 (e.g., BRD4 vs. IgG IP, or cpd vs. DMSO) using duplicate datasets analyzed by Principal Component Analysis (PCA) with emphasis on nuclear-localized proteins according to Human Protein Atlas and GeneCards (nucleus confidence > 3). Significant BRD4-L and BRD4-S interactors were then analyzed for common and unique isoform interactors and enriched biological processes, pathways, and networks in GO, GSEA, and STRING datasets.

[0111] These results showed > 3-fold of BRD4-L interactors were identified in differentiated (395) vs. proliferating (100) W12E cells (Figure 5B, Venn diagrams), unlike a nearly constant number of BRD4-S interactors in both cell states (70 vs. 76). In GO analysis of all interactors using the STRING web platform (Figure 5B), BRD4-L interactors found in differentiated W12E cells (green bars) are highly enriched in biological processes such as gene expression and mRNA processing, much more than those in proliferating cells (blue bars) and significantly higher than BRD4-S interactors in both cell states (pink and red bars). Interestingly, BRD4-L but not BRD4- S interactors showed a link to DDR with 20 out of 100 proteins found in proliferating W12E and 50 out of 395 proteins identified in differentiated W12E cells.

[0112] Further analysis of 308 BRD4-L-unique interactors in differentiated W12E cells uncovered highly enriched association with DDR networks, such as global genome nucleotide excision repair (GG-NER), transcription-coupled nucleotide excision repair (TC-NER), and DNA double-strand break (DSB) repair pathways, while only GG-NER is modestly enriched in 52 L- unique interactors in proliferating cells (Figures 5C). The L-unique link to GG-NER, TC-NER, and DSB is also evident when comparing all interactors of BRD4-L and -S in differentiated W12E cells (Figure 5D), or when differentiation vs. proliferation of BRD4-L interactors or BRD4-S interactors are similarly analyzed. It is important to note that the Mediator complex is predominantly present in the L-Diff-unique interactome (Figure 5D, L > S) likely contributing to differentiation-induced gene expression. Intriguingly, STRING network profiling highlights acentral role of BRD4-L in connecting all three DDR networks through interaction with: a) DSB factors 53BP1 (p53-binding protein 1), BARD1 (BRC Al -associated RING domain 1), WHSC1 / NSD2, and cohesion component RAD21 and its loading factor NIPBL; b) TC-NER factors CDK9 and various Pol II components; and c) GG-NER factor INO80 (Figure 5E). With similar analyses, BRD4-S Diff-unique interactors were found to be involved in biological processes mediated by chromatin remodeling (HSF90a, RSF1), proteosomes (PSMB5, PSMD12), and RNA binding (YBX1, LRRC59) (Figure 5F), as well as differentiation-enhanced association with SRCAP, SNRPE, and RTRAF, even though the identification of BRD4-S nuclear interactors may be potentially limited by its low abundance, typically < 3% of BRD4-L in cells.

[0113] Example 6. pNPS-Targeting Compounds Disrupt BRD4-L Association with 53BP1 and BARD1

[0114] To identify BRD4 isoform -interacting networks disrupted by 14 and 90, compound-treated and mock-treated (i.e., DMSO only) proteomics datasets were compared and 53BP1 and BARD1 were found, among others, to be reduced in BRD4-L interactomes from differentiated W12E cells treated by 14 or 9, with 90 but not 14 specifically inhibiting BRD4-S and polo-like kinase 1 (PLK1) association. Since many DDR factors contain phosphoserine / threonine (pS / T)-binding domains such as breast cancer gene 1 (BRCA1) C-terminal (BRCT) domain, Forkhead-associated (FHA) domain, polo-box domain (PBD), FF domain, and phosphopeptide-binding 14-3-3 family proteins (Reinhardt and Yaffe, 2013), pS / T-binding proteins in BRD4 IP-proteomics datasets were searched to identify potential pNPS binders whose interaction with pBRD4 could be disrupted by 14 / 90 (Figure 6A). Intriguingly, among twenty pS / T-binding (reader) proteins present in the datasets, only 53BP1 and BARD1 interaction with BRD4-L in differentiated W12E cells was reduced by 14 and 90, while PLK1 interaction with BRD4-S but not BRD4-L was uniquely suppressed by 90 with no obvious effect by 14, indicating interaction between BRD4-L / S and their common or unique pNPS binders could be selectively targeted by 14 and 90.

[0115] Using IP followed by IB detection, 53BP1 and BARD 1 were confirmed to have association with BRD4-L but not BRD4-S in nuclear extracts isolated from differentiated W12E cells and that both 14 and 90 disrupted endogenous BRD4-L interaction with 53BP1 and BARD1 (Figure 6B, lanes 1-5). Similar results were also obtained with ZM0145. No 53BP1 / BARD1-BRD4-L / S interaction and compound disruption could be detected in proliferating W12E cells (data not shown). The induced BRD4-S association with BARD1 in 14 / 90 treatment (Figure 6B, lanes 6-8) is consistent with the proteomics data showing compound-induced BRD4-S interaction with BARD1 (Figure 6A, 1st row). It is likely that BARD1 is limited in nuclear extracts for interactionwith less abundant BRD4-S, so BARD1 released from BRD4-L following 1 pM 14 / 90 treatment fortuitously contacts BRD4-S in solution, which could then be disrupted by an increased concentration (2 pM) of compound treatment.

[0116] Since BARD1 and 53BP1 both contain two BRCT domains that typically function in pairs to control DDR and cell cycle progression, it was postulated the dual BRCT domains in each protein could interact directly with BRD4 isoforms. Using comparable amounts of purified FLAG-tagged BRD4-L and BRD4-S for interaction with GST-tagged tandem BRCT1 / 2, 53BP1 BRCTs were found to interact specifically with BRD4-L but not BRD4-S, while BARD1 BRCTs contact both BRD4-L and BRD4-S (lanes 3 and 4). Furthermore, BRCT1 / 2 from either 53BP1 or BARD1 only interacts with purified Sf9-pPDID but not Bac-PDID, and alanine-substituted mutations in single (mutant d) or multiple S / T residues (mutants a, b, and c) in pNPS selectively abolish BRD4-L interaction with respective BRCT1. These results indicate that 53BP1 and BARD1 BRCT domains indeed bind pNPS in a phosphorylation-dependent manner and their interaction with pNPS-containing pPDID could be effectively disrupted by 14, 90, and ZM0145 as evidenced by AlphaScreen interaction analysis showing compound inhibition IC50 ranging from -1.0-1.5 pM.

[0117] To examine whether pBRD4-targeting compounds might regulate pNPS-binding DDR factor function at the transcriptional level as seen with BBDz, RNA-seq datasets were analyzed in proliferating and differentiated W12E cells with or without 14, 90, or JQ1 treatment (Figure 4E) by normalizing pNPS binder RNAs to that of untreated proliferating cell samples (Figure 6C, 1st column). While expression of many DDR factors was perturbed by JQ1 in both proliferating and differentiated W12E cells, it was found that none of these 20 pNPS binders’ RNA levels were affected by 14 and 90, highlighting the direct action of 14 and 90 in disrupting pBRD4 interaction with pNPS binders without transcriptionally regulating their gene expression.

[0118] The uncovering of 53BP1 and BARD1 as compound-disrupted partners of pBRD4 in differentiated W12E cells indicates their potential role in HPV genome amplification. To investigate this, siRNA knockdown of 53BP1 and BARD1 was performed in proliferating and differentiated W 12E cells and 53BP1 and BARD1 were both found to be important for ecHPV16 genome amplification in differentiated cells but not for ecHPV16 genome maintenance in proliferating cells, with knockdown of either protein showing little effect on cell viability in both cell states (Figure 6D). In comparison, BRD4-L and BRD4-S are involved in both replication stages, as knockdown of either isoform significantly impaired HPV replication in proliferatingand differentiated cells (Figure 6E). Interestingly, severe diminishment of HPV replication was only observed in the maintenance but not amplification stage when combined (pan) knockdown of BRD4-L and BRD4-S was performed, suggesting that BRD4-L and BRD4-S may regulate different pathways to control HPV genome maintenance but act through the same pathway in genome amplification. Ectopic expression of wild-type but not phosphorylation-defective NPS 7A BRD4-L or BRD4-S could rescue the loss of HPV genome amplification in differentiated W12E cells upon respective BRD4 isoform knockdown, consistent with the requirement of NPS phosphorylation for pBRD4 isoform-dependent HPV genome replication.

[0119] Example 7. pNPS-Targeting PPI / Do Not Globally Alter BRD4 Chromatin Landscape

[0120] The finding that 14 and 90 disrupt pBRD4 interaction with select DDR factors (Figure 6A) without inhibiting DDR gene expression (Figure 6C) prompted us to examine whether pNPS- targeting PPIz would prevent BRD4 binding to its target chromatin as seen with BBDz. Because of the low abundance of BRD4-S, its profiling of global binding could not be easily achieved by standard ChlP-seq. CUT&RUN (Cleavage Under Targets and Release Using Nuclease; Skene and Henikoff, 2017) was thus used to determine genome-wide BRD4-S and BRD4-L binding to chromatin with or without 90 or JQ1 treatment. By analyzing overlapping BRD4-L or -S-binding peaks obtained from proliferating and differentiated W12E cells, it was found that 45-55% of BRD4-L binds to similar locations while only 16-20% of BRD4-S peaks are preserved in the two cell states, suggesting BRD4 isoforms respond differentially to signaling cues during the growth- to-differentiation transition. Genome-wide promoter binding (within 3 kb upstream of the transcription start site, TSS) by BRD4-L increases from 12,626 (proliferation) to 14,492 (differentiation) peaks, contrasting with reduced (from 10,855 to 9,683) BRD4-S promoter occupancy. Nevertheless, both isoforms show differentiation-enhanced binding to introns and distal intergenic regions. GSEA analysis of potential target genes based on promoter-binding peaks showed BRD4-L remains engaged with Pol II promoters during the proliferation-to- differentiation transition, while BRD4-S mainly associates with promoters of c-Myc targets in proliferation and G2 / M checkpoint genes in differentiation (S-Prol and S-Diff). Similar analysis in distal intergenic regions identified differentiation-enhanced BRD4-L and BRD4-S occupying respectively the enhancers of Cornified Envelope Formation and STAT5 pathway gene sets that are both important for HPV genome amplification in differentiated W12E cells.

[0121] When H3K27ac-marked enhancer association was analyzed, BRD4-L but not BRD4-S was found to show increased binding to cellular enhancers upon differentiation (Figure 7A, leftfour heatmaps and plots). As expected, JQ1 significantly diminishes BRD4-L and completely abolishes BRD4-S binding to enhancers, while 90 only partially reduces BRD4-L but has no effect on BRD4-S occupancy (Figure 7A), indicating 90 does not globally perturb BRD4-L / S association with host chromatin as seen with JQ1. To identify BRD4 direct target genes and associated transcription factors (TFs) perturbed by 90 or JQ1, RNA-seq and CUT&RUN datasets were integrated to obtain BRD4-L / S promoter-binding peaks in compound down (Dn) or up (Up)- regulated. By comparing BRD4-binding peaks in 90- or JQ1 -repressed DEGs with >17,000 TF and chromatin modifier ChlP-seq datasets available from Cistrome Data Browser, JQ1 was found to prevent BRD4-S from binding to promoters coregulated by differentiation-promoting master regulator MAFB (MAF bZIP transcription factor B) in JQl-Dn DEGs, while 90 showed marginal disruption of BRD4-L binding in the same MAFB-coregulated promoters in 90-Dn DEGs. This result indicates 90 action is distinct from that of JQ1 with more target- selective inhibition by PPI / vs. global suppression by BBDz.

[0122] Example 8. pBRD4-Dependent Recruitment of 53BP1 and BARD1 to the HPV Ori in a Temporal and Isoform-Specific Manner

[0123] To examine whether 90 / JQ1 also differentially regulate BRD4-L and -S binding to HPV chromatin, BRD4 isoform binding to the ecHPV16 genomes in W12E cells was analyzed and BRD4-L and BRD4-S were found to maintain most of their binding to the viral genome during cell proliferation and differentiation, except specific differentiation-reduced binding of both isoforms at the late promoter (PL) region and the 3’ end of the E2ZE4 gene body. Interestingly, BRD4-L but not BRD4-S is uniquely present in the HPV16 LCR where viral enhancer and early promoter are located (blue box), suggesting BRD4-L is more important than BRD4-S in regulating viral early gene expression. 90 did not alter BRD4-L and -S binding to ecHPV16 chromatin in differentiation. Unexpectedly, JQ1 significantly enhances BRD4-L occupancy at the HP VI 6 LCR (tracks 3 vs. 7), distinct from its downregulation of BRD4 isoform binding to host chromatin (Figure 7A). This analysis supports a minor disruption of BRD4 binding to ecHPV16 chromatin by PPIz (90) and provides a potential explanation for JQ1 -enhanced HPV genome amplification (Figures 2F and 4D).

[0124] It was then analyzed whether 53BP1 / BARD1 involvement in HPV genome replication (Figure 6D) relies on pBRD4-dependent recruitment to the HPV ori by performing qPCR analysis to detect factor-bound DNA in CUT&RUN-released fragments using four primer pairs binding respectively to (" / / ' / -proximal (#1-3) and -distal (#4) regions. In differentiated W12E cells, 53BP1 and BARD1 were detected in orz-flanking regions (#1 and #3) with much reduced occupancy atthe ori (#2) and ~3.5 kb downstream (#4), unlike BRD4-L / S binding to all four loci with also slightly reduced signals in the ori. Notably, while BRD4 isoform binding to the orz-proximal region is evident (shown by locus #3 with highest signal) in both proliferating and differentiated cells, 53BP1 and BARD1 association was only detected in the differentiated but not proliferating cell state.

[0125] BARD1 binding near the ori was reduced by 14 and 90, distinct from 53BP1 association that was diminished by 90 and its analog ZM0145, but not 14 (Figures 7B). This is consistent with stronger inhibition of differentiation-associated HPV genome amplification by 90 (IC50 = 1.1 pM) and ZM0145 (IC50 = 0.8 pM) vs. 14 (IC50 = 8.7 pM; see Figures 4D, Hirt-qPCR panels) and more effective blocking (i.e., lower IC50) of pPDID interaction with 53BP1 BRCT1 / 2 by 90 and ZM0145 vs. 14. Interestingly, recruitment of 53BP1 and BARD1 to the ori region was significantly enhanced by JQ1, providing a molecular basis for JQ1 -stimulated HPV genome amplification (Figures 2F and 4D). The altered 53BP1 and BARD1 binding following compound treatment is not caused by the loss (for PPIz) or increase (for BBDz) of orz-associated BRD4 isoform levels as BRD4-L and BRD4-S occupancy remains generally constant without showing corresponding changes as seen in compound-perturbed 53BP1 and BARD1 binding (Figure 7B).

[0126] Since compound treatment mainly alters 53BP1 and BARD1 but not BRD4-L / S association with the HPV ori, it is likely that 53BP1 and BARD1 recruitment depends on their interactions with BRD4-L or -S. To determine the temporal order of recruitment, factor binding by CUT&RUN-qPCR following individual knockdown of BRD4-L, BRD4-S, 53BP1, and BARD1 was analyzed. BRD4-L binding was only found to be reduced by its own depletion and not by BRD4-S, 53BP1, or BARD1 knockdown (Figure 7C), indicating BRD4-L binds earlier than the other three factors to the HPV ori. BRD4-S binding was decreased by knockdown of BRD4-L or itself, but not 53BP1 or BARD1, showing BRD4-S entry is dependent on BRD4-L. Likewise, 53BP1 occupancy was diminished by knockdown of BRD4-L, not BRD4-S or BARD1, indicating 53BP1 is recruited separately by BRD4-L independent of BRD4-S. BARD1 occupancy was strongly diminished by BRD4-L / S knockdown, but only partially by 53BP1 knockdown, suggesting BARD1 entry could be mediated by BRD4-L or BRD4-S but less so by 53BP1. A likely model for sequential recruitment of these factors to the HPV ori is summarized in the schematic drawing.

[0127] To determine whether BRD4-L recruits 53BP1 and BARD1 in a sequential or parallel manner, a temporal recruitment assay was conducted by monitoring ori association of these factors at days 1, 3, and 5 post high-Ca2+-induced differentiation. Maximal 53BP1 binding was found atday 1 which was diminished at day 3 and barely detectable at day 5, while BARD1 association stayed constant at days 1 and 3 but increased by day 5, seemingly compensating for the loss of 53BP1 even though BRD4-L / S occupancy remained stable throughout the entire time course (Figure 7D, left). Since BARD1 only associates with BRD4-L but not BRD4-S in nuclear extracts prepared from differentiated W12E cells (Figure 6A and 6B) yet shows BRD4-S-dependent recruitment to the HPV ori (Figure 7C), it was pondered whether BARD1 associates with ori- bound BRD4-S. Using CUT&RUN-isolated BRD4-L / S-bound chromatin for BARD 1 IP, BARD1 was found to indeed bind BRD4-S at days 3 and 5 post differentiation while BARD1 associates with BRD4-L less at day 3 but more at day 5 (Figure 7D, right), indicating BRD4-S binds stably with BARD1 at the HPV ori. Using the same CUT&RUN / re-IP, 14 and 90 were shown to indeed disrupt BARD1 association with chromatin-bound BRD4-L and BRD4-S. These kinetic studies indicate 53BP1 and BARD1 recruitment by BRD4-L occurs in a sequential manner during the transition from early to late phases of HPV genome amplification.

[0128] Example 9. Experimental Model and Subject Details

[0129] Bacterial strains

[0130] DH5a (Thermo Fisher Scientific) was used for regular plasmid-based DNA cloning. One Shot™ Stbl3™ Chemically Competent E. coli (Thermo Fisher Scientific) was used for constructing lentiviral delivery vectors. ArcticExpress (DE3) RIL Competent Cells from Agilent Technologies were used for protein expression of BRD4 PDID (wild-type and mutants), 16E2 DBD and BRCT domains.

[0131] Insect cell line

[0132] Sf9 (Expression Systems, LLC) was maintained in ESF-921 medium (Expression Systems, LCC) and used for expression and purification of truncated PDID and full-length BRD4- L and BRD4-S proteins.

[0133] Mammalian cell lines

[0134] W12E human cervical keratinocytes containing ~ 300 copies of ecHPV16 genomes per cell (20863 subline of W12, used before reaching passage #23) were cultured in F-medium containing 3: 1 [v / v] of F-12:DMEM, 5% heat-inactivated FBS, 0.66 mM Ca2+, 0.4 pg / ml hydrocortisone, 5 pg / ml insulin, 0.01 pg / ml cholera toxin, 0.01 pg / ml EGF, 24.2 pg / ml adenine, 100 units / ml penicillin, and 100 pg / ml streptomycin, and grown on top of mitomycin C (4 pg / ml)- pretreated 3T3-J2 feeder cells as described. 3T3-J2 mouse fibroblasts were maintained in high- glucose DMEM (Sigma-Aldrich) supplemented with 10% calf serum, 20 mM glutamate, 10 mMsodium pyruvate, 100 units / ml penicillin, and 100 pg / ml streptomycin. Proliferating W12E cells were maintained at sub-confluence without feeder cells in low Ca2+(0.05 mM) F-medium by mixing 3:1 [v / v] of Ca2+-free DMEM:F-12 and 5% Chelex® 100-treated FBS with the other supplements as described above for F-medium. To induce cell differentiation, confluent W12E cells without feeders were incubated either in EGF-free high Ca2+(1.4 mM) F-medium or regular EGF-free F-medium containing 1.5% methylcellulose and 20% heat-inactivated FBS. HEK Lenti- X 293T, HPV-negative C-33A and its derived lines containing an integrated HPV18 LCR-Luc reporter with or without exogenously expressed HPV16 E2 (C33A / 18LCR / 16E2) or BPV-1 E2 (C33A / 18LCR / BE2), and HeLa cervical cancer cells were cultured in high-glucose DMEM with 10% heat-inactivated FBS. All cell lines were routinely tested for mycoplasma using MycoAlert Detection Kit (Lonza) and authenticated by STR profiling at the University of Texas Southwestern Medical Center (UTSW) Genomic Core. Sources of cell lines and reagents are listed in the Key Resources Table.

[0135] Three-dimensional (3D) organotypic raft culture and H&E staining

[0136] Preparation and transfection of primary human keratinocytes (PHKs, derived from neonatal foreskin) used for subsequent raft cultures and HPV18 genome amplification assay were performed as described. In brief, discarded and de-identified neonatal foreskin tissues were collected from Neonatal Nursery of the University of Alabama at Birmingham (UAB) under IRB- approved protocol IRB-300005595 and processed to isolate PHKs. Plasmids pNeo-loxP-HPV18 and pCAGGS-nlsCre were co-transfected using FUGENE 6 (Promega) into early-passage (P0 or Pl) PHKs which were then selected with G418 (110 pg / ml) for 3 days. Surviving PHKs containing HPV18 replicons excised from pNeo-loxP-HPV18 by Cre recombinase were seeded on a dermal equivalent consisting of rat tail collagen (Corning, 3-4 mg / ml) and 3T3-J2 mouse fibroblasts. After 48-hour incubation in submerged cultures, the assembly (raft) was placed onto a stainless- steel grid, with the raft lifted to the air-medium interface and grown into fully stratified and differentiated epithelium wherein productive HPV18 DNA amplification took place. Independent experiments were conducted with different batches of PHKs and recombinant plasmids at different times.

[0137] For determining compound inhibition efficacy, HP VI 8 rafts were treated with compound 14 (3.0 or 10.0 pM), 90 (3.3 pM), or vehicle DMSO-only from day 6 to day 14 and harvested on day 14, day 16 or day 18. Bromodeoxyuridine (BrdU, Sigma- Aldrich) was added 6 hours before harvest at a final concentration of 100 pg / ml to mark replicating host DNA. One set of raft culturesalong with the dermal equivalent was fixed in 10% buffered formalin and paraffin embedded. A parallel set of raft cultures was detached from the underlying dermal equivalent, briefly washed in lx PBS, flash-frozen in liquid nitrogen, and stored at -80°C until DNA extraction. The formalin- fixed-paraffin-embedded (FFPE) tissue was cut into 4-pm thin sections using manual microtome. The FFPE sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) (Fisher Scientific) following standard histology protocols. Brightfield images of the tissue sections were captured at 20X magnification with a BX63 microscope and BH73 colored camera (Olympus) using cellSens software (Olympus). Representative images of control and treatment rafts were cropped at identical scale to make composite images using the Photoshop image processor. Sources of cell lines and reagents are listed in the Key Resources Table.

[0138] Example 10. Preparation of Compounds

[0139] High-throughput compound screen

[0140] Chemical compound library screening was conducted at High-Throughput Screening (HTS) Core using the UTSW Chemical Library consisting of -200K compounds purchased from ChemBridge Corporation (75,000), Chemical Diversity Labs (100,000), ComGenex (22,000), TimTek (1,200), Prestwick (1,100), NIH clinical collection (450), and UTSW chemistry labs (2,500). All compounds are drug-like molecules and satisfy a relaxed set of Lipinski’s rules, with 99% having a molecular weight less than 550 (average 250-300) Daltons.

[0141] For the primary screen, C33A / 18LCR-Luc / 16E2 cells were seeded onto 384-well tissue culture plates (Greiner, 781098) at a density of 3,500 cells in 60 pL culture medium and then incubated at 37 °C with 5% CO2 overnight (~14 hours). Individual compounds from the UTSW Chemical Library were added to cells in columns 3 to 22 of each assay plate, resulting in a final screening concentration of 5 pM for each compound. The positive control, JQ1, was added to column 1 at a final concentration of 0.5 pM. DMSO (vehicle control) was added to columns 2 and 23 on each assay plate. Following library addition, the assay plates were incubated for another 48 hours at 37 °C with 5% CO2 after which the medium was removed from each plate by centrifugation at 40 x g at room temperature. Bright-Glo (20 pL, diluted 1 :6 in lx PBS / 1% Triton X-100) was subsequently added to each assay plate. Plates were incubated at room temperature for 10 minutes and then scanned on the Envision reader (PerkinElmer, Inc.). Data from the primary screen were analyzed as previously described with Z’ values > 0.5 for all assay plates, resulting in 14,218 hits with increased luciferase activity relative to the library test population (Z-score > 3). After hit selection to cover all compound clusters based on their chemical scaffolds, 1,021compounds, in triplicate, were cherry-picked for subsequent confirmation and counter- screen using three cell lines, C33A / 18LCR-Luc (no E2), C33A / 18LCR-Luc / 16E2, and C33A / 18LCR- Luc / BE2. The final compound selection was based on the following: 1) increased luciferase activity (relative to DMSO only) by HR-16E2 in both primary and secondary screens, and 2) more than 1.5-fold increase of luciferase activity in HR-16E2 vs. no E2. Those compounds conforming to the above selection criteria were further analyzed by comparing their luciferase activity between HR-16E2 and LR-BE2 to classify three groups of compounds that are HR-E2 unique (Luc activity HR-E2 > 0 and LR-E2 < 0), HR-E2-preferred (Luc activity HR-E2 / LR-E2 > 1.5, HR-E2 > 0, and LR-E2 > 0), and HR&LR-Common (Luc activity HR-E2 / LR-E2 < 1.5, HR-E2 > 0, and LR- E2 > 0). Eight-three compounds were identified. Sources of cells and reagents are listed in the Key Resources Table.

[0142] Compound synthesis — General Information

[0143] All commercial chemicals and solvents are ACS reagent grade and used directly without further purification unless otherwise stated. All reactions were performed in dry glassware with magnetic stirring under the atmosphere of nitrogen. Preparative column chromatography was performed using silica gels (100-200 mesh). Analytical TLC was carried out by using silica gel 60 GF254 plates (Merck, Darmstadt) with visualization of the chromatograms detected by UV (254 nm).JH and13C NMR spectra were recorded on a Bruker-300 / 400 spectrometer (1H, 300 / 400 MHz;13C, 75 MHz) using the internal reference of TMS. Chemical shifts were expressed in ppm, and J values were given in Hz with the following abbreviations indicating the multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; and m, multiples. Purity of all final compounds, including 14, 90, ZM0145, and the biotinylated derivatives, was determined by analytical HPLC equipped with UV detectors on the Shimadzu HPLC system (model: CBM-20A LC-20AD SPD-20A UV / VIS) using a Waters pBondapak C18 (300 x 3.9 mm) column at a flow rate of 0.5 mL / min with UV detection at 270 and 254 nm and a linear gradient from 10% acetonitrile in water to 100% acetonitrile over 20 min, followed by 30 min of the last-named solvent (0.1% TFA was added into both acetonitrile and water). All biologically evaluated compounds have a minimum of 95% purity. Schemes for the synthesis of compounds 14, 90, ZM0145, and the biotinylated derivatives are listed in Supplemental Information (SI).

[0144] Example 10(a). Synthesis of compound 14 (Scheme 1)

[0145] 2-Chloro-l-(2-methyl-lH-indol-3-yl)ethan-l-one (2)

[0146] Chloroacetyl chloride (5.17 g, 45.74 mmol) was added to a solution of 2-m ethyl- I / / -indole (1) (5.00 g, 38.12 mmol) and pyridine (3.02 g, 38.12 mmol) in anhydrous 1,4-dioxane (100 ml)with ice bath under an argon atmosphere. The mixture was warmed to 40°C and stirred for 5 hours. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (150 mL x 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (Hexane / EA = 2: 1) to produce2-chloro-l-(2-methyl-l / / -indol-3-yl)ethan-l-one (2) (6.50 g, yield 82.1%) as a purple solid.JH NMR (300 MHz, DMSO ) 8 12.05 (s, 1H), 8.01 (dd, J = 6.0, 3.1 Hz, 1H), 7.40 (dd, J= 6.1, 2.9 Hz, 1H), 7.17 (dd, J= 6.0, 3.2 Hz, 2H), 4.94 (s, 2H), 2.72 (s, 3H).

[0147] lH-Indole-3-carbothioamide (4)

[0148] (NH4)2S (80% in water) (71.90 g, 211.03 mmol) was added to a solution of EbN (3.92 g, 38.69 mmol) and I / / -indole-3 -carbonitrile (3) (5.00 g, 35.17 mmol) in pyridine (23.47 g, 296.73 mmol). The mixture was heated to 80°C and stirred for 12 hours. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (Hexane / EA = 5: 1, v / v) to generate l / T-indole-3 -carbothioamide (4) (5.00 g, yield 80.7%) as a white solid. 'H NMR (300 MHz, DMSO-tL) 5 11.80 (s, 1H), 8.98 (s, 1H), 8.85 (s, 1H), 8.64 (dd, J = 6.3, 2.3 Hz, 1H), 8.11 (d, J = 3.0 Hz, 1H), 7.45 (dd, J = 6.3, 2.4 Hz, 1H), 7.21-7.13 (m, 2H).

[0149] 2-(lH-Indol-3-yl)-4-(2-methyl-lH-indol-3-yl)thiazole (compound 14)

[0150] 2-Chloro-l-(2-methyl-l / / -indol-3-yl)ethan-l-one (2) (5.00 g, 24.08 mmol) was added to a solution of l / T-indole-3 -carbothioamide (4) (4.24 g, 24.08 mmol) in EtOH (80 ml). The mixture was heated to 80°C and stirred for 12 hours. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (150 mL x 3). The combined organic layers were washed with brine, dried over anhydrous ISfeSCU, concentrated, and purified by silica gel chromatography (Hexane / EA = 2: 1, v / v) to produce 14 (6.00 g, yield 75.6%) as a white solid. 'H NMR (300 MHz, DMSO-d6) 5 12.18 (s, 1H), 11.50 (s, 1H), 8.39 (d, J = 2.9 Hz, 1H), 8.25-8.19 (m, 1H), 7.94-7.89 (m, 1H), 7.61-7.57 (m, 1H), 7.54 (s, 1H), 7.42-7.38 (m, 1H), 7.32-7.26 (m, 2H), 7.15-7.06 (m, 2H), 2.72 (s, 3H).13C NMR (75 MHz, DMSO-d6) 5 162.94, 148.01, 137.18, 135.76, 135.57, 128.51, 127.22, 124.62, 123.18, 121.75, 121.37, 120.26, 120.00, 119.42, 113.11, 111.33, 109.58, 109.28, 105.58, 13.78. HPLC purity: 98.8%.

[0151] Example 10(b). Synthesis ofbiotinylated-14 (Scheme 2)

[0152] 6-Hydroxy-lH-indole-3-carbothioamide (6)

[0153] BBrs (12.15 g, 48.48 mmol) was added dropwise to a solution of 6-m ethoxy- 1 / T-indole-3 -carbothioamide (5) (2.00 g, 9.70 mmol) in anhydrous dichloromethane (30 mL) at 0°C. Themixture was warmed to room temperature after stirring for 10 minutes at 0°C. The reaction was quenched by saturated NaHCCL solution and then extracted with dichloromethane (60 mL x 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated to obtain 6-hydroxy-l 7-indole-3 -carbothioamide (6) (1.64 g, yield 88.2%) as a brown solid which was directly used for the next step without further purification.

[0154] 3-(4-(2-Methyl-lH-indol-3-yl)thiazol-2-yl)-lH-indol-6-ol (7)

[0155] 2-Chloro-l-(2-methyl-l / / -indol-3-yl)ethan-l-one (2) (1.77 g, 8.53 mmol) was added to a solution of 6-hydroxy- l / / -indole-3 -carbothioamide (6) (1.64 g, 8.53 mmol) in EtOH (30 ml).The mixture was heated to 80°C and stirred for 12 hours. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (Hexane / EA = 1 : 1) to produce 3-(4-(2-methyl-l / / -indol-3- yl)thiazol-2-yl)-l / / -indol-6-ol (7) (2.16 g, yield 73.4%) as a white solid. 'H NMR (400 MHz, DMSO-t / e) 5 11.72 (s, 1H), 11.45 (s, 1H), 8.15 (s, 1H), 7.96 (d, J= 8.6 Hz, 1H), 7.89 (d, J= 7.5 Hz, 1H), 7.50 (s, 1H), 7.41 - 7.34 (m, 1H), 7.14-7.08 (m, 2H), 6.93 (d, J= 2.2 Hz, 1H), 6.80 (dd, J= 8.6, 2.2 Hz, 1H), 2.70 (s, 3H).

[0156] 3-(4-(2-Methyl-lH-indol-3-yl)thiazol-2-yl)-lH-indol-6-yl 5-((3aS, 4S, 6aR)-2- oxohexahydro-lH-thieno[ 3, 4-d]imidazol-4-yl)pentanoate (biotinylated- 14)

[0157] 3-(4-(2-Methyl-l / / -indol-3-yl)thiazol-2-yl)-l / / -indol-6-ol (7) (1.00 g, 2.89 mmol), biotin (0.71 g, 2.89 mmol), DCC (0.60 g, 2.89 mmol) and DMAP (0.42 g, 3.47 mmol) were dissolved in anhydrous DMF solution at room temperature and the mixture was stirred for 12 hours at 0°C. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (DCM / MeOH = 10: 1) to generate biotinylated-14 (1.14 g, yield 69.2%) as a white solid. 'H NMR (300 MHz, DMSO-d6) 5 11.79 (s, 1H), 11.24 (s, 1H), 8.17 (d, J= 2.8 Hz, 1H), 8.09-8.03 (m, 2H), 7.51 (d, J = 8.7 Hz, 1H), 7.40 (s, 1H), 7.36 (d, = 7.0 Hz, 1H), 7.13-7.03 (m, 2H), 6.98 (dd, J= 8.7, 2.3 Hz, 1H), 6.46 (s, 1H), 6.37 (s, 1H), 4.33-4.26 (m, 1H), 4.18-4.11 (m, 1H), 3.14-3.06 (m, 1H), 2.85-2.77 (m, 1H), 2.73 (s, 3H), 2.65-2.56 (m, 3H), 1.75-1.67 (m, 3H), 1.58-1.43 (m, 3H).13C NMR (75 MHz, DMSO-d6) 5 172.78, 163.20, 161.56, 151.22, 145.39, 135.57, 134.85, 134.79, 128.10, 127.19, 125.05, 121.15, 119.88, 119.75, 117.19, 113.30, 113.05, 111.60, 111.17, 108.00, 107.67, 61.51, 59.68, 55.78, 33.95, 28.47, 25.13, 14.10. HPLC purity: 98.7%.

[0158] Example 10(c). Synthesis of compound 90 (Scheme 3)

[0159] 2-chloro-l-(5-methoxy-2-methyl-lH-indol-3-yl)ethan-l-one (9)

[0160] Chloroacetyl chloride (4.20 g, 37.22 mmol) was added to a solution of 5-methoxy-2- m ethyl- 1 / T-indole (8) (5.00 g, 31.02 mmol) and pyridine (2.45 g, 31.02 mmol) in anhydrous 1,4- dioxane (50 ml) with ice bath under an argon atmosphere. The mixture was warmed to 40°C and stirred for 5 hours. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (150 mL x 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (Hexane / EA = 2: 1) to obtain 2-chloro-l-(5-methoxy-2-methyl-l / / -indol-3-yl) ethan-l-one (9) (6.00 g, yield 81.4%) as a purple solid. 'H NMR (300 MHz, DMSO-tL) 8 11.91 (s, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 8.7 Hz, 1H), 6.82-6.78 (m, 1H), 4.92 (s, 2H), 3.79 (s, 3H), 2.69 (s, 3H).

[0161] 6-Methoxy-lH-indole-3-carbonitrile (11)

[0162] Chlorosulfonyl isocyanate (8.75 g, 61.83 mmol) was added to a solution of 6-methoxy- 1 / T-indole (10) (7.00 g, 47.56 mmol) in DMF (2.1 mL) and MeCN (21 mL) under an argon atmosphere. The mixture was warmed to room temperature and stirred for 5 hours. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (200 mL x 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (Hexane / EA = 5: 1) to generate 6- methoxy- l / / -indole-3 -carbonitrile (11) (6.50 g, yield 79.4%) as a white solid. 'H NMR (300 MHz, DMSO-t / e) 6 12.00 (s, 1H), 8.12 (d, J = 2.9 Hz, 1H), 7.51 (d, = 8.7 Hz, 1H), 7.03 (d, J= 2.1 Hz, 1H), 6.88 (dd, J= 8.7, 2.2 Hz, 1H), 3.80 (s, 3H).

[0163] 6-Methoxy-lH-indole-3-carbothioamide (12)

[0164] (NH4)2S (80% in water) (59.36 g, 174.23 mmol) was added to a solution of EtsN (3.23 g, 31.94 mmol) and 6-methoxy-l / / -indole-3 -carbonitrile (11) (5.00 g, 29.04 mmol) in pyridine (19.02 g, 240.45 mmol). The mixture was heated to 80°C and stirred for 12 hours. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (150 mL x 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (Hexane / EA = 5: 1) to give 6-methoxy- l / T-indole-3 -carbothioamide (12) (4.50 g, yield 75.1%) as a white solid. 'H NMR (300 MHz, DMSO-t / e) 6 12.00 (s, 1H), 8.12 (d, J = 2.9 Hz, 1H), 7.51 (d, = 8.7 Hz, 1H), 7.03 (d, J= 2.1 Hz, 1H), 6.88 (dd, J= 8.7, 2.2 Hz, 1H), 3.80 (s, 3H).

[0165] 2-(6-Methoxy-lH-indol-3-yl)-4-(5-methoxy-2-methyl-lH-indol-3-yl)thiazole (compound 90)

[0166] 2-Chloro- l -(5-methoxy-2-methyl- IT / -indol-3-yl)ethan- l -one (9) (5.00 g, 21.04 mmol) was added to a solution of 6-methoxy-U / -indole-3 -carbothioamide (12) (4.34 g, 21.04 mmol) in EtOH (80 ml). The mixture was heated to 80°C and stirred for 12 hours. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (150 mL x 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (Hexane / EA = 2: 1) to obtain compound 90 (5.50 g, yield 67.1%) as a white solid. 'HNMR (300 MHz, DMSO ) 5 11.48 (s, 1H), 11.09 (s, 1H), 8.28 (d, J= 8.7 Hz, 1H), 7.97 (s, 1H), 7.66 (s, 1H), 7.32 (s, 1H), 7.24 (d, J= 8.8 Hz, 1H), 7.00 (s, 1H), 6.84 (dd, J = 8.8, 2.3 Hz, 1H), 6.74 (dd, J = 8.7, 2.4 Hz, 1H), 3.82 (s, 6H), 2.70 (s, 3H).13C NMR (75 MHz, DMSO-tL) 8 161.95, 156.69, 154.17, 151.36, 137.91, 135.27, 130.48, 127.67, 125.47, 121.56, 119.23, 111.74, 111.55, 111.13, 110.99, 107.79, 107.40, 102.15, 95.46, 55.69, 14.28. HPLC purity: 95.9%.

[0167] Example 10(d). Synthesis of biotinylated-90 (Scheme 4)

[0168] 5-Methoxy-lH-indole-3-carbonitrile (14)

[0169] Chlorosulfonyl isocyanate (8.75 g, 61.83 mmol) was added to a solution of 5-methoxy- UT-indole (13) (7.00 g, 47.56 mmol) in DMF (2.1 mL) and MeCN (21 mL) under an argon atmosphere. The mixture was warmed to room temperature and stirred for 5 hours. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (200 mL><3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated, to produce 5-methoxy-U / -indole-3-carbonitrile (14) (6.82 g, yield 83.3%) as a white solid, which was directly used for the next step without further purification.

[0170] 5-Hydroxy-lH-indole-3-carbonitrile (15)

[0171] BBrs (29.77 g, 118.80 mmol) was added dropwise to a solution of 5-methoxy-U / -indole- 3 -carbonitrile (14) (6.82 g, 39.60 mmol) in anhydrous dichloromethane (100 mL) at 0°C. The mixture was warmed to room temperature after stirring for 10 minutes at 0°C. The reaction was quenched by addition of saturated NaHCCL solution and then extracted with ethyl acetate (150 mL x 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated, to yield 5-hydroxy-177-indole-3-carbonitrile (15) (5.00 g, yield 79.9%) as a brown solid, which was directly used for the next step without further purification.

[0172] 5-Hydroxy-lH-indole-3-carbothioamide (16)

[0173] (NH4)2S (80% in water) (59.36 g, 174.23 mmol) was added to a solution of EtsN (3.23 g, 31.94 mmol) and 5-hydroxy-U / -indole-3-carbonitrile (15) (5.00 g, 29.04 mmol) in pyridine (19.02 g, 240.45 mmol). The mixture was heated to 80°C and stirred for 12 hours. The reactionwas quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (150 mL x 3). The combined organic layers were washed with brine, dried over anhydrous ISfeSCU, concentrated, and purified by silica gel chromatography (Hexane / EA = 5: 1) to generate 5- hydroxy-l / f-indole-3 -carbothioamide (16) (4.45 g, yield 73.2%) as a white solid. 'H NMR (400 MHz, DMSO-t / e) 5 11.53 (s, 1H), 8.87 (s, 1H), 8.74 (s, 1H), 8.68 (s, 1H), 8.07 (d, = 2.4 Hz, 1H), 7.99 (d, J= 3.2 Hz, 1H), 7.21 (d, J= 8.6 Hz, 1H), 6.66 (dd, J= 8.6, 2.4 Hz, 1H).

[0174] 3-(4-(5-Methoxy-2-methyl-lH-indol-3-yl)thiazol-2-yl)-lH-indol-5-ol (17)

[0175] 2-Chloro-l-(5-methoxy-2-methyl-U / -indol-3-yl)ethan-l-one (9) (2.00 g, 8.41 mmol) was added to a solution of 5-hydroxy-177-indole-3-carbothioamide (16) (1.62 g, 8.41 mmol) in EtOH (50 ml). The mixture was heated to 80°C and stirred for 12 hours. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (60 mL / 3). The combined organic layers were washed with brine, dried over anhydrous ISfeSCU, concentrated, and purified by silica gel chromatography (Hexane / EA = 1 : 1) to produce 3-(4-(5-methoxy-2- methyl-U / -indol-3-yl)thiazol-2-yl)-U / -indol-5-ol (17) (2.01 g, yield 63.6%) as a white solid. 'H NMR (300 MHz, DMSO-d6) 5 11.30 (d, J = 2.1 Hz, 1H), 11.10 (s, 1H), 9.17 (s, 1H), 8.18 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 2.6 Hz, 1H), 7.66 (d, J = 2.4 Hz, 1H), 7.30 (s, 1H), 7.24 (d, J = 8.7 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 6.71 (td, J = 8.4, 2.3 Hz, 2H), 3.83 (s, 3H), 2.69 (s, 3H).

[0176] 3-(4-(5-Methoxy-2-methyl-lH-indol-3-yl)thiazol-2-yl)-lH-indol-5-yl 5-((3aS,4S, 6aR)-2- oxohexahydro-lH-thieno[ 3, 4-d]imidazol-4-yl)pentanoate (biotinylated-90)

[0177] 3-(4-(5-Methoxy-2-methyl-U / -indol-3-yl)thiazol-2-yl)-U / -indol-5-ol (17) (1.22 g, 3.25 mmol), biotin (0.79 g, 3.25 mmol), DCC (0.67 g, 3.25 mmol) and DMAP (0.48 g, 3.90 mmol) were added to anhydrous DMF (10 mL) at room temperature. The mixture was stirred at 0°C for 12 hours. The reaction was quenched by addition of saturated NH4CI solution and then extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, concentrated, and purified by silica gel chromatography (DCM / MeOH = 10: 1) to obtain biotinylated-90 (1.32 g, yield 67.3%) as a white solid. 'H NMR (300 MHz, DMSO-d6) 5 11.80 (s, 1H), 11.09 (s, 1H), 8.17 (d, J= 2.8 Hz, 1H), 8.09 (d, J= 2.5 Hz, 1H), 7.59 (d, J = 2.3 Hz, 1H), 7.51 (d, .7= 8.7 Hz, 1H), 7.35 (s, 1H), 7.24 (d, = 8.7 Hz, 1H), 6.98 (dd, J = 8.8, 2.3 Hz, 1H), 6.74 (dd, J = 8.8, 2.5 Hz, 1H), 6.44 (s, 1H), 6.36 (s, 1H), 4.32-4.25 (m, 1H), 4.16-4.09 (m, 1H), 3.79 (s, 3H), 3.12-3.04 (m, 1H), 2.84-2.76 (m, 1H), 2.68 (s, 3H), 2.64-2.54 (m, 3H), 1.76- 1.62 (m, 3H), 1.57-1.38 (m, 3H).13C NMR (75 MHz, DMSO-d6) 5 172.83, 163.19, 161.65, 154.23, 151.38, 145.44, 135.31, 134.81, 130.50, 128.13, 127.64, 125.04, 117.40, 113.14, 113.07,111.75, 111.64, 110.98, 107.73, 102.08, 61.48, 59.67, 55.76, 55.69, 33.90, 28.44, 25.04, 14.18. HPLC purity: 96.2%.

[0178] Example 10(e). Synthesis of compound ZM0145 (Scheme 3)

[0179] 3-(2-( 6-Hydroxy-lH-indol-3-yl)thiazol-4-yl)-2-methyl-lH-indol-5-ol (ZM0145)

[0180] To a solution of compound 90 (30 mg, 77 pmol) in anhydrous DCM (5.0 mL), BBrrCEhCh (1.0 M, 29 pL, 308 pmol) was added slowly at 0°C, and then stirred at room temperature for 2 hours. Upon the completion of the reaction, saturated NaHCCL was added slowly to quench the reaction and extracted with DCM (20 mL x 3). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure to leave a residue, which was purified by silica gel chromatography to obtain compound ZM0145 (13 mg, yield 47%) as a white solid. 'H NMR (300 MHz, MeOD) 3 8.04 (d, J= 9.3 Hz, 1H), 7.82 (s, 1H), 7.33 (d, J= 1.8 Hz, 1H), 7.17-7.13 (m, 2H), 6.89-6.85 (m, 1H), 6.80 (dd, J= 8.6, 2.2 Hz, 1H), 6.67 (dd, J = 8.6, 2.4 Hz, 1H), 2.67 (s, 3H).13C NMR (75 MHz, MeOD) 3 164.79, 155.33, 152.77, 152.18, 139.81, 136.47, 132.17, 129.91, 125.39, 122.11, 120.20, 113.02, 112.31, 112.18, 111.88, 109.48, 108.74, 105.21, 98.42, 13.77. HPLC purity: 99.0%.

[0181]

[0182] Example 11. Plasmid construction

[0183] For expression of GST-P3C-fused PDID recombinant proteins, human BRD4 cDNA encoding aa 287-530 containing wild-type (WT), NPS deletion (ANPS) or eight glutamate (8E)- substituted S / T residues was PCR-amplified from pF:PDID-l ld, pF:PDID-l ld NPS and pF:PDID-l ld 8E, respectively, and subcloned into pGEX-6P-l vector between AzzzzHI and TzoI sites to generate corresponding pGST-P3C:PDID, pGST-P3C:PDID A NPS, and pGST- P3C:PDID 8E as previously described. The cDNA inserts from pGST-P3C:PDID and pGST- P3C:PDIDANPS were subsequently transferred to pVL1392 between Not! and Smal sites to create pVL-GST-P3C:PDID and pVL-GST-P3C:PDID A NPS, individually. Plasmid pGST: 16E2-DBD was constructed by inserting the PCR fragment encoding 16E2 aa 279-365 amplified from pGEX-16E2 to pGEX-TL(+) between Ndel and AzzzzHI sites. For lentiviral delivery, plasmids pLKO-3F :BRD4-L 7A and pLKO-3F :BRD4-S 7A were generated by replacing the wild-type BRD4 5 ’-fragment in pLKO-3F :BRD4-L and pLKO-3F:BRD4-S respectively with the same 5’ fragment containing NPS 7A from pcDNA3-3F:BRD4-L 7A between BamHI and EcoRI sites. The HPV16 orz-containing plasmid pLucl6orz was constructed by inserting the HPV16 fragment spanning nucleotides 7710-7904 / 1-98 (GenBank K02718.1), obtained by PCRamplification from pUR41 using primer pair HPV16mOrNgoMIV-F and HPV16mOrNgoMIV-R (see the Key Resources Table), into pLuc vector at the AgoMIV site. pLuc was constructed by inserting the luciferase cDNA excised from pGL3 -Basic (Promega) into pCI (Promega) between Xhol and Xbal. Plasmids previously published are listed in the Key Resources Table.

[0184] Example 12. Protein purification

[0185] Recombinant FLAG-tagged (f:) and GST-tagged proteins, including f:BRD4-L WT and NPS S / T mutants a-d, f:BRD4-S, Sf9-f:PDID, and GST-SfO-pPDID WT and ANPS, were expressed in and purified from insect Sf9 cells, with Bac-fPDID, GST: 16E2 DBD, and GST- BRCT1 / 2 of 53BP1 or BARD1 similarly expressed in and purified from bacteria, according to the established protocols. GST-P3C-fused PDID proteins were likewise purified from insect Sf9 or bacteria with some modifications. GSH bead-bound proteins were first washed three times with buffer containing 20 mM Tris-HCl (pH 7.9 at 4°C), 500 mM NaCl, 20% glycerol, and 1 mM EDTA (pH 8.0), and two times more in P3C digestion buffer containing 20 mM Tris-HCl (pH 7.9 at 4°C), 100 mM NaCl, 20% glycerol, 5 mM EDTA (pH 8.0), and 0.2 mM DTT. PDID proteins were then released from GST / GSH beads by GST-P3C protease digestion (enzyme vs. substrate ratio according to manufacturer’s instructions) at 4°C for 2 hours or overnight. Tag-free PDID proteins were collected and loaded into a HiTrap Q HP column using AKTA FPLC (GE Healthcare) and eluted in a 0.1-1.0 M NaCl gradient. PDID-containing fractions between 300-450 mM NaCl were concentrated and buffer-exchanged by centrifugation in a centrifugal filter with MW cutoff of 10 kDa (Millipore, UFC900324) to final protein concentrations between 1~3 mM (27.1-81.4 mg / ml) in 10 mM HEPES (pH 7.4) and 150 mMNaCl. Sources of reagents for protein purification are listed in the Key Resources Table.

[0186] Example 13. Amplified luminescent proximity homogeneous assay screen (AlphaScreen)

[0187] AlphaScreen reactions were set up in PROXIPL ATE-384-PLUS plates (PerkinElmer) using assay buffer containing 20 mM Tris-HCl (pH 7.3), 100 mM NaCl, 0.2 mM EDTA (pH 8.0), 0.01% NP40 (or IGEPAL), and 1% BSA (Sigma, A7030). For the assay conducted by the HTS Core (Fig. ID), the protein mix in 15 pl containing GST: 16E2 DBD (400 nM) and f: Sf9-pPDID (200 nM) was first added into the plates using an automated reagent dispenser (Agilent BioTek MicroFlo™) and preincubated at room temperature for one hour, followed by addition of 10 pl bead mix containing AlphaScreen GSH donor beads (final 10 pg / ml, PerkinElmer) and AlphaLISA anti-FLAG acceptor beads (final 5 pg / ml, PerkinElmer). Each assay plate wasincubated overnight at room temperature in a humidified chamber without room light and then read on a EnVision™ plate reader (PerkinElmer) with 680-nm wavelength illumination followed by light emission at the 615 nm wavelength. The raw data was read into Genedata’s Screener™ Software Suite (v.10) and analyzed by the Condoseo module. Curve fits were carried out with the logistics equation using a four-parameter fit model as previously described. Four parameters were reported for each curve: Sinf (the asymptotic activity predicted for infinitely high compound concentration), So(the asymptotic activity predicted for infinitely low compound concentration), the Hill slope, and the IC50 value (half-maximal inhibitory concentration). For validation of AlphaScreen results in the lab, the assay was similarly conducted except using 25 nM of Sf9- f: pPDID for incubation with each GST-tagged 16E2 DBD (50 nM), p53BPl-BRCTl / 2 (500 nM), or BARD1 BRCT1 / 2 (1 pM) in assay buffer containing 20 mM Tris-HCl (pH 7.3) for 16E2 DBD or HEPES (pH 6.5) for BRCT1 / 2, 100 mM NaCl, 0.2 mM EDTA (pH 8.0), 0.01 % NP40, and 1% BSA. Compounds in final 1% DMSO were titrated down from 40 pM in 2-10-fold dilution, each in triplicate, for a total of 4-7 doses. IC50 was determined by GraphPad PRISM (Dotmatics, Inc.) using a nonlinear regression curve fit with inhibitor concentrations on the x-axis and the normalized response on the y-axis. Sources of proteins and reagents are listed in the Key Resources Table.

[0188] Example 14. Thermal shift assay (TSA) with purified proteins and cellular TSA (CETSA)

[0189] TSA was performed as described. Briefly, PDID / compound mix (each in final 25 pM, 10 pl per well) was added into a 384-well PCR plate (Bio-Rad, HSP3801) in assay buffer containing 20 mM HEPES (pH 6.5) for untagged PDID or 20 mM Tris-HCl (pH 6.5) for FLAG-tagged PDID, 100 mM NaCl, 2.5% glycerol, and 5x SYBRO Orange (Invitrogen, S6650). After sealed with adhesive films (Bio-Rad, MSB 1001) and quick centrifugation, the plates were heated in a CFX384 Touch Real-Time PCR Detection machine (Bio-Rad) following the melt curve protocol: step 1, 25°C for 1 minute; and step 2, temperature increase at the rate of 0.5°C every 30 seconds from 25°C to 95°C with fluorescent signal recorded every 0.5°C. The resulting melt curves were analyzed by Bio-Rad CFX Manager software (v3.1) with Tm (melting temperature) indicating the temperature at which 50% of protein is unfolded and bound by SYBRO Orange. ATm is the difference of Tm comparing compound treated vs. untreated (i.e., DMSO-only) samples.

[0190] For compound binding to endogenous BRD4-L and BRD4-S in CETSA, differentiated W12E (5xl06) and proliferating HeLa (IxlO6) cells were treated respectively with compoundsnear their viability IC50 for 4 hours. Cells were harvested by a scraper, collected in lx PBS at room temperature, spun, and resuspended in 120 pl of lx PBS at room temperature. 100 pl of cells were incubated at 49.2 °C for 1 minute, with the remaining 20 pl incubated at 40 °C for 1 minute. Cells were then lysed by 3x freeze-thaw cycles alternating between liquid nitrogen and a 25 °C thermal cycler. After spinning at 20,000 x g for 30 minutes at 4 °C, the supernatant was collected for immunoblotting.

[0191] Example 15. Microscale thermophoresis (MST)

[0192] MST measuring compound binding affinity (Kd) to fluorescent-labeled proteins was conducted as previously described (Tso and Brautigam, 2021). In brief, untagged PDID proteins, including phosphorylated Sf9-pPDID, unphosphorylated Bac-PDID, and XPPase- dephosphorylated Sf9-pPDID, were first incubated with Sulfo-Cyanine5.5 NHS ester (Lumiprobe, 23020) in buffer containing 50 mM HEPES (pH 7.4) and 150 mM NaCl at 4°C for 2 hours according to the manufacturer’s instruction. Unlabeled dyes were removed by passing protein samples through a Sephadex G-25 column (Cytiva, 28918007) with labeled proteins concentrated as described in protein purification and quantified by UV absorbance at 280 nm. Fifteen compound doses ranging from 1 pM to 1 mM were each incubated with 200 nM of a Cy5-labeled PDID protein in the labeling buffer supplemented with 0.01% TWEEN-20 for 30 minutes at room temperature in the dark. MST measurements were then conducted at 22°C in standard-coated capillaries at 10% LED power and 20% IR laser power (or MST power) using the Monolith™ NT.115 instrument (NanoTemper Technologies). Kd was calculated by Monolith Affinity Analysis v.2.2.4. software (NanoTemper Technologies).

[0193] Example 16. Immobilized peptide and compound binding assays

[0194] For immobilized peptide binding assay (Fig. 3D), biotinylated NPS peptides (0.01 mg / ml) containing unphosphorylated, monophosphorylated, or diphosphorylated S492 and / or S494 were first immobilized onto Streptavidin MagneSphere (Promega, Z5481) according to the manufacturer’s instruction. GST-tagged 16E2 DBD (3 pM) was incubated with 1 pl of NPS peptide beads for 2 hours with gentle rotation at 4°C in buffer containing 20 mM Tris-HCl (pH7.9 at 4° C), 100 mM NaCl, 5% glycerol, and 0.1% NP40, followed by three washes with the binding buffer. IB detection of bead-bound proteins was performed with anti-GST antibody. For immobilized compound binding assay (Fig. 3G), Streptavidin MagneSphere with or without prebound biotinylated-14 or -90 (0.4 mM) was incubated overnight at 4°C with 10 pg of respective GST-tagged PDID proteins in the same binding buffer and, after 3x binding buffer washes, bead-bound proteins were spotted on a nitrocellulose membrane using a 96-well Bio-Dot microfiltration system (Bio-Rad) following the manufacturer’s instruction with IB detection conducted as described for peptide binding assay.

[0195] Example 17. Cell-based HPV replication, compound inhibition, and viability assays

[0196] Transient transfection of 16orz-containing and E1ZE2 expression plasmids was performed as described with some modification. C-33A cells in 1.2 ml suspension (2xl05 / ml) were transfected with 12.5 ng of pLucl6orz or pUR41, 200 ng of each pMT2-16El and pCN- Myc: 16E2, and pcDNA3 (to final total 600 ng) using 2 pl of Fugene HD (Promega, E2311). 100 pl of the above cell solution was added to each well in a 96-well plate for subsequent HPV ori replication analysis by luciferase assay or Hirt-qPCR and 25 pl of the cell solution was added to each well in a 384-well plate for cell viability assay after 48-hour incubation. Compounds were added at 24 hours post transfection in the same culture volume and incubated for an additional 24 hours. Luciferase assay was conducted by adding 100 pl of Bright-Glo solution to medium-free cells, and cell viability was performed by directly adding CellTiter-Glo to medium-containing cells following the manufacturer’s instruction. Hirt DNA was isolated as described except using 80 pl of lysis buffer (0.6% SDS and 10 mM EDTA, pH 8.0) per well in a 96-well plate. Hirt lysates were transferred into a 1.5-ml microcentrifuge tube and spun at 20,000 x g for 30 minutes at 4°C. The supernatant (i.e., Hirt lysate) was treated with 10 pg of RNase A (Thermo Scientific, FEREN0531) for 30 minutes at 37°C, followed by dilution with 5-fold NTB binding buffer (MACHEREY-NAGEL, 740595.1). DNA was isolated by using a PCR clean-Up column (MACHEREY-NAGEL, 740609).

[0197] For HPV genome maintenance assay, sub-confluent W12E cells grown on top of 3T3-J2 feeder cells in F-medium were harvested in low Ca2+F-medium and seeded at lxl04 / 100 pl per well in a 96-well plate with compound in final 0.15% DMSO added 24 hours post seeding for a 3-day treatment. For HPV genome amplification assay in suspension cultures, 5xl05of W12E cells were cultured in 1 ml of EGF-free F-medium supplemented with 1.5% methylcellulose and 20% FBS for 5 days. Compounds in final 0.5% DMSO were added to cells in suspension. For high Ca2+-induced differentiation in monolayer culture, 5xl04of W12E cells in 100 pl were first seeded in a 96-well plate for 24 hours prior to medium switch from low (0.05 mM) to high (1.4 mM) Ca2+F-medium with compound addition the following day for a total 3-day inhibitor treatment. Cells in methylcellulose were harvested by adding 10-fold volume of cold lx PBS, in which 90% of the harvested cells were used for HPV genome amplification assay analyzed byHirt-qPCR and 10% of cells for Calcein-AM cell viability assay. Cells in 2D differentiation were harvested for Hirt-qPCR or labeled by Calcein-AM for cell viability assay. W12E cells were kept below passage #23.

[0198] HPV replication assays analyzed for all three stages with or without compound treatment were each performed with at least three biological replicates.

[0199] Example 18. Fluorescence in situ hybridization coupled immunofluorescence assay (FISH-IFA)

[0200] Dual detection of BrdU and HPV genome in the raft culture was performed as described with, first, indirect IF probing for BrdU incorporation and then HPV18 DNA detection by FISH. In brief, the biotinylated DNA probe was prepared beforehand by nick-translation of HPV18 genomic DNA (from pNeo-loxP-HPV18) with Biotin-Nick Translation Mix (Roche, marketed by Sigma) per the manufacturer’s protocol. The ideal probe size distribution between 200-500 bp was confirmed by agarose gel electrophoresis before the nick-translated probe was ethanol- precipitated and resuspended at a concentration of 1 pg per 100 pl in Hybrisol VII (MP Biomedicals).

[0201] For IF detection, the deparaffinized tissue sections were subjected to heat-induced epitope retrieval (HER) in 10 mM citrate buffer (pH 6.0) for 10 minutes at sub-boiling temperature (95°C) followed by slow cooling to room temperature. Tissue sections were incubated with mouse anti- BrdU antibody which was then bound by horseradish peroxidase (HRP)-conjugated goat antimouse secondary antibody (Invitrogen) followed by Cyanine 3 (Cy3)-labeled tyramide treatment using Tyramide Signal Amplification (TSA) kit (Akoya Biosciences). For subsequent FISH detection, the tissue sections after three washes in lx PBS were treated with RNase A (100 Kunitz units / ml) for 1 hour at 37°C, dehydrated through graded ethanol (2 minutes each in 70, 90 and 100% ethanol), dried on a hot plate at 50°C, denatured at 74°C for 2 minutes in 70% formamide / 2x SSC (pH 7.2,), quickly chilled in 70% ethanol for 2 minutes, dehydrated by treating with 90% and 100% ethanol for 2 minutes each, and dried on a hot plate at 50°C. Next, biotin-labeled HPV18 DNA probe in Hybrisol VII after denatured at 74°C for 10 min was added to the tissue sections, which were then covered by glass cover slips, sealed by rubber cement (Walmart), and incubated overnight (12-16 h) at 37°C. Tissue sections were then sequentially washed at 37°C with: 1) 50% formamide in lx SSC (pH 7.2), 3 times for 10 minutes each; 2) lx SSC, one time for 30 minutes; 3) 3% H2O2 (hydrogen peroxide) in lx SSC, one time for 15 minutes; and 4) 2x SSC, 3 times each 15 minutes. After blocking buffer (1% casein in 4x SSC) treatment for 30 minutes at 37°C, tissue sections in the blocking buffer were incubated at 37°C with 1 :100 diluted HRP-labeledstreptavidin (provided in TS A-Fluorescein System Kit, Akoya Biosciences) for 1 hour and washed at room temperature sequentially with 4x SSC / 0.1% Triton X100 twice for 5 minutes each and 4x SSC once for 5 minutes. For Tyramide signal amplification (TSA), rinsed tissue sections were treated with Tyramide-Fluorescein (from TSA-Fluorescein System Kit) for 10 minutes. After this step, the tissue sections were again rinsed in 4x SSC / 0.1% Triton X100 and 4x SSC as above to remove excess unbound fluorophores. Finally, the tissue sections were mounted with DAPI- containing media (Vectashield, H1200) and stored at -20°C.

[0202] Images were captured with a 20x objective in an Olympus BX63 microscope fitted with appropriate fluorescence filters (Chroma) using an Olympus DP73 camera and cellSens software. Fluorescence excitation / emission wavelengths are 550 / 570 nm for Cy3 and 494 / 517 nm for fluorescein. All images in each set of experiments were collected at the same setting. Images were processed using Adobe Photoshop software. Sources of reagents are listed in the Key Resources Table.

[0203] Example 19. HPV DNA replication analysis by real-time quantitative polymerase chain reaction (qPCR)

[0204] For real-time qPCR analysis of HPV genomes in raft cultures, total DNA was extracted from rafts using DNA lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM EDTA, pH 8.0, 100 mM NaCl, and 0.5% SDS) by homogenization and treated with 10 pg / ml Proteinase K at 37°C overnight. Total DNA was recovered by phenol / chloroform extraction and ethanol precipitation. HPV18 DNA was determined in 25 ng of the total DNA by real-time qPCR (Applied Biosystems vii A7 machine) using SYBR GreenER qPCR SuperMix (ThermoFisher) with the forward primer annealed to nucleotides 820-839 in the E7 region and the reverse primer annealed to nucleotides 912-931 in the El region. In a parallel set, 10-fold serial dilutions of the pNeo-LoxP-HPV-18 reference plasmid, mixed in 25 ng of total DNA from an uninfected PHK raft culture, were PCR amplified to generate a standard reference curve. The Ct values were then computed to yield HPV18 DNA copies per pg of the standard curve samples using a formula, (A x 106x 1 / 660 x 1 / B) x IO’12x (6.022 x 1023), where A is the microgram of the HPV-18 plasmid in the dilution, B is the number of nucleotide pairs in the pNeo-LoxP-HPV-18 reference plasmid (11,314 bp), 660 is the molecular mass of a pair of nucleotides and 6.022 x 1023is the Avogadro’s number (molecules per mole). This standard curve was then used to determine HPV18 DNA present in 25 ng of raft culture samples. Finally, HPV18 DNA copies per cell were calculated by considering one human 2N nucleus (representing one cell) containing 6.6 pg genomic DNA.

[0205] For quantification of newly replicated HPV16 DNA in cell-based assays, two pl of column-purified DNA from a total of 30 pl was digested overnight with 10 units of Dpnl in 10 pl at 37°C and then 20 minutes at 85°C, followed by qPCR in SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) using primer pairs detecting regions spanning ori or the E6 TSS in a BioRad CFX384 Cl 000 thermal cycler. HP VI 6 DNA from the qPCR reaction was quantified according to a standard curve generated by 10-fold serial titration of pUR41 from 2 to 2x10'5ng with R2> 0.98 for curve fitting using the built-in CFX analysis software. IC50 was obtained by a nonlinear regression curve fit of logic (inhibitor) vs. the response ratio, which was normalized with DMSO first and then with the average of the lowest dose (i.e., 0.01 pM) for each group, with the setting of 4-parameter variable slope and fixed bottom (= 0) and top (= 1.0) values using GraphPad PRISM 9. Primer sequences are listed in the Key Resources Table.

[0206] Example 20. siRNA transfection and lentiviral delivery

[0207] siRNA transfection was performed in C-33A and W12E cells using universal negative siRNA control or target-specific siRNA for BRD4 pan, BRD4-S, BRD4-L, 53BP1, or BARD1 following the protocols as described. Gene delivery using lentivirus was conducted as described except using lentiviral transfer plasmids expressing wild-type 3f:BRD4-L / S, their phosphorylation-deficient 7A mutants, or vector alone. Approximately 50% of collected viruses were added to IxlO6of W12E cells in low Ca2+F-medium without the feeder cells which were then added back the next day for cell expansion. W12E cells ectopically expressing 3f:BRD4 proteins were used for endogenous BRD4 knockdown. All siRNA sequences are listed in the Key Resources Table.

[0208] Example 21. RNA-seq, reverse transcription (RT)-qPCR, BRD4 gene target and pathway / GO analyses

[0209] In RNA-seq transcriptome analysis, total RNAs were extracted from proliferating and differentiated W12E cells with or without compound treatment (5 pM of 14, or 1 pM of 90 or JQ1) for 4 hours, each in triplicate, by miRNeasy Mini Kit (Qiagen #217004) as previously described. RNA integrity with a RIN score > 9.6 was verified using Agilent 2100 Bioanalyzer. Library construction using NEBNext Ultra II directional RNA Library Prep Kit, together with Illumina Ribo-zero Plus, and paired-end sequencing using HiSeq 2x150 were both conducted by Admera Health. Raw reads in fastq files were analyzed by BICF RNA-seq Analysis Workflow as previously described except keeping reads with base quality score > 25 and sequence length > 35 bp. Trimmed reads were mapped to hg38 using HiSAT2 with duplicated reads marked bySAMBAMBA and removed. RNA counts generated by FeatureCounts and StringTie were used to identify differentially expressed genes (DEGs) by edgeR with isoform expression calculated using Ballgown software. Statistical cutoffs based on p < 0.05 and fold change > 1.5 or < -1.5 were used to filter DEGs between the cell states and compound treatments.

[0210] Average values of RNA abundance measured by FPKM (Fragments Per Kilobase of transcript per Million mapped reads) were used for heatmap clustering analyzed by ClustVis web platform with the following settings: no transformation, rows centered, unit variance scaling applied to rows, and rows clustered using correlation distance and average linkage. Pathway and GO analyses were conducted as described except FPKM in differentiation vs. proliferation and compound vs. DMSO only (-) were compiled for gene set enrichment analysis (GSEA) with false discovery rate (FDR) < 0.05 using local GSEA software and resources from MSigDB (Molecular Signature Database, 7.0, Broad Institute).

[0211] The abundance of HPV16-encoded transcripts was obtained by Bowtie2 mapping of high- quality reads (see above) aligned to the HPV16 genome (K02718.1) starting at the L2 open reading frame using the default setting followed by removal of PCR duplicates by SAM / BAM RmDuP as previously described. Reads in non-overlapping ORF regions (except E4) were collected by MultiCovBed in Galaxy with the following criteria: proper paired, MAPQ > 25, and 150 bp overlap. FPKM of HPV transcripts were calculated after normalization with sequencing depth and mapped fragment length, and used to generate heatmaps in PRIZM. RT-qPCR reactions were conducted as described with total RNA isolated by using NucleoSpin RNA II Kit (Clontech) coupled to on-column DNase I treatment and subsequently used for one-step RT-qPCR reactions. Primer pairs for HPV16 E6, E2, and cellular IVL are listed in the Key Resources Table.

[0212] Example 22. IP-Proteonncs

[0213] For identification of BRD4-L / S interactors, proliferating W12E cells were seeded without feeders at IxlO7cells per 150-mm plate in low Ca2+F-medium for 24 hours and then treated with 0.05% DMSO, 5 pM of 14, or 1 pM of 90 or JQ1, each in triplicate, for additional 24 hours before harvest. For identification of differentiation-associated BRD4-L / S interactors, proliferating W12E cells were similarly seeded in low Ca2+F-medium for 24 hours and then induced to differentiation in high Ca2+F-medium for 72 hours before compound treatment. Nuclear extracts (3 mg per sample) prepared as described were supplemented with DMSO or 10 pM of compounds during preparation to sustain the compound effect. BRD4 complexes were isolated by immunoprecipitation (IP) with purified normal IgG (2 pg), a-BRD4-L (Bethyl, 1 pg), or a-BRD4-S (2 pg) in 100 mM NaCl-containing mRIPA at 4°C overnight. The antibody / protein complex was subsequently incubated with 10 pl of Protein A-conjugated Dynabeads pre-blocked with B SA (1 mg / ml) for 90 minutes. After three 10-minute washes using the same buffer, proteins were eluted with 50 pl of 2x protein sample buffer. 40 pl of eluates were run 10 mm into a 4-15% premade gradient gel (Bio-Rad). The protein-containing gels, after cutting into smaller pieces, were sent for protein complex identification in UTSW Proteomics Core as described below.

[0214] Proteins samples were digested overnight with trypsin (Pierce) following reduction and alkylation with DTT and iodoacetamide (Sigma-Aldrich). The samples then underwent solidphase extraction cleanup with an Oasis HLB plate (Waters). The resulting samples were injected onto an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Samples were injected onto a 75 pm i.d., 75-cm long EasySpray column (Thermo Fisher) in buffer A and eluted with a gradient from 1-28% buffer B over 90 minutes. Buffer A contains 2% (v / v) ACN and 0.1% formic acid in water, and buffer B has 80% (v / v) ACN, 10% (v / v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 1.5 kV and an ion transfer tube temperature of 275°C. Mass spectrometry (MS) scans were acquired at 120,000 resolutions in the Orbitrap and up to 10 MS / MS spectra were obtained in the ion trap for each full spectrum acquired using higher- energy collisional dissociation (HCD) for ions with charges 2-7. Dynamic exclusion was set for 25 s after an ion was selected for fragmentation. Raw MS data files were analyzed using Proteome Discoverer v2.4 SP1 (Thermo Fisher), with peptide identification performed using Sequest HT searching against the human protein database from UniProt. Fragment and precursor tolerances of 10 ppm and 0.6 Da were specified, and three missed cleavages were allowed. Carbamidomethylation of Cys was set as a fixed modification, with oxidation of Met set as a variable modification. The FDR cutoff for all peptides was set at High (1%), Medium (5%), or Low (>5%). FDR in this analysis is the probability of correct peptide identification by comparing the number of peptides identified relative to the number of peptides identified from a decoy database, which is the protein database but with reversed sequences. Number of PSM (Peptide Spectrum Match, from the sum of all samples) is the number of spectra assigned to peptides that contribute to the inference of the protein. If more than one protein in a group has the same score, an equal number of PSMs, and an equal number of peptides, the protein with the longest sequence is designated as the master protein. Protein accession number and description is according to UniProt database. Coverage percentage is the percentage of amino acids in that protein's sequencethat were covered by observed peptides. Protein abundance is the sum of the peak intensities for each peptide identified for that protein and can be used to compare relative abundance of a protein between samples.

[0215] Example 23. PPI network analysis

[0216] BRD4-L / S interactors with PSM > 2 and high protein ID confidence (1% FDR) were evaluated by principal component analysis (PCA) with ClustVis (a web tool for visualizing clustering of multivariate data) for consistency in the replicates. Fold change (FC) and significance p value of protein abundance were calculated by using raw data log2 transformation and normalization, followed by missing data imputation, F-test, log2(FC), and p value determination. Venn diagrams analyzed at the Bioinformatics & Evolutionary Genomics website were used to identify overlapping significant interactors (FC > 2, / ? < 0.05) from replicated samples after PCA analysis and manually sorting only nuclear proteins (according to Human Protein Atlas and GeneCards with nuclear localization confidence > 3) following removal of ribosomal and mitochondrial proteins. Volcano plots were prepared using PRIZM. Pathway s / networks analysis was performed using Gene Ontology (GO), GSEA, and STRING webtools. DDR factors in TC- NER (transcription-coupled nucleotide excision repair), GG-NER (global genome nucleotide excision repair), and double-strand break (DSB) identified in BRD4 interactomes were analyzed together with BRD4 in STRING based on confidence of data support from experiments, coexpression, databases, gene co-occurrence, text-mining, and repeatedly occurring neighborhood with thickness of solid lines indicating the degree of confidence. K-means clustering was used to group input proteins, with dashed lines connecting proteins in the three DDR pathways. The heatmap for pS / T readers (Figure 6 A) was generated in Excel using FC normalized to that of IgG in respective proliferation and differentiation groups.

[0217] Example 24. BRD4 immunoblotting (IB) and in vitro GST pulldown

[0218] IB detection of BRD4 protein isoforms was conducted as described. Highly sensitive ECL reagents (e.g., Thermo Scientific™ SuperSignal™ West Femto Chemiluminescent Substrate) were used for detection of low-abundant proteins such as BRD4-S, 53BP1, and BARD1 in W12E cells. Direct interaction between BRD4 and BRCT domains was conducted by GST pulldown using 10 pg of immobilized GST-tagged BRCT1 / 2 domains of 53BP1 or BARD1 for respective incubation with Sf9-purified f:BRD4-L (1 pg), f:BRD4-S (0.4 pg), and fPDID (0.2 pg), or with bacteria-purified Bac-f:PDID (0.2 pg) in BC100 buffer, followed by IB detection with anti-FLAG antibody. All antibodies used for this study are listed in the Key Resources Table.

[0219] Example 25. CUT&RUN (Cleavage Under Targets and Release Using Nuclease) and re-IP

[0220] CUT&RUN was performed in W12E cells using the described protocol. Briefly, 3xl06of cells in duplicate were collected from six 100-mm plates of low Ca2+proliferative cells or eighteen 100-mm plates of high Ca2+3-day differentiated cells pretreated with DMSO or compound (1 pM) for 4 hours before harvest. Cells immobilized on Concanavalin A-coated magnetic beads were permeabilized in 300 pl of CUT&RUN wash buffer plus 0.05% digitonin and 2 mM EDTA with addition of IgG (2 pg), a-BRD4-L (Bethyl, 1 pg), or a-BRD4-S (2 pg) antibodies. Cell / nuclei retained on the beads were incubated with Protein A / G-fused micrococcal nuclease (pA / G- MNase, 700 ng / ml) at 4°C for 1 hour. Antibody-associated protein-DNA complex was released from chromatin by adding CaCh to digitonin-containing wash buffer in final 2 mM CaCh and incubated at 0°C (i.e., ice-water bath) for 30 min. An equal volume of 2x stop buffer containing spike-in yeast DNA was then added to the reaction with an additional 30-min incubation at 37°C. The released DNA was purified (see below) and used for library preparation or qPCR analysis. For subsequent BARD1 immunoprecipitation (i.e., re-IP) of the released BRD4 / DNA complex, 1 pg of normal mouse IgG or a-BARDl antibody was incubated with the BRD4 / DNA complex after 5-fold dilution in digitonin-containing wash buffer at 4°C overnight and later captured with BSA-blocked Protein G Dynabeads. After 3x washes with digitonin-containing wash buffer, the re-IP DNA was eluted from beads following incubation with 100 pl of ChIP elution buffer (1% SDS, 0.1 M NaHCCh) at room temperature for 1 hour.

[0221] Example 26. CUT&RUN library preparation, high-throughput sequencing, andqPCR analysis

[0222] DNA fragments from CUT&RUN were isolated after proteinase K treatment, phenol / chloroform extraction, and ethanol precipitation, and then used for library preparation with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) as previously described. Barcoded libraries were pooled for paired-end sequencing using HiSeq 2x150 performed by Admera Health. DNA fragments from CUT&RUN and re-IP were isolated as described for Hirt- qPCR assay in HPV replication analysis. The four primer pairs for HPV locus detection are listed in the Key Resources Table.

[0223] Example 27. Genome mapping, peak calling, and quality control for high-throughput CUT&RUN sequencing

[0224] Raw sequencing data from CUT&RUN followed by high-throughput sequencing were processed by BICF ChlP-seq Analysis Workflow using BioHPC Astrocyte web interface. In general, raw reads from fastq files were trimmed by TrimGal ore and aligned to hg38 using BWA, followed by Samtools to sort and convert the mapped reads to bam files. Duplicate reads in the resulting bam files were marked with Sambamba and subsequently removed by Samtools. Consensus peaks were compiled according to ENCODE (ENCODE Project Consortium, 2012) and annotated using ChlPseeker. For alignment to the HP VI 6 genome, raw reads were processed by FASTP in Galaxy and mapped to the HPV16 genome (K02718.1) starting at the L2 open reading frame by Bowtie2 using the default setting. PCR duplicates in the sorted bam files were removed by SAM / BAM RmDuP. Percent unique-mapped reads is defined as the ratio of deduplicated reads divided by total input reads. NRF and PBC1 / 2 for evaluating library complexity and NSC / RSC for measuring enrichment were generated by BICF ChlP-seq Analysis Workflow according to the standards set by ENCODE.

[0225] MACS2 was used for peak calling from deduplicated bam files with IgG as control using BICF ChlP-seq Analysis Workflow as previously described. FRiP (percent fraction of reads in peaks) evaluating the signal-to-noise ratio was calculated by Deeptool plotEnrichment in Galaxy using read counts in the consensus peaks after removing chrM (mitochondria) peaks. For peaks overlapping with DNase I hypersensitive sites (DHS), summits of top 5,000 peaks were used to intersect ENCODE DHS sites compiled from 125 human cell types and tissues (wgEncodeAwgDnaseMasterSites.bed) after converting their genome coordinates from hgl9 to hg38. Genomic distributions of BRD4-L / S-bound promoter, exon, intron, and intergenic regions (i.e., not found in promoter, exon, and intron regions) were analyzed by using the ChlPseeker results. PhastCons-based conservation analysis was conducted at Cistrome using top 5,000 peak summits.

[0226] Example 28. Integration of RNA-seq and CUT&RUN for direct target identification

[0227] Identification of overlapping BRD4-L / S binding peaks and RNA-seq DEGs in the Venn diagrams was conducted as described in the PPI network analysis. W12E ChlP-seq peaks of H3K27ac were obtained by intersecting hg38 non-promoter peaks from GSM5550313 and GSM5550314 using Galaxy bedtools. Signal tracks for BRD4 isoform binding to host and HPV chromatin with signal fold change of BRD4 antibody IP vs. IgG IP were generated by using BICF ChlP-seq Analysis Workflow and MACS2 bdgcmp tool in Galaxy, respectively, and were displayed using Integrative Genomics Viewer (IGV 2.4.8). Heatmaps of BRD4-binding signals enriched in W12E enhancers were generated by plotHeatmap based on computMatrix scorescalculated from BRD4 signals within + / - 1-kb regions centered at H3K27ac peaks. Since high levels (> 20%) of BRD4-L and BRD4-S are found in promoters, direct transcription targets (p < 0.05, no FC cutoff) were identified by integrating RNA-seq and CUT&RUN datasets using Cistrome-GO in Cistrome-DB with settings in the promoter mode (half decay distance = 1 kb). BRD4-L / S peaks within 15 kb relative to the transcription start site (TSS) were then overlapped with ChlP-seq transcription factor (TF) datasets available from Cistrome DB Toolkit. GIGGLE scores show the degree of overlap between TFs and BRD4-L or -S. Compound 90- or JQ1- perturbed BRD4 occupancy was similarly analyzed as above described.

[0228] Example 28. Synthetic procedures

[0229] Example 28(e). General procedure A

[0230] To a solution of compound 90 (30 mg, 77 pmol) in anhydrous DMF (2.0 mL), NaH (9.2 mg, 231 pmol) was added at 0 °C and stirred for 30 min. Halides (231 pmol) was added and then stirred at room temperature for 1 h. Water (20 mL) was added to the mixture and extracted with ethyl acetate (20 mL x 3). The organic layers were combined and washed with water and brine, and dried over anhydrous Na2SO4. Filtrated, and evaporated under reduced pressure, and then purified by pTLC to afford target compounds.

[0231] Example 28(f). General procedure B

[0232] To a solution of compound 90 (30 mg, 77 pmol) in anhydrous DMF (2.0 mL), NaH was added at r.t. and stirred for 30 min. RX-HX (X = Cl, Br, or I) (231 pmol) and KI (3.9 mg, 23.5 pmol) was added, and then stirred at 600C for 12 h. Water (20 mL) was added to the mixture and extracted with ethyl acetate (20 mL x 3). The organic layers were combined, washed with water and brine, and dried over anhydrous Na2SO4. Filtrated, evaporated under reduced pressure, and then purified by pTLC to afford desired compounds.

[0233] Example 28(g). General procedure C

[0234] To a solution of ZM0124 (15 mg, 24 pmol) or ZM0144 (15 mg, 28 pmol) in DCM (5.0 mL), TFA (0.5 mL) was added and then stirred at room temperature for 2.0 h. The solvent was removed under reduced pressure and then freeze-dried to afford ZM0138 (10 mg, 81%) and ZM0149 (10 mg, 75%).

[0235] To a solution of ZM0149 (20 mg, 0.04 mmol) in anhydrous DMF (2.0 mL), was added HATU (30.4 mg, 0.08 mmol), DIPEA (28 pL, 0.16 mmol), and amine (0.04 mmol). The mixture was stirred at room temperature for 12 h. Water was added and extracted with ethyl acetate (30 mL x 3). The combined organic layers were washed with water, brine, dried over anhydrous Na2SO4, concentrated and purified by silica chromatography to afford the desired compounds.

[0236] Example 28(h). General procedure D

[0237] To a solution of compound 20 (1.2 g, 4.92 mmol) and arylboronic acid or arylpinacoly boronate ester (2.46 mmol) in dioxane (10 mL) and H2O (2.0 mL), was added K2CO3 (0.85 g, 6.15 mmol) and Pd(dppf)C12 (0.18 g, 0.25 mmol). The mixture was stirred at 80 °C under a nitrogen atmosphere for 12 h. After cooling to room temperature, water was added and extracted with DCM (50 mL><3). The combined organic layer was washed with brine, dried over anhydrous ISfeSCU, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to afford compound 21 as a white solid with a yield of 60-70%.

[0238] To a solution of compound 21 (1.42 mmol) and 22 (0.66 g, 1.70 mmol) in dioxane (10 mL) and H2O (2.0 mL), was added K2CO3 (0.49 g, 3.55 mmol) and Pd(dppf)C12 (0.10 g, 0.14 mmol). The mixture was stirred at 80 °C under a nitrogen atmosphere for 12 h. After cooling to room temperature, water was added and extracted with DCM (50 mL><3). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to afford compound 23 as a white solid with a yield of 60-70%.

[0239] To a solution of 23 (0.71 mmol) in DCM (5.0 mL) was added TFA (1.0 mL) and stirred at r.t. overnight. The solvent was removed under reduced pressure and the residue was dissolved in MeOH (5.0 mL). NHs-MeOH (7.0 M, 3.0 mL) was added and stirred for 30 min. The solvent was removed under reduced pressure to give a crude product, which was further purified by silica chromatography to give target compounds with a yield of 50-90%.

[0240] Example 28(h). 4-(5-methoxy-l,2-dimethyl-lH-indol-3-yl)-2-(6-methoxy-l-methyl-lH-

[0242] General procedure A. White solid; 20 mg; yield 62%.JH NMR (300 MHz, CDCI3) 8 8.33 (d, J= 8.8 Hz, 1H), 7.65 (s, 1H), 7.58 (d, J= 2.4 Hz, 1H), 7.20 (d, J= 8.8 Hz, 1H), 7.05 (s, 1H), 6.90 (ddd, J= 16.4, 8.8, 2.4 Hz, 2H), 6.81 (d, J= 2.2 Hz, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.80 (s, 3H), 3.72 (s, 3H), 2.73 (s, 3H).13C NMR (75 MHz, CDCI3) 6 161.73, 156.97, 154.55, 151.00,138.16, 135.96, 132.09, 127.87, 127.16, 122.23, 119.86, 111.56, 111.01, 110.68, 109.36, 108.33,108.16, 102.09, 93.16, 56.01, 55.74, 33.17, 29.73, 11.90.

[0243] Example 28(i). l-(6-methoxy-3-(4-(5-methoxy-2-methyl-lH-indol-3-yl)thiazol-2-yl)~ lH-indol-l-yl)ethan-l-one (ZM0110-1).

[0245] General procedure A. White solid; 2.0 mg; yield 6.0%. 'H NMR (300 MHz, CDCh) 8 9.39 (s, 1H), 8.29 (d, J = 8.3 Hz, 1H), 8.14 (d, J= 2.2 Hz, 1H), 7.99 (s, 1H), 7.40 (s, 2H), 7.03 (m, 3H), 3.94 (s, 3H), 3.89 (s, 3H), 2.89 (s, 3H), 2.75 (s, 3H).

[0246] Example 28(j). l-(3-(4-(l-acetyl-5-methoxy-2-methyl-LH-indol-3-yl)thiazol-2-yl)-6- methoxy-lH-indol-l-yl)ethan-l-one (ZM0110-2).

[0248] General procedure A. White solid; 10 mg; yield 27%. 'H NMR (300 MHz, CDCI3) 6 8.30 (d, J = 8.7 Hz, 1H), 8.14 (d, J = 2.4 Hz, 1H), 7.98 (d, J= 9.6 Hz, 2H), 7.39 (d, J= 2.7 Hz, 1H), 7.35 (s, 1H), 7.06 (dd, J= 8.8, 2.4 Hz, 1H), 6.96 (dd, J= 9.1, 2.7 Hz, 1H), 3.94 (s, 3H), 3.87 (s, 3H), 2.86 (s, 3H), 2.81 (s, 3H), 2.74 (s, 3H).13C NMR (75 MHz, CDCh) 6 170.27, 168.83, 160.51, 159.09, 156.45, 149.30, 137.32, 135.88, 130.53, 130.32, 123.02, 121.72, 120.96, 117.48, 116.21, 115.95, 114.94, 113.85, 112.69, 102.81, 100.75, 55.77, 55.70, 27.57, 24.13, 15.96.

[0249] Example 28(k). 4-(2-(6-methoxy-3-(4-(5-methoxy-2-methyl-lH-indol-3-yl)thiazol-2- yl)-lH-indol-l-yl)ethyl)morpholine (ZM0111)

[0251] General procedure B. White solid; 2.0 mg; yield 5.2%. 'H NMR (300 MHz, CDCh) 68.36 (d, J= 8.7 Hz, 1H), 7.92 (s, 1H), 7.75 (s, 1H), 7.63 (d, J= 2.4 Hz, 1H), 7.23 (d, J= 8.7 Hz,1H), 7.08 (s, 1H), 6.96 - 6.90 (m, 1H), 6.88 - 6.81 (m, 2H), 4.25 (t, J= 6.7 Hz, 2H), 3.91 (s, 3H), 3.90 (s, 3H), 3.73 (t, J= 4.7 Hz, 4H), 2.82 (t, J= 6.7 Hz, 2H), 2.75 (s, 3H), 2.54 (t, J= 4.7 Hz, 4H).

[0252] Example 28(1). 2-(6-methoxy-l-(2-(piperidin-l-yl)ethyl)-lH-indol-3-yl)-4-(5-methoxy- 2-methyl-lH-indol-3-yl)thiazole (ZM0113).

[0254] General procedure B. White solid; 5.0 mg; yield 13%. 'H NMR (300 MHz, CDCh) 8 8.35 (d, J= 8.6 Hz, 1H), 7.92 (s, 1H), 7.74 (s, 2H), 7.64 (d, J= 2.5 Hz, 2H), 7.21 (d, J= 8.7 Hz, 1H), 7.07 (s, 2H), 6.95 - 6.88 (m, 3H), 6.84 (dd, J = 8.7, 2.5 Hz, 2H), 4.27 (t, J= 7.5 Hz, 2H), 3.91 (s, 5H), 3.90 (s, 5H), 2.79 (t, J= 7.2 Hz, 2H), 2.74 (s, 5H), 2.51 (s, 3H), 1.62 (q, J= 5.3 Hz, 4H), 1.51 - 1.42 (m, 1H).13C NMR (75 MHz, CDCh) 6 161.81, 156.92, 154.63, 150.89, 137.52, 134.25, 130.18, 128.11, 127.19, 122.28, 119.92, 111.81, 111.42, 110.84, 110.62, 108.98, 108.16, 102.33, 93.43, 58.19, 55.94, 55.80, 54.85, 44.32, 25.76, 24.07, 13.87.

[0255] Example 28(m). 4-(l-ethyl-5-methoxy-2-methyl-lH-indol-3-yl)-2-(l-ethyl-6-methoxy-

[0257] General procedure A. White solid; 15 mg; yield 44%. 'H NMR (300 MHz, CDCh) 6 8.34 (d, J= 8.3 Hz, 1H), 7.72 (s, 1H), 7.58 (d, J= 2.5 Hz, 1H), 7.22 (d, J= 8.8 Hz, 1H), 7.04 (s, 1H), 6.93 (dd, J= 8.8, 2.2 Hz, 1H), 6.89 - 6.82 (m, 2H), 4.22 - 4.13 (m, 4H), 3.91 (s, 3H), 3.88 (s, 3H), 2.73 (s, 3H), 1.53 (t, J= 7.2 Hz, 3H), 1.37 (t, J= 7.2 Hz, 3H).13C NMR (75 MHz, CDCh) 5 161.81, 156.82, 154.49, 151.02, 137.21, 135.07, 130.89, 127.42, 126.13, 122.29, 120.05, 111.67, 110.99, 110.51, 109.38, 108.35, 108.29, 102.18, 93.36, 56.00, 55.76, 41.28, 37.94, 15.30, 15.18, 11.64.

[0258] Example 28(n). 4-(l-(2-fluoroethyl)-5-methoxy-2-methyl-lH-indol-3-yl)-2-(l-(2- fluoroethyl)-6-methoxy-lH-indol-3-yl)thiazole (ZM0122).

[0260] General procedure A. White solid; 16 mg; yield 43%. 'H NMR (300 MHz, CDCh) 8 8.36 (d, J= 8.8 Hz, 1H), 7.74 (s, 1H), 7.59 (d, J= 2.5 Hz, 1H), 7.19 (d, J= 8.8 Hz, 1H), 7.09 (s, 1H), 6.95 (dd, J= 8.8, 2.2 Hz, 1H), 6.88 (dd, J= 8.8, 2.6 Hz, 1H), 6.83 (d, J= 2.2 Hz, 1H), 4.86 (t, J= 4.9 Hz, 1H), 4.79 (t, J= 5.3 Hz, 1H), 4.70 (t, J= 5.0 Hz, 1H), 4.63 (t, J= 5.3 Hz, 1H), 4.50 - 4.44 (m, 2H), 4.39 (m, 2H), 3.90 (s, 3H), 3.88 (s, 3H), 2.73 (s, 3H).13C NMR (75 MHz, CDCh) 6 161.57, 157.14, 154.81, 150.77, 137.61, 135.69, 131.35, 127.66, 127.04, 122.46, 120.03, 112.46,111.38, 110.79, 109.26, 109.18, 109.01, 102.34, 93.30, 83.15 (d, J = 1.9 Hz), 80.85 (d, J = 1.7 Hz), 55.97, 55.76, 46.81 (d, J = 21.9 Hz), 43.53 (d, J = 22.7 Hz), 11.82 (d, J= 1.8 Hz).

[0261] Example 28(o). tert-butyl 2-(3-(4-(l-(2-(tert-butoxy)-2-oxoethyl)-5-methoxy-2-methyl- lH-indol-3-yl)thiazol-2-yl)-6-methoxy-lH-indol-l-yl)acetate (ZM0124).

[0263] General procedure A. White solid, 19 mg, 40% yield. 'H NMR (300 MHz, CDCh) 6 8.36(d, J= 8.7 Hz, 1H), 7.68 (s, 1H), 7.59 (d, J= 2.5 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.10 (s, 1H), 6.93 (dd, J= 8.8, 2.2 Hz, 1H), 6.87 (dd, J= 8.8, 2.5 Hz, 1H), 6.74 (d, J = 2.2 Hz, 1H), 4.73 (s, 4H), 3.90-3.86 (m, 6H), 2.70 (s, 3H), 1.47-1.43 (m, 18H).13C NMR (75 MHz, CDCh) 6 167.66,167.02, 161.47, 157.18, 154.80, 150.85, 137.98, 135.67, 131.90, 127.59, 127.55, 122.49, 120.00,112.64, 111.32, 110.83, 109.18, 109.12, 102.44, 93.21, 82.98, 82.52, 55.97, 55.71, 49.01, 46.01, 28.02, 11.75.

[0264] Example 28(p). 2-(l-isopropyl-6-methoxy-lH-indol-3-yl)-4-(5-methoxy-2-methyl-lH- indol-3-yl)thiazole (ZM0125).

[0266] General procedure A. White solid; 4.0 mg; yield 12%. 'H NMR (300 MHz, CDCI3) 6 8.34 (d, J= 8.8 Hz, 1H), 7.92 (s, 1H), 7.82 (s, 1H), 7.64 (d, J= 2.3 Hz, 1H), 7.22 (d, J= 8.6 Hz, 1H), 7.07 (s, 1H), 6.97 - 6.80 (m, 3H), 4.70-4.60 (m, 1H), 3.96-3.87 (m, 6H), 2.73 (s, 3H), 1.59 (d, .7= 6.6 Hz, 6H).13C NMR (75 MHz, CDCh) 6 162.05, 156.73, 154.63, 150.79, 137.09, 134.21, 130.19, 128.17, 123.02, 122.16, 120.01, 111.72, 111.49, 110.84, 110.51, 108.99, 108.18, 102.28, 93.71, 55.91, 55.79, 47.43, 22.68, 13.85.

[0267] Example 28(q). 2-(6-methoxy-l-(methylsulfonyl)-lH-indol-3-yl)-4-(5-methoxy-2- methyl-lH-indol-3-yl)thiazole (ZM0128).

[0270] General procedure A. White solid; 8.0 mg; yield 22%. 'H NMR (300 MHz, CDCh) 6 8.47 (d, J= 8.9 Hz, 1H), 7.96 (s, 2H), 7.61 (d, J= 2.5 Hz, 1H), 7.49 (d, J= 2.3 Hz, 1H), 7.25-7.20 (m, 2H), 7.06 (dd, J= 8.8, 2.3 Hz, 1H), 6.86 (dd, J= 8.8, 2.5 Hz, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.19 (s, 3H), 2.74 (s, 3H).13C NMR (75 MHz, CDCh) 8 159.18, 158.85, 154.79, 151.79, 136.46, 134.46, 130.13, 127.92, 123.62, 123.26, 121.64, 118.03, 113.61, 111.64, 111.01, 110.17, 108.51, 102.11, 97.09, 55.91, 55.83, 40.89, 13.95.

[0271] Example 28(r). (3-(4-(l-(cyclopropanecarbonyl)-5-methoxy-2-methyl-lH-indol-3- yl)thiazol-2-yl)-6-methoxy-lH-indol-l-yl)(cyclopropyl)methanone (ZM0129-1).

[0273] General procedure A. White solid; 6.0 mg; yield 15%. 'H NMR (300 MHz, CDCh) 88.32 (d, J= 8.8 Hz, 1H), 8.27 (s, 1H), 8.11 (d, J= 2.3 Hz, 1H), 7.91 (d, J= 9.1 Hz, 1H), 7.47 (d,J = 2.6 Hz, 1H), 7.32 (s, 1H), 7.03 (dd, J= 8.8, 2.4 Hz, 1H), 6.92 (dd, J= 9.0, 2.6 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H), 2.84 (s, 3H), 2.49 - 2.34 (m, 2H), 1.49 - 1.42 (m, 2H), 1.41 - 1.35 (m, 2H), 1.23 - 1.13 (m, 4H).13C NMR (75 MHz, CDC13) 5 174.51, 172.54, 160.57, 158.91, 156.22, 149.63, 137.42, 135.78, 130.32, 129.96, 122.74, 121.74, 121.00, 117.41, 115.08, 114.87, 114.07, 113.97, 112.53, 102.76, 100.43, 55.74, 55.68, 17.95, 15.16, 14.18, 11.27, 10.18.

[0274] Example 28(s). cyclopropyl(6-methoxy-3-(4-(5-methoxy-2-methyl-LH-indol-3- yl)thiazol-2-yl)-lH-indol-l-yl)methanone (ZM0129-2).

[0276] General procedure A. White solid; 13 mg; yield 37%. 'H NMR (300 MHz, CDCI3) 8 8.36 (d, J= 8.7 Hz, 1H), 8.23 (s, 1H), 8.10 (d, J= 2.4 Hz, 1H), 8.00 (s, 1H), 7.60 (d, J= 2.5 Hz, 1H), 7.24 - 7.17 (m, 2H), 7.03 (dd, J= 8.7, 2.4 Hz, 1H), 6.85 (dd, J = 8.7, 2.5 Hz, 1H), 3.93 - 3.87 (m, 6H), 2.71 (s, 3H), 2.40 - 2.31 (m, 1H), 1.40 - 1.33 (m, 2H), 1.18 - 1.10 (m, 2H).13C NMR (75 MHz, CDCI3) 6 172.54, 159.82, 158.83, 154.74, 151.67, 137.40, 134.46, 130.17, 127.98, 122.54, 121.87, 121.17, 117.73, 113.85, 111.56, 111.00, 110.09, 108.52, 102.15, 100.41, 55.92, 55.67, 14.14, 13.83, 10.12.

[0277] Example 28(t). 2-(6-methoxy-l-(prop-2-yn-l-yl)-lH-indol-3-yl)-4-(5-methoxy-2-

[0279] General procedure A. White solid; 14 mg; yield 39%. 'H NMR (300 MHz, CDCI3) 6 8.35 (d, J= 8.5 Hz, 1H), 7.79 (s, 1H), 7.58 (d, J= 2.4 Hz, 1H), 7.29 (d, J= 8.8 Hz, 1H), 7.09 (s, 1H), 6.97 - 6.88 (m, 3H), 4.87 (t, J= 2.5 Hz, 4H), 3.91 (s, 3H), 3.88 (s, 3H), 2.77 (s, 3H), 2.47 (t, J = 2.5 Hz, 1H), 2.28 (t, J= 2.5 Hz, 1H).13C NMR (75 MHz, CDCI3) 8 161.55, 157.15, 154.89, 150.70, 137.22, 135.14, 131.11, 127.65, 126.19, 122.48, 120.16, 112.44, 111.45, 111.09, 109.53, 109.28, 109.25, 102.41, 93.43, 78.20, 76.93, 74.40, 72.40, 55.98, 55.75, 36.19, 32.70, 11.69.

[0280] Example 28(u). 4-(3-(4-(l-(3-cyanopropyl)-5-methoxy-2-methyl-lH-indol-3-yl)thiazol- 2-yl)-6-methoxy-lH-indol-l -yl)butanenitrile (ZMO 143).

[0282] General procedure A. White solid; 11 mg; yield 27%. 'H NMR (300 MHz, CDCh) 8 8.33 (d, J= 8.7 Hz, 1H), 7.68 (s, 1H), 7.56 (d, J= 2.4 Hz, 1H), 7.22 (s, 1H), 7.10 (s, 1H), 6.98 - 6.86 (m, 3H), 4.37 - 4.25 (m, 4H), 3.91 (s, 3H), 3.88 (s, 3H), 2.75 (s, 3H), 2.40 - 2.30 (m, 4H), 2.29 - 2.12 (m, 4H).13C NMR (75 MHz, CDCh) 6 161.48, 157.29, 154.81, 150.67, 137.42, 135.02, 131.24, 127.52, 126.20, 122.49, 119.87, 118.96, 118.65, 112.54, 111.49, 111.17, 109.38, 109.30, 102.37, 93.04, 55.97, 55.76, 44.47, 41.46, 26.10, 25.97, 14.75, 14.70, 11.86.

[0283] Example 28(v). tert-butyl 4-(6-methoxy-3-(4-(5-methoxy-2-methyl-lH-indol-3- yl)thiazol-2-yl)-lH-indol-l-yl)butanoate (ZM0144).

[0285] General procedure A. White solid; 16 mg; yield 39%. 'H NMR (300 MHz, CDCh) 6 8.36 (d, J= 9.3 Hz, 1H), 7.94 (s, 1H), 7.67 (s, 1H), 7.64 (d, J= 2.6 Hz, 1H), 7.20 (d, J= 92 Hz, 1H), 7.06 (s, 1H), 6.93 (dd, J = 8.7, 2.3 Hz, 1H), 6.89 - 6.87 (m, 1H), 6.83 (dd, J= 8.7, 2.5 Hz, 1H), 4.19 (t, J= 6.8 Hz, 2H), 3.92 - 3.87 (m, 6H), 2.72 (s, 3H), 2.30 - 2.23 (m, 2H), 2.21 - 2.11 (m, 2H), 1.46 (s, 9H).13C NMR (75 MHz, CDCh) 8 172.04, 161.76, 156.97, 154.63, 150.91, 137.51, 134.28, 130.19, 128.11, 126.87, 122.33, 119.99, 111.82, 111.43, 110.86, 110.68, 108.93, 108.19, 102.30, 93.52, 80.80, 77.45, 77.23, 77.02, 76.60, 55.95, 55.76, 45.63, 32.14, 28.13, 25.24, 13.88.

[0286] Example 28(w). 2-(6-methoxy-3-(4-(5-methoxy-2-methyl-lH-indol-3-yl)thiazol-2-yl)- IH-indol-l -y I) acetonitrile (ZMO 146).

[0288] General procedure A. White solid, 13 mg, yield 39%. 'H NMR (300 MHz, CDCh) 6 8.38 (d, J= 8.8 Hz, 1H), 7.96 (s, 1H), 7.64 - 7.59 (m, 2H), 7.21 (d, J= 8.9 Hz, 1H), 7.11 (s, 1H), 6.98(dd, J= 8.8, 2.2 Hz, 1H), 6.87 - 6.80 (m, 2H), 4.95 (s, 2H), 3.93 - 3.88 (m, 6H), 2.70 (s, 3H).13C NMR (75 MHz, CDCh) 6 160.52, 157.85, 154.72, 151.27, 137.23, 134.40, 130.18, 128.02, 125.50, 122.96, 120.12, 114.26, 113.79, 111.81, 111.49, 110.95, 109.09, 108.72, 102.29, 92.89, 55.97, 55.80, 34.51, 13.89.

[0289] Example 28(u). 4-(l-(but-l-yn-l-yl)-5-methoxy-2-methyl-lH-indol-3-yl)-2-(l,2-di(but-

[0291] General procedure A. White solid; 4.0 mg, yield 10%. 'H NMR (300 MHz, CDCh) 6 8.43 (d, J = 8.7 Hz, 1H), 7.54 (s, 1H), 7.39 (d, J = 2.5 Hz, 1H), 7.19 (d, J = 9.0 Hz, 1H), 6.95 - 6.88 (m, 2H), 6.77 (dd, J= 8.8, 2.5 Hz, 1H), 4.96 - 4.89 (m, 1H), 4.86 - 4.80 (m, 3H), 4.72 (q, J = 2.4 Hz, 2H), 3.90 (s, 3H), 3.43 (s, 3H), 2.56 (s, 3H), 1.95 (t, J= 3.0 Hz, 3H), 1.87 (t, J= 2.4 Hz, 3H), 1.74 (t, J = 2.4 Hz, 3H).13C NMR (75 MHz, CDCh) 8 206.64, 206.34, 157.51, 155.75, 153.97, 137.55, 135.67, 131.40, 131.00, 127.38, 123.66, 120.16, 112.25, 111.68, 111.09, 109.38, 106.31, 103.09, 100.86, 93.61, 91.14, 82.91, 79.95, 73.67, 71.88, 55.76, 55.30, 36.86, 32.81, 16.46, 11.74, 3.60, 3.51.

[0292] Example 28(x). 4-(l-(but-l-yn-l-yl)-5-methoxy-2-methyl-lH-indol-3-yl)-2-(l-(but-l- yn-l-yl)-6-methoxy-lH-indol-3-yl)thiazole (ZM0148-2).

[0294] General procedure A. White solid; 11 mg, yield 29%. 'H NMR (300 MHz, CDCh) 88.35 (d, J= 8.6 Hz, 1H), 7.81 (s, 1H), 7.58 (d, J= 2.5 Hz, 1H), 7.29 (d, J= 8.8 Hz, 1H), 7.07 (s,1H), 6.96 - 6.87 (m, 3H), 4.84 - 4.79 (m, 3H), 3.91 (s, 3H), 3.88 (s, 3H), 2.77 (s, 3H), 1.87 (t, J= 2.4 Hz, 3H), 1.76 (t, = 2.4 Hz, 3H).13C NMR (75 MHz, CDC13) 5 161.66, 156.96, 154.72, 150.82, 137.18, 135.25, 131.14, 127.51, 126.35, 122.37, 120.16, 112.02, 111.24, 110.87, 109.63, 108.89, 108.85, 102.27, 93.50, 82.29, 80.05, 73.67, 72.40, 55.96, 55.74, 36.58, 33.02, 11.69, 3.60, 3.48.

[0295] Example 28(y). 4-(l-(cyclopropylmethyl)-5-methoxy-2-methyl-lH-indol-3-yl)-2-(l- (cyclopropylmethyl)-6-methoxy-lH-indol-3-yl)thiazole (ZM0190-1).

[0297] General procedure A. White solid; 6.0 mg, yield 15.7%. 'H NMR (300 MHz, CDCI3) 8 8.34 (d, J= 8.7 Hz, 1H), 7.83 (s, 1H), 7.59 (d, J= 2.5 Hz, 1H), 7.22 (s, 1H), 7.06 (s, 1H), 6.93 (dd, J= 8.8, 2.3 Hz, 1H), 6.88 - 6.83 (m, 2H), 4.06 (d, J= 6.3 Hz, 2H), 3.98 (d, J= 6.8 Hz, 2H), 3.91 (s, 3H), 3.89 (s, 3H), 2.74 (s, 3H), 1.39 - 1.28 (m, 2H), 0.73 - 0.66 (m, 2H), 0.60 - 0.52 (m, 2H), 0.45 - 0.36 (m, 4H).13C NMR (75 MHz, CDCI3) 6 161.83, 156.82, 154.45, 151.04, 137.62, 135.31, 131.68, 127.34, 126.53, 122.21, 119.96, 111.62, 111.04, 110.53, 109.82, 108.47, 108.42, 102.02, 93.51, 55.96, 55.75, 50.98, 47.23, 12.08, 11.60, 10.91, 4.26, 3.98.

[0298] Example 28(z). 2-(l-(cyclopropylmethyl)-6-methoxy-lH-indol-3-yl)-4-(5-methoxy-2- methyl-lH-indol-3-yl)thiazole (ZM0190-2).

[0300] General procedure A. White solid; 12 mg, yield 35%. 'H NMR (300 MHz, CDCI3) 6 8.36 (dd, J= 8.8, 0.5 Hz, 1H), 7.98 - 7.88 (m, 1H), 7.81 (s, 1H), 7.65 (d, J= 2.5 Hz, 1H), 7.21 (dd, J= 8.7, 0.6 Hz, 1H), 7.07 (s, 1H), 6.93 (dd, J= 8.7, 2.3 Hz, 1H), 6.87 - 6.81 (m, 2H), 3.98 (d, J= 6.8 Hz, 2H), 3.92 - 3.88 (m, 6H), 2.73 (s, 3H), 1.37 - 1.31 (m, 1H), 0.73 - 0.66 (m, 2H), 0.46 - 0.40 (m, 2H).13C NMR (75 MHz, CDCI3) 5 161.91, 156.83, 154.62, 150.86, 137.63, 134.20, 130.18, 128.13, 126.56, 122.20, 119.96, 111.59, 111.47, 110.83, 110.56, 109.00, 108.13, 102.30, 93.51, 55.91, 55.76, 50.98, 13.86, 10.92, 4.26.

[0301] Example 28(aa). 4-(l-benzyl-5-methoxy-2-methyl-lH-indol-3-yl)-2-(l-benzyl-6- methoxy-lH-indol-3-yl) thiazole (ZM0191-1).

[0303] General procedure A. White solid; 15 mg, yield 34%. 'H NMR (300 MHz, CDCh) 8 8.36 (d, J= 8.8 Hz, 1H), 7.72 (s, 1H), 7.64 (d, J= 2.5 Hz, 1H), 7.40 - 7.25 (m, 5H), 7.23 - 7.11 (m, 4H), 7.09 (s, 1H), 7.06 - 7.01 (m, 2H), 6.92 (dd, J= 8.8, 2.2 Hz, 1H), 6.82 (dd, J = 8.8, 2.5 Hz, 1H), 6.77 (d, J= 2.2 Hz, 1H), 5.33 (d, J= 14.9 Hz, 4H), 3.87 (s, 3H), 3.82 (s, 3H), 2.67 (s, 3H).13C NMR (75 MHz, CDCh) 6 161.72, 156.99, 154.70, 151.00, 137.84, 137.66, 136.55, 135.73, 131.82, 128.93, 128.80, 127.93, 127.43, 127.33, 127.25, 126.96, 126.06, 122.28, 120.09, 112.20, 111.33, 110.75, 109.87, 108.83, 108.80, 102.23, 93.82, 55.93, 55.67, 50.41, 46.72, 11.92.

[0304] Example 28(bb). 2-(l,2-dibenzyl-6-methoxy-lH-indol-3-yl)-4-(5-methoxy-2-methyl- lH-indol-3-yl)thiazole (ZMO 191-2).

[0307] General procedure A. White solid; 7.0 mg, yield 18%. 'H NMR (300 MHz, CDCh) 6 8.40 (d, J= 8.8 Hz, 1H), 7.70 (s, 1H), 7.44 - 7.33 (m, 4H), 7.27 - 7.22 (m, 2H), 7.16 - 7.08 (m, 3H), 7.01 (dd, J= 8.8, 2.3 Hz, 1H), 6.86 (ddd, J= 14.3, 8.8, 2.4 Hz, 4H), 6.48 (s, 1H), 6.44 (d, J = 2.6 Hz, 1H), 5.36 (s, 2H), 4.20 (d, J= 13.2 Hz, 1H), 3.86 (s, 3H), 3.72 (s, 3H), 3.23 (d, J= 13.3 Hz, 1H), 2.56 (s, 3H).13C NMR (75 MHz, CDCh) 8 181.99, 163.03, 157.31, 157.17, 153.39, 148.97, 142.13, 137.90, 136.43, 129.96, 128.99, 128.01, 127.71, 127.43, 126.92, 126.69, 122.21, 120.32, 120.02, 113.66, 111.99, 111.82, 111.06, 110.91, 94.01, 66.12, 55.72, 55.62, 50.48, 41.67, 17.81.

[0308] Example 28(cc). 2-(6-methoxy-l-(methoxymethyl)-lH-indol-3-yl)-4-(5-methoxy-2- methyl-lH-indol-3-yl)thiazole (ZM0195).

[0310] General procedure A. White solid; 3.0 mg, yield 9%. 'HNMR (300 MHz, CDCh) 8 9.38 (s, 1H), 8.31 (d, J= 8.6 Hz, 1H), 7.80 (s, 1H), 7.45 (d, J= 2.5 Hz, 1H), 7.29 (s, 2H), 7.05 - 6.97 (m, 3H), 5.50 (s, 2H), 3.93 (s, 3H), 3.89 (s, 3H), 3.35 (s, 3H), 2.90 (s, 3H).13C NMR (75 MHz, CDCh) 6 158.40, 157.42, 157.34, 148.14, 137.76, 130.58, 127.09, 122.06, 120.18, 116.47, 113.04, 112.80, 112.34, 111.75, 103.53, 94.07, 77.95, 56.13, 55.74, 14.10.

[0311] Example 28(dd). 2-(6-methoxy-l-(3-methoxypropyl)-lH-indol-3-yl)-4-(5-methoxy-l- (3-methoxypropyl)-2-methyl-lH-indol-3-yl)thiazole (ZMO 196).

[0313] General procedure A. White solid; 16 mg, yield 39%. 'H NMR (300 MHz, CDCh) 6 8.34 (d, J= 8.7 Hz, 1H), 7.69 (s, 1H), 7.59 (d, J= 2.5 Hz, 1H), 7.28 - 7.23 (m, 1H), 7.05 (s, 1H), 6.94 - 6.84 (m, 3H), 4.25 (t, J= 6.7 Hz, 4H), 3.91 - 3.87 (m, 6H), 3.37 - 3.31 (m, 10H), 2.73 (s, 3H), 2.17 - 1.99 (m, 4H).13C NMR (75 MHz, CDCh) 6 161.78, 156.89, 154.51, 151.06, 137.63, 135.56, 131.53, 127.30, 127.05, 122.24, 119.90, 111.76, 111.03, 110.64, 109.70, 108.39, 102.05, 93.39, 69.10, 68.76, 58.75, 58.61, 55.96, 55.72, 43.09, 40.01, 30.30, 30.09, 11.68.

[0314] Example 28(ee). tert-butyl 6-(3-(4-(l-(6-(tert-butoxy)-6-oxohexyl)-5-methoxy-2-methyl-

[0316] General procedure A. White solid; 20 mg, yield 36%. 'H NMR (300 MHz, CDCh) 68.36 (d, J= 8.8 Hz, 1H), 7.71 (s, 1H), 7.60 (d, J= 2.5 Hz, 1H), 7.22 (d, J= 8.8 Hz, 1H), 7.07 (s,1H), 6.96 - 6.84 (m, 3H), 4.14 (t, J= 7.3 Hz, 4H), 3.94 - 3.88 (m, 6H), 2.74 (s, 3H), 2.27 - 2.21 (m, 4H), 1.98 - 1.89 (m, 2H), 1.87 - 1.75 (m, 2H), 1.73 - 1.58 (m, 8H), 1.46 - 1.43 (m, 18H).13C NMR (75 MHz, CDC13) 5 172.91, 172.83, 161.74, 156.83, 154.46, 151.01, 137.44, 135.28, 131.33, 127.32, 126.81, 122.30, 120.00, 111.64, 111.00, 110.46, 109.56, 108.40, 108.33, 102.11, 93.48, 80.19, 80.15, 55.98, 55.76, 46.48, 43.24, 35.33, 35.26, 29.69, 28.40, 28.12, 28.09, 26.53, 26.40, 24.83, 24.64, 11.87.

[0317] Example 28(ff). tert-butyl 6-(6-methoxy-3-(4-(5-methoxy-2-methyl-lH-indol-3- yl)thiazol-2-yl)-lH-indol-l-yl)hexanoate (ZM0219-2).

[0319] General procedure A. White solid; 13 mg, yield 30%. 'H NMR (300 MHz, CDCI3) 8 8.37 (d, J= 8.8 Hz, 1H), 7.96 (s, 1H), 7.71 (s, 1H), 7.66 (d, J= 2.5 Hz, 1H), 7.23 (d, J= 8.7 Hz, 1H), 7.09 (s, 1H), 6.95 (dd, J= 8.8, 2.2 Hz, 1H), 6.86 (d, J= 9.1 Hz, 2H), 4.14 (t, J= 7.1 Hz, 2H), 3.92 (d, J = 2.8 Hz, 6H), 2.75 (s, 3H), 2.23 (t, J= 7.3 Hz, 2H), 1.99 - 1.88 (m, 2H), 1.75 - 1.59 (m, 4H), 1.43 (s, 9H).13C NMR (75 MHz, CDCI3) 6 172.85, 161.83, 156.85, 154.62, 150.86, 137.46, 134.24, 130.19, 128.12, 126.87, 122.29, 120.00, 111.60, 111.43, 110.84, 110.49, 108.97, 108.14, 102.31, 93.49, 80.21, 55.93, 55.76, 46.48, 35.26, 29.68, 28.09, 26.39, 24.64, 13.87.

[0320] Example 28(gg). tert-butyl 8-(3-(4-(l-(8-(tert-butoxy)-8-oxooctyl)-5-methoxy-2-methyl-

[0322] General procedure A. White solid; 22 mg, yield 36%. 'H NMR (300 MHz, CDCI3) 6 8.36 (d, J= 8.7 Hz, 1H), 7.71 (s, 1H), 7.60 (d, J= 2.5 Hz, 1H), 7.22 (d, J= 8.8 Hz, 1H), 7.07 (s, 1H), 6.97 - 6.84 (m, 3H), 4.12 (t, J= 7.3 Hz, 4H), 3.95 - 3.88 (m, 6H), 2.74 (s, 3H), 2.25 - 2.17 (m, 4H), 1.97 - 1.86 (m, 2H), 1.84 - 1.75 (m, 2H), 1.72 - 1.50 (m, 8H), 1.48 - 1.44 (m, 18H), 1.40 - 1.35 (m, 8H).13C NMR (75 MHz, CDCI3) 6 173.20, 173.16, 161.79, 156.79, 154.44, 151.05,137.46, 135.33, 131.36, 127.30, 126.86, 122.26, 120.00, 111.56, 110.96, 110.39, 109.60, 108.35, 108.26, 102.07, 93.54, 79.98, 55.97, 55.77, 46.68, 43.41, 35.52, 35.48, 30.26, 29.89, 29.69, 29.11, 29.00, 28.95, 28.91, 28.12, 26.97, 26.82, 25.01, 24.97, 11.87.

[0323] Example 28(hh). tert-butyl 8-(6-methoxy-3-(4-(5-methoxy-2-methyl-lH-indol-3- yl)thiazol-2-yl)-lH-indol-l-yl)octanoate (ZM0220-2).

[0325] General procedure A. White solid; 15 mg, yield 33%. 'H NMR (300 MHz, CDCh) 8 8.35 (d, J= 8.7 Hz, 1H), 7.98 (s, 1H), 7.72 - 7.59 (m, 2H), 7.20 (d, J= 8.7 Hz, 1H), 7.06 (s, 1H), 6.92 (dd, J= 8.8, 2.2 Hz, 1H), 6.83 (dt, J= 4.7, 2.5 Hz, 2H), 4.10 (d, J= 7.2 Hz, 2H), 3.93 - 3.87 (m, 6H), 2.71 (s, 3H), 2.19 (t, J= 7.4 Hz, 2H), 1.91 - 1.82 (m, 2H), 1.62 - 1.54 (m, 2H), 1.40 - 1.44 (m, 9H), 1.37 - 1.29 (m, 6H).13C NMR (75 MHz, CDCh) 6 173.19, 161.89, 156.81, 154.60, 150.87, 137.48, 134.28, 130.20, 128.11, 126.92, 122.24, 119.99, 111.51, 111.41, 110.86, 110.43, 108.93, 108.12, 102.30, 93.55, 80.01, 55.93, 55.77, 46.68, 35.49, 29.89, 28.95, 28.92, 28.12, 26.81, 24.97, 13.84.

[0326] Example 28(ii). tert-butyl 4-(3-(4-(l-(4-(tert-butoxy)-4-oxobutyl)-5-methoxy-2-methyl-

[0328] General procedure A. White solid; 8.0 mg, yield 15%. 'H NMR (300 MHz, CDCh) 5 8.35 (d, J= 8.7 Hz, 1H), 7.68 (s, 1H), 7.58 (d, J= 2.5 Hz, 1H), 7.23 (s, 1H), 7.06 (s, 1H), 6.95 - 6.84 (m, 3H), 4.25 - 4.14 (m, 4H), 3.92 - 3.87 (m, 6H), 2.73 (s, 3H), 2.34 - 2.25 (m, 4H), 2.22 - 2.14 (m, 2H), 2.10 - 2.02 (m, 2H), 1.48 - 1.43 (m, 18H).13C NMR (75 MHz, CDCh) 8 172.21, 172.01, 161.71, 156.96, 154.55, 150.99, 137.50, 135.32, 131.44, 127.35, 126.80, 122.34, 119.99, 111.86, 111.12, 110.65, 109.64, 108.55, 102.11, 93.50, 80.78, 80.67, 55.98, 55.75, 45.63, 42.38, 32.29, 32.14, 29.69, 28.13, 25.35, 25.23, 11.83.

[0329] Example 28(jj). 2-(3-(4-(l-(carboxymethyl)-5-methoxy-2-methyl-lH-indol-3-yl)tliiazol- 2-yl)-6-methoxy-lH-indol-l-yl)acetic acid (ZM0138).

[0331] General procedure C. Yellow solid; 10 mg; yield 81%. 'H NMR (300 MHz, MeOD) 5 8.00 (d, J= 8.7 Hz, 1H), 7.91 (s, 1H), 7.36 - 7.19 (m, 3H), 6.97-6.94 (m, 1H), 6.92 (dd, J= 8.7, 2.3 Hz, 1H), 6.86 (dd, J= 8.7, 2.5 Hz, 1H), 5.01 (s, 2H), 4.93 (s, 2H), 3.86 (s, 3H), 3.82 (s, 3H), 2.53 (s, 3H).13C NMR (75 MHz, MeOD) 5 172.44, 171.79, 165.16, 159.34, 156.71, 148.15,140.23, 138.59, 133.57, 131.48, 128.88, 122.29, 120.51, 113.24, 112.75, 111.06, 110.22, 107.00,102.96, 95.28, 56.64, 56.44, 50.15, 49.87, 49.58, 49.30, 49.02, 48.96, 48.73, 48.45, 45.76, 11.81.

[0332] Example 28(kk). 4-(6-methoxy-3-(4-(5-methoxy-2-methyl-lH-indol-3-yl)thiazol-2-yl)- lH-indol-l-yl)butanoic acid (ZM0149).

[0334] General procedure C. Yellow solid; 10 mg, yield 75%. 'H NMR (300 MHz, MeOD) 5 7.97 (d, J= 8.8 Hz, 1H), 7.89 (s, 1H), 7.33 - 7.18 (m, 2H), 7.15 (d, J= 8.7 Hz, 1H), 7.03 (d, J= 2.3 Hz, 1H), 6.85 (dd, J= 8.9, 2.3 Hz, 1H), 6.70 (dd, J= 8.7, 2.5 Hz, 1H), 4.20 (t, J= 7.2 Hz, 2H), 3.80 (s, 3H), 3.75 (s, 3H), 2.52 (s, 3H), 2.27 (t, J = 6.9 Hz, 2H), 2.10 - 2.01 (m, 2H).13C NMR (75 MHz, MeOD) 5 176.70, 165.33, 159.27, 156.25, 148.69, 139.71, 137.33, 132.36, 130.49, 129.40, 122.34, 120.67, 113.39, 112.83, 112.72, 112.55, 109.66, 106.14, 102.58, 95.38, 56.65,56.46, 47.12, 31.73, 26.60, 13.37.

[0335] Example 28(11). 4-(6-methoxy-3-(4-(5-methoxy-2-methyl-lH-indol-3-yl)thiazol-2-yl)- lH-indol-l-yl)-l-morpholinobutan-l-one (ZM0283).

[0337] General procedure C. White solid; 15 mg, yield 69%. 'H NMR (300 MHz, CDCh) 88.36 (d, J= 8.7 Hz, 1H), 7.94 (s, 1H), 7.69 - 7.60 (m, 2H), 7.22 (d, J= 8.7 Hz, 1H), 7.08 (s, 1H),6.95 - 6.82 (m, 3H), 4.27 (t, J = 6.2 Hz, 2H), 3.90 (s, 6H), 3.70 - 3.60 (m, 4H), 3.50 (t, J = 4.8 Hz, 2H), 3.23 (t, J= 4.8 Hz, 2H), 2.74 (s, 3H), 2.29 - 2.14 (m, 4H).13C NMR (75 MHz, CDCh) 5 170.26, 161.73, 157.00, 154.68, 150.97, 137.73, 134.28, 130.22, 128.13, 126.81, 122.33, 119.92, 111.92, 111.43, 110.87, 110.68, 108.94, 108.23, 102.40, 93.73, 66.83, 66.41, 55.98, 55.79, 45.65, 45.47, 41.99, 28.73, 24.95, 13.91.

[0338] Example 28(mm). 4-(6-methoxy-3-(4-(5-methoxy-2-methyl-lH-indol-3-yl)thiazol-2-yl)- IH-indol-l -yl)-l-(4-methylpiperazin-l -yl)butan-l -one (ZM084).

[0340] General procedure C. White solid; 16 mg, yield 72%. 'H NMR (300 MHz, CDCh) 8 8.36 (d, J= 8.6 Hz, 1H), 7.98 (s, 1H), 7.68 - 7.61 (m, 2H), 7.21 (d, J= 8.7 Hz, 1H), 7.07 (s, 1H), 6.94 - 6.88 (m, 2H), 6.84 (dd, J= 8.7, 2.5 Hz, 1H), 4.27 (t, J= 5.2 Hz, 2H), 3.90 (s, 6H), 3.65 (t, J = 5.2 Hz, 2H), 3.27 (t, J= 5.1 Hz, 2H), 2.73 (s, 3H), 2.36 (t, J= 5.1 Hz, 2H), 2.28 - 2.19 (m, 9H).13C NMR (75 MHz, CDCh) 6 169.93, 161.74, 156.96, 154.64, 150.96, 137.68, 134.25, 130.19, 128.09, 126.85, 122.28, 119.88, 111.83, 111.40, 110.85, 110.76, 108.93, 108.13, 102.35, 93.62, 55.95, 55.75, 54.78, 54.56, 45.80, 45.50, 45.05, 41.49, 28.82, 24.94, 13.88.

[0341] Example 28(nn). 4-(6-methoxy-3-(4-(5-methoxy-2-methyl-lH-indol-3-yl)thiazol-2-yl)- IH-indol-l -yl)-N,N-dimethylbutananude (ZM0296).

[0343] General procedure C. White solid; 12 mg, yield 60%. 'H NMR (300 MHz, CDCh) 5 8.36 (d, J= 8.5 Hz, 1H), 7.92 (s, 1H), 7.67 (s, 1H), 7.64 (d, J= 2.5 Hz, 1H), 7.22 (d, J= 8.7 Hz, 1H), 7.07 (s, 1H), 6.94 - 6.89 (m, 2H), 6.84 (dd, J = 8.7, 2.5 Hz, 1H), 4.31 - 4.23 (m, 2H), 3.90 (s, 6H), 2.97 (s, 3H), 2.82 (s, 3H), 2.28 - 2.21 (m, 4H).13C NMR (75 MHz, CDCh) 8 171.64, 161.84, 156.98, 154.67, 150.93, 137.73, 134.26, 130.21, 128.14, 126.87, 122.28, 119.91, 111.82, 111.44, 110.85, 108.98, 108.18, 102.39, 93.49, 55.97, 55.76, 45.62, 37.05, 35.45, 29.11, 25.10, 13.92.

[0344] Example 28(oo). tert-butyl 4-(4-(6-methoxy-3-(4-(5-methoxy-2-methyl-lH-indol-3- yl)thiazol-2-yl)-lH-indol-l-yl)butanoyl)piperazine-l-carboxylate (ZM0297).

[0346] General procedure C. White solid; 18 mg, yield 70%. 'H NMR (300 MHz, CDCI3) 68.36 (d, J= 8.7 Hz, 1H), 7.96 (s, 1H), 7.66 (s, 1H), 7.63 (d, J= 2.5 Hz, 1H), 7.22 (d, J= 8.7 Hz, 1H), 7.07 (s, 1H), 6.95 - 6.88 (m, 2H), 6.84 (dd, J= 8.7, 2.5 Hz, 1H), 4.32 - 4.22 (m, 2H), 3.92 - 3.87(m, 6H), 3.60 (t, J= 5.3 Hz, 2H), 3.44 - 3.39 (m, 2H), 3.28 - 3.19 (m, 4H), 2.74 (s, 3H), 2.28 - 2.18 (m, 4H), 1.44 (s, 9H).13C NMR (75 MHz, CDC13) 6 170.23, 161.71, 156.97, 154.67, 154.52,150.98, 137.74, 134.28, 130.22, 128.11, 126.78, 122.33, 119.91, 111.93, 111.42, 110.86, 110.59,108.93, 108.21, 102.37, 93.75, 80.31, 55.97, 55.77, 45.45, 45.04, 41.45, 28.97, 28.37, 25.01, 13.90.

[0347] Example 29 (pp). 4-(5-methoxy-2-metbyl-lH-indol-3-yl)-2-phenylthiazole (ZM0493).General procedure D. Yellow solid; 213 mg, yield 93%. 'H NMR (300 MHz, CDCI3) 6 8.10 - 7.95 (m, 3H), 7.56 (d, J= 2.5 Hz, 1H), 7.43 - 7.31 (m, 3H), 7.10 (s, 1H), 6.96 (d, J= 8.7 Hz, 1H), 6.77 (dd, J= 8.8, 2.5 Hz, 1H), 3.85 (s, 3H), 2.39 (s, 3H).13C NMR (75 MHz, CDCI3) 6 166.91, 154.63, 152.30, 135.13, 134.09, 130.39, 129.94, 129.06, 127.90, 126.56, 111.68, 111.33, 111.15, 108.20, 102.38, 56.08, 13.48.

[0349] Example 30 (qq). 4-(5-methoxy-2-methyl-lH-indol-3-yl)-2-(4-methoxyphenyl)thiazole (ZM0497).General procedure D. Yellow solid; 147.4 mg, yield 59%. 'HNMR (300 MHz, CDCI3) 6 8.04 - 7.95 (m, 3H), 7.55 (d, J= 2.5 Hz, 1H), 7.14 - 7.08 (m, 2H), 6.97 - 6.91 (m, 2H), 6.81 (dd, J= 8.8, 2.5 Hz, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 2.58 (s, 3H).13C NMR (75 MHz, CDCI3) 8 166.63, 160.99, 154.62, 151.88, 134.77, 130.28, 127.96, 127.13, 114.28, 111.15, 111.03, 110.70, 108.47, 102.36, 56.01, 55.44, 13.65.

[0350] Example 31 (rr). 4-(5-methoxy-2-methyl-lH-indol-3-yl)-2-(pyridin-4-yl)thiazole(ZM0506).General procedure D. Yellow solid; 164.4 mg, yield 72%. 'HNMR (300 MHz, CDCh+MeOD) 5 10.06 (s, 1H), 8.65 (d, J = 5.7 Hz, 2H), 8.04 - 7.91 (m, 2H), 7.52 (d, J= 2.5 Hz, 1H), 7.41 (s, 1H), 7.27 (d, J= 8.7 Hz, 1H), 6.83 (dd, J= 8.7, 2.5 Hz, 1H), 3.91 (s, 3H), 2.70 (s, 3H).13C NMR (75 MHz, CDCh+MeOD) 5 163.14, 154.31, 153.74, 149.80, 141.46, 135.67, 130.63, 127.51, 1 20. 60 , 1 1 3 .20 , 1 1 1 .26 , 1 1 0.73 , 1 06. 99 , 1 0 1 .98 , 5 5 .95 , 1 3 . 30 , 1 3 .25 .

[0351] Example 32 (ss). 4-(5-methoxy-2-methyl-lH-indol-3-yl)-2-(6-methoxypyridin-3- yl)thiazole (ZM0519).General procedure D. Yellow solid; 161.5 mg, yield 64.7%.XH NMR (300 MHz, CDCh+MeOD) 5 8.80 (d, J = 2.4 Hz, 1H), 8.20 (dd, J = 8.2, 2.5 Hz, 1H), 7.52 (d, J= 2.5 Hz, 1H), 7.24 - 7.13 (m, 2H), 6.86 - 6.77 (m, 2H), 3.98 (s, 3H), 3.88 (s, 3H), 2.61 (s, 3H).13C NMR (75 MHz, CDCh+MeOD) 5 164.93, 163.54, 154.23, 152.50, 144.87, 136.87, 135.34, 130.60, 127.65, 124.10, 111.16, 111.00, 1 10.74, 1 10.68, 107.35, 102.1 1, 55.92, 53.77, 13.16.

[0353] Example 33. Anti-tumor and anti-metastasis experiments of phosph-BRD4 inhibitors were carried out as shown in Figure 8. (A) 14 suppresses mammary tumor development in syngeneic mouse Py8119 and xenograft human PDX BCM-3887 models with no overt toxicity. Mouse Py8119 cells (IxlO5) and a 2-mm BCM-3887 PDX tumor fragment were implanted into each side of the fourth mammary fat pads in female C57BL / 6J and SCID / NSG mice, respectively (n = mice number with both sides injected). When tumors reached -100 mm3, cpd 14 (50 mg / kg)or DMSO only was administered via daily intraperitoneal (IP) injection since 14 has excellent bioavailability (F= 78%). Tumor volume and mouse weight were monitored twice a week for 27 days for Py8119 and 42 days for BCM-3887 PDX. Compound efficacy was determined by differences in tumor weights between 14- vs. solvent-treated mice. Body weight (mean + s.e.m.) was plotted against treatment time. Significance calculated by unpaired two-tailed Student’ s t-test using GraphPad Prism. (B) 14 and 90 suppress breast cancer brain metastasis (BrM) better than JQ1. (left) Brain BLI signals from mice treated with 14, 90, JQ1, or vehicle control with representative images of dissected mouse brains, (right) Brain metastasis-triggered mouse body weight loss was alleviated completely by 14 and 90, but only partially by JQ1. Brain-prone MDA- MB-231 BrM2 cells (IxlO4) were IC-injected into 10 female 6- to 8-week-old athymic mice. Compounds (50 mg / kg) or DMSO carrier were IP -injected daily starting at day 7 post BrM2 cell injection, with BLI and body weight monitored weekly. Statistics: mean + s.e.m., significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, two-way ANOVA with Tukey’s multiple comparison test.

[0354] Example 34. Efficacy of 14 / 90 in Inhibiting Cancer Cell Growth

[0355] That suppressed HPV replication upon knockdown of BRD4 in human C-33A cervical cancer cells could only be rescued by re-expression of wild-type BRD4, but not a phosphorylationdefective pNPS mutant, highlights the importance of BRD4 phosphorylation in virus-induced cancer progression. Indeed, pBRD4 IDR-targeting compounds 14 / 90 could effectively inhibit cancer cell growth in viability assay performed in HeLa and CaSki cervical cancer cells as well as parental and brain-metastasized (BrM) MDA-MB-468, HCC1806 and MDA-MB-231 TNBC cells (Table 3). 90 exhibits low micromolar to sub-micromolar anticancer potency (IC50) as that seen with the widely used BBD inhibitor JQ1. 14 / 90 significantly inhibited cell viability of JQ1- resistant SUM159R cells.23 14 / 90 also had less cytotoxicity against the noncancerous human mammary MCF10A epithelial cells than that of JQ1 (Table 8).

[0356] Table 3. Cell viability efficacy of 14, 90, and JQ1. AlphaScreen was performed to analyze pPDID interaction with 16E2 DBD and p53 C-terminal regulatory domain (REG; aa 309-393). In cell viability analysis, 600 cells in 25 pl per well were seeded in 384-well plates 24 hours prior to compound treatment for four days, with viability measured by CellTiter-Glo.

[0357] Example 35. In vivo Anti-tumor and Anti-metastasis Effects of 14 / 90

[0358] Given the overall favorable PK and toxicity profiles, in vivo anti -turn or efficacy and antimetastasis effects of 14 / 90 was further evaluated using three primary tumors and three BrM mouse models (Fig. 8). Intriguingly, 14 suppressed mammary tumor development in both syngeneic mouse Py8119 and human PDX BCM-3887 xenograft models with no overt toxicity (Fig. 8A). Furthermore, 90, with higher binding affinity to pBRD4 than 14, is capable of suppressing JQ1- resistant SUM159R tumor growth better than 14 and JQ1 (Fig. 8B). Brain BLI signals from mice treated with 14, 90, JQ1, or vehicle control with representative brain images were analyzed. 14 / 90 were found to effectively suppress brain metastasis better than JQ1 in multiple TNBC animal models, including two xenograft (MDA-MB-231 and -468) and one immunocompetent syngeneic (4T1) mouse model (Fig. 8C). 14 significantly prolonged mouse life span against metastasis- triggered death with higher disease-free percentage compared to JQ1 that did not inhibit brain metastasis (Fig. 8D and 8E) and is more effective than 90 likely due to its more favorable in vivo PK profile. Collectively, these in vivo data provide a proof of concept that targeting pBRD4 IDR using proprietary pNPS-binding compounds provides a novel therapeutic approach in inhibiting TNBC progression / metastasis and extending survival with potential to overcome drug resistance.

[0359] Example 36. Hit-to-Lead Optimization of 14 / 90 to Establish Preliminary Structure- Activity Relationships and Yield Improved New Analogs.

[0360] New batches of 14 / 90 analogs were designed and synthesized to establish preliminary SAR information and the feasibility of lead optimization. The chemical structures of ten representative analogs are depicted below, with their in vitro pharmacological activities and cytotoxicity as well as microsome stability in comparison with 14 / 90 and JQ1 shown in Table 8. These new analogs with side chains containing basic nitrogen atoms have enhanced druglike properties, including aqueous solubility and oral bioavailability, and showed comparable or improved PPI modulation capability in AlphaScreen analysis. Some of these new analogs, such as ZM0146, ZM0489, ZM0616, ZM0619, and ZM0552, exhibited submicromolar to low nanomolar IC50 potency against TNBC MDA-MB-468 and HCC1806 parental cells and their BrM lines. Some displayed remarkably low cytotoxicity with a high selectivity IC50 ratio between MCF10A and TNBC cells.Moreover, many of them exhibit comparable or significantly improved microsome stability withTl / 2 more than 60 or 120 minutes.

[0361] Example 37. Further Compounds which Effectively Inhibit HPV genome Replication

[0362] Compounds identified in the HTS AlphaScreen (Fig. ID) were then tested in this establishment replication assay, with results showing cpds 14 and 22 effectively reducing El / E2 / ori-dependent luciferase activity without affecting cell viability. Compound 22 is the following:2214 and 22, when tested in proliferating and differentiated W12E, exhibited stage-specific inhibition: 14 suppresses genome amplification with no effect on cell viability but does not inhibit maintenance replication while reducing cell proliferation; in contrast, 22 specifically hinders maintenance replication but not genome amplification with no effect on cell viability. It is likelythat 22, with a distinct chemical scaffold, mainly targets pBRD4 interaction with E2, while 14 may inhibit pBRD4 interaction with cellular partners, particularly DNA damage response (DDR) factors, other than viral E2 (16,32,46-51).

[0363] Example 38. The efficacy of compounds inhibiting BRD4 pPDID interaction with HPV16 E2C

[0364] Table 4 summarizes that efficacy of compounds inhibiting BRD4 pPDID interactions with HPV16 E2C. In the table, a is Sf9-f:PDID = 25 nM, Bac-GST: 16E2C = 50 nM; b is IC50 : Concentration needed to inhibit 50% interaction; IC50 (calculated in Prism 9.0): Nonlinear Regression (curve fit), [Inhibitor] vs normalized response; c is S.E. (calculated in Prism 9.0); and tZis N: Number of biological replicates each with technical triplicates.

[0365] Table 4. The efficacy of compounds inhibiting BRD4 pPDID interaction with HPV16E2C

[0366] Example 39. The efficacy of compounds inhibiting full-length (FL) BET protein interaction with FL HP VI 6 E2 or 16E2C-DBD.

[0367] Table 5 shows the efficacy of compounds inhibiting FL BET protein interaction with FL HPV16 E2 or 16E2C-DBD, where a is IC50: Concentration needed to inhibit 50% interaction; IC50 (calculated in Prism 9.0): Nonlinear Regression (curve fit). [Inhibitor] vs normalized response.

[0368] Table 5. The efficacy of compounds inhibiting full-length (FL) BET protein interaction with FL HPV16 E2 or 16E2C-DBD

[0369] Example 40. The efficacy of compounds inhibiting FL BET protein interaction withFL p53 or p53-REG

[0370] Table 6 shows the efficacy of compounds inhibiting FL BET protein interaction with FL p53 or p53-REG, where a is IC50: Concentration needed to inhibit 50% interaction; IC50(calculated in Prism 9.0): Nonlinear Regression (curve fit). [Inhibitor] vs normalized response.

[0371] Table 6. The efficacy of compounds inhibiting FL BET protein interaction with FL p53 or p53-REG

[0372] Example 41. Pharmacokinetics of compound ADME (absorption, distribution, metabolism, and excretion) properties.

[0373] Table 7 shows the pharmacokinetics of compound absorption, distribution, metabolism, and excretion properties for 14 and 90, in comparison to ZM0145. In the table, LogP is log 1 ©(Partition Coefficient), compound lipophilicity (the permeability of the drugs to reach the target tissue in the body); tPSA is topological polar surface area, CLogP: calculated LogP; IV is intravenous; IP is intraperitoneal; PO is oral (per os); Terminal Tl / 2 is the terminal phase half-life (the true elimination half-life of the drug); Tmax is the time to achieve maximal concentration; Cmax (mg / L) is maximal plasma concentration; AUC(h*mg / L) is the area under the plasma drug concentration-time curve (AUC) reflecting the actual body exposure to drug after administration of a specific drug dose; VSS (L / kg) is steady state volume of distribution [the larger # means mainly in tissues (> 0.7 L / kg for intracellular target), little in plasma; <0.3 L / kg is mainly in plasma for vascular or extracellular target]; Vz (L / kg) is volume of distribution during terminal phase (the larger # means mainly in tissues, little in plasma); CL (L / h / kg) is serum clearance (the volume in liter of blood completely cleared of the drug per hour when it passes through a clearing organ), CL <1.782 L / h / kg in mice is considered low clearance; MRTlast (h, mean residence time) is mean residence time (MRT) from the time of dosing to the time of the last measurable concentration; and F (Bioavailability) is the systematic availability of the administered drug.

[0374] Table 7. Pharmacokinetics of compound ADME (absorption, distribution, metabolism, and excretion) properties.Compound Pharmacokinetics (CD1 Mice)PK Parameter i 14 i 90 ; ZM0145

[0375] Example 42. In vitro biological evaluation of pBRD4-IDR analogs. Table 8 shows in vitro biological evaluation of ten representative new pBRD4-IDR analogs in comparison with those of 14, 90, and JQ1. Compound analyses were similarly performed as described in Table 3. Red indicates viability IC50 in the sub-micromolar to low-micromolar range. Green marks higher selectivity for TNBC than noncancerous MCF10A cells. Magenta marks more stable analogs. ND, not determined.

[0376] Table 8. In vitro biological evaluation of ten representative new pBRD4-IDR analogs in comparison with those of 14, 90, and JQ1.

Claims

CLAIMSWe claim:

1. A compound of Formula (I) or a pharmaceutically acceptable salt, tautomer, or isotopologue thereof:Formula (I)R1is selected from hydrogen, alkyl (e.g., methyl, ethyl, isopropyl), cycloalkyl (e.g., cyclopropyl, cyclobutyl), alkoxy (e.g., methoxy, ethoxy), -OCD3, -CD3, CD2CD3, haloalkyl (e.g., trifluoromethyl, difluoromethyl), haloalkoxy (e.g., trifluoromethoxy, difluoromethoxy), halo, cyano, nitro, amino, etc.R2is selected from hydrogen, alkyl, cycloalkyl, alkoxy, -OCD3, -CD3, CD2CD3, haloalkyl, haloalkoxy, halo, cyano, nitro, amino, carboxy, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, alkoxyalkyl, aminoalkyl, aminoalkoxy, hydroxy, hydroxyalkyl, wherein alkoxycarbonyl, alkylcarbonyl, alkylaminocarbonyl are unsubstituted or substituted with halo, hydroxy, cycloalkyl, alkoxy, alkylsulfonyl; etc.Ring A is a unsubstituted or substituted aryl (e.g., phenyl, naphthyl), heteroaryl (e.g., pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, furanyl, thiophenyl, imidazolyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl), fused heteroaryl (e.g., quinolinyl, indolyl, benzofuranyl, benzothiophenyl, benzothiazolyl, benzoimidazolyl, indazolyl), heterocyclicyl (e.g., morpholinyl, piperazinyl, homopiperazinyl, piperdinyl, thiomorpholinyl, pyrrolidinyl), spirocyclicyl, bridged bicyclicyl, etc. m is 1, 2, 3 or 4;R3is independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, haloalkyl, haloalkoxy, halo, -OCD3, -CD3, CD2CD3, amide, cyano, nitro, carboxy, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, alkoxyalkyl, aminoalkyl, aminoalkoxy, hydroxy, hydroxyalkyl, wherein alkoxycarbonyl, alkylcarbonyl, alkylaminocarbonyl, amide, or alkyl isoptionally substituted with halo, hydroxy, cycloalkyl, alkoxy, alkylsulfonyl, ester, alkynyl, or substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroaryl, or Boc.X1is CH, O, S or NR4, wherein R4is hydrogen, alkyl, cycloalkyl, -OCD3, -CD3, CD2CD3, alkoxyalkyl, alkylcarbonyl, alkyl sulfonyl; etc. X2is CH, O, S or NR5, wherein R5is hydrogen, alkyl, cycloalkyl, -OCD3, -CD3, CD2CD3, alkoxyalkyl, alkylcarbonyl, alkyl sulfonyl; etc.X3is CH, O, S or NR6, wherein R6is hydrogen, alkyl, cycloalkyl, -OCD3, -CD3, CD2CD3, alkoxyalkyl, alkylcarbonyl, alkyl sulfonyl; etc.Yis O, S or NR7, wherein R7is hydrogen, alkyl, cycloalkyl, -OCD3, -CD3, CD2CD3, alkylcarbonyl, alkylsulfonyl, aminoalkyl, etc., wherein, alkyl, cycloalkyl, alkylcarbonyl, alkylsulfonyl, aminoalkyl are unsubstituted or substituted with halo, hydroxy, alkyl, haloalkyl, hydroxyalkyl, cycloalkyl, alkyloxy, carboxy, aminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, cycloaminecarbonyl, etc.

2. The compound of claim 1, wherein said compound of Formula I is chosen from:Formula laFormula IcFormular Id wherein R8, R9, and R9are independently selected from hydrogen, alkyl, cycloalkyl, alkoxy, haloalkyl, haloalkoxy, halo, -OCD3, -CD3, CD2CD3, amide, cyano, nitro, carboxy, alkoxycarbonyl, alkylcarbonyl, aminocarbonyl, alkylaminocarbonyl, alkoxyalkyl, aminoalkyl, aminoalkoxy, hydroxy, hydroxyalkyl, wherein alkoxycarbonyl, alkylcarbonyl, alkylaminocarbonyl, amide, or alkyl is optionally substituted with halo, hydroxy, cycloalkyl, alkoxy, alkylsulfonyl, ester, alkynyl, or substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroaryl, or Boc.

3. The compound of claim 1, wherein said compound is any one of:

4. The compound of claim 1, wherein said compound is any one of the following:

5. The compound of any of the preceding claims, wherein said compound is a phospho-BRD4 inhibitor.

6. The compound of any of the preceding claims, wherein said compound is chosen from:

7. A compound or a pharmaceutically acceptable salt, tautomer, or isotopologue thereof:

228. A method for inhibiting phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication, said method comprising contacting one or more cells with of a subject with a compound of any of the preceding claims.

9. A method for treating a disease or condition, said method comprising administering an effective amount of a compound of any of the preceding claims to a subject.

10. The method of claim 9, wherein said disease or condition involves inhibition of bromodomain-containing protein 4 (BRD4) via upstream inhibition of protein-protein interaction inhibitor (PPIz).

11. The method of claim 9, wherein said method involves targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that effectively inhibits phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication, and / or disturbingthe interaction between phospho-BRD4 and its partners including HPV E2, p53, and DNA damage response factors, such as 53BP1 and BARD1.

12. The method of claim 10, wherein said disease or condition is chosen from cancer, inflammation, viral infection and neurodegeneration disease / condition.

13. The method of claim 10, wherein said cancer is chosen from breast cancer, prostate cancer, cervical cancer, lung cancer, GI cancer, neuroblastoma, acute myeloid leukemia, mantle cell lymphoma.

14. The method of claim 12, wherein said breast cancer is triple negative breast cancer (TNBC).

15. The method of claim 10, wherein said inflammation involves repressing LPS-induced proinflammatory cytokine / chemokine production.

16. The method of claim 10 or 15, wherein said inflammation is Crohn’s disease, ulcerative colitis, airway inflammation and / or fibrosis.

17. The method of claim 10, wherein said viral infection is chosen from HPV, HIV, and SARS- CoV-2 infection.

18. The method of claim 10, wherein said neurodegeneration is chosen from Alzheimer's disease, Parkinson’s disease, and Huntington’s disease.

19. The method of claim 10, wherein said method involves selectively inhibiting bromodomain-containing protein 4 (BRD4) via upstream inhibition of protein-protein interaction inhibitor (PPIz) without disrupting BBDz.

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