IDR-targeting compounds suppress HPV genome replication via disruption of phospho-BRD4 association with DNA damage response factors

Abstract

Compounds binding to the bromodomains of bromodomain and extra-terminal (BET) family proteins, particularly BRD4, are promising anticancer agents. Nevertheless, side effects and drug resistance pose significant obstacles in BET-based therapeutics development. Using high-throughput screening of a 200,000-compound library, we identified small molecules targeting a phosphorylated intrinsically disordered region (IDR) of BRD4 that inhibit phospho-BRD4 (pBRD4)-dependent human papillomavirus (HPV) genome replication in HPV-containing keratinocytes. Proteomic profiling identified two DNA damage response factors-53BP1 and BARD1-crucial for differentiation-associated HPV genome amplification. pBRD4-mediated recruitment of 53BP1 and BARD1 to the HPV origin of replication occurs in a spatiotemporal and BRD4 long (BRD4-L) and short (BRD4-S) isoform-specific manner. This recruitment is disrupted by phospho-IDR-targeting compounds with little perturbation of the global transcriptome and BRD4 chromatin landscape. The discovery of these protein-protein interaction inhibitors (PPIi) not only demonstrates the feasibility of developing PPIi against phospho-IDRs but also uncovers antiviral agents targeting an epigenetic regulator essential for virus-host interaction and cancer development.

Keywords: 53BP1; BARD1; BET; BRD4; DDR; HPV; IDR; PPI inhibitors; antiviral; compound.

Figure 1.. High-Throughput Screening (HTS) of pBRD4-Targeting Compounds

(A) Domain features of BRD4-L/S and HPV16-encoded E2. (B) Rationale for compound screening and predicted outcomes. (top) Cell-based luciferase (Luc) assay. (bottom) Model for compound-derepressed Luc activity unique to HR-16E2 but not LR-BE2. (C) Outline of HTS compound screen. (D) Compound IC₅₀ in AlphaScreen calculated from a 5-dose titration, each in five replicates, with IC₅₀ of each compound listed in Table S1.

Figure 2.. Cpd 14 Inhibits HPV ori Replication and Genome Amplification But Not Genome Maintenance

(A) Model for three stages of HPV replication. BM, basal membrane; ECM, extracellular matrix; trx, transcription; rep, replication. Copy numbers of ecHPV are based on published estimation.^,, (B) Cpd 14-reduced HPV18 genome amplification in 3D raft cultures. Images were captured with 20X objective magnification. (C) H&E staining of HPV18 raft tissue with or without 14 treatment. (D) E1/E2-dependent HPV16 ori replication inhibited by 14 but not JQ1. (E) Reduced E1/E2-dependent HPV16 ori replication in C-33A cells following knockdown of BRD4-L or BRD4-S. (F) 14-suppressed vs. JQ1-stimulated ecHPV16 genome amplification. Data represent mean ± SEM, compound vs. DMSO-only (−) or siBRD4 vs. siControl (−). Significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using unpaired two-tailed Student’s t-test.

Figure 3.. Target Validation of Cpd 14 and Its Analog 90

(A) Table summarizing △Tm 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). (B) TSA showing 14 does not bind GST-tagged 16E2 DBD. (C) Kd of 14 measured by microscale thermophoresis (MST) in > 3 independent experiments. (D) Immobilized peptide binding assay. (E) Chemical Structure of 14/90 and mean AlphaScreen IC₅₀ of 14 and 90. (F) TSA violin plots of 14, 90 and JQ1 △Tm values. (G) PDID binding assay with biotinylated 14 and 90. (H) Determination of the optimal temperature for cellular TSA (CETSA). (I) CETSA showing 14/90/JQ1 bind endogenous BRD4-L/S in differentiated W12E cells. (J) CETSA conducted in proliferating HeLa cells as (I). (K) AlphaScreen analyzing 14/90/JQ1 disruption of BRD4-L binding to acH4 peptide. (L) IP-IB analyzing 14/90/JQ1 inhibition of BRD4-L binding to acetylated cellular chromatin. (M) CETSA showing 14/90 binding to BRD4-L in HEK293 cells. Data are mean ± SEM. Significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using unpaired two-tailed Student’s t-test for (A) and (E), and 2-way ANOVA followed by Tukey’s multiple comparison test for (F) and (K).

Figure 4.. pBRD4 PPIi Suppress HPV DNA Replication without Perturbing Global Cellular Transcriptome

(A) 3D raft cultures showing 14/90 suppression of HPV18 genome amplification. (B) IC₅₀ of 90/14-inhibited HPV16 ori replication analyzed by Hirt-qPCR and viability assay. (C) Hirt-qPCR analyzing 90/14-inhibited HPV16 genome maintenance and viability assay. (D) IC₅₀ of 90/14-inhibited HPV16 genome amplification analyzed by Hirt-qPCR and viability assay. (E) RNA-seq MA plots. RNA-seq DEGs are listed in Table S2. (F) PRO-seq MA plots. The read counts were normalized against the library size and total spike-in reads, then used for edgeR DEG analysis. PRO-seq DEGs are listed in Table S3. Data represent mean ± SEM, cpd vs. DMSO-only (−). Significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using unpaired two-tailed Student’s t-test for (B) and (C), or 2-way ANOVA followed by Tukey’s multiple comparison test for (D).

Figure 5.. Unique BRD4 Isoform-Associated DDR Factors Identified by IP-Proteomics

(A) Outline of BRD4-L/S IP-Proteomics with the identified proteins listed in Table S4. (B) Key biological processes identified in BRD4-L/S interactomes. See Table S4 for lists of BRD4-interacting proteins shown in Venn diagrams and identified in STRING analysis. (C) DDR pathways enriched in BRD4-L-Unique interactomes. See Table S4 for lists of proteins identified in Venn diagrams and the three repair pathways. (D) Volcano plot showing BRD4 interactors enriched in BRD4-L (L > S) or BRD4-S (L < S) interactomes in differentiated W12E cells. (E) STRING network of 41 BRD4-L-Diff-Unique DDR interactors in DSB, GG-NER, and TC-NER pathways. (F) STRING network of 14 S-Diff-Unique interactors.

Figure 6.. 14/90-Disrupted BRD4 Association with 53BP1 and BARD1 Impairs Differentiation-Dependent HPV Genome Amplification

(A) Heatmap showing 14/90-disrupted BRD4 isoform interaction with pS/T-binding proteins identified in IP-Proteomics. (B) IP-IB validation of endogenous BRD4-L/S interaction with 53BP1 and BARD1 in differentiated W12E cells. (C) Heatmap shows compound-treated RNA changes in RNA-seq and PRO-seq datasets. (D) Knockdown of 53BP1 and BARD1 reduces HPV genome amplification but not genome maintenance in W12E cells. (E) BRD4-L and BRD4-S are both important for HPV genome amplification and genome maintenance. (F) BRD3, but not BRD2, is critical for HPV genome amplification but not genome maintenance. (G) CETSA showing 14/90 bind endogenous BRD3 but not BRD2 in differentiated W12E cells. (H) Knockdown of BRD4-L, BRD4-S, BRD3 or BARD1, but not BRD2 or 53BP1, alleviates 14/90 inhibition of HPV genome amplification in differentiated W12E cells. Data represent mean ± SEM, siRNA vs. siControl (−). Significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using unpaired two-tailed Student’s t-test.

Figure 7.. PPIi Prevent BRD4 Isoform-Dependent Recruitment of 53BP1 and BARD1 without Globally Inhibiting BRD4 Binding to Cellular and Viral Chromatin

(A) Heatmaps show BRD4-L and BRD4-S binding signals in W12E enhancers and flanking 1 kb regions with the average signal plot shown below. (B) 14/90 selectively disrupt 53BP1 and BARD1 binding to HPV ori without reducing corresponding BRD4-L/S occupancy in differentiated W12E cells. (C) Knockdown analysis showing BRD4-L-dependent recruitment of 53BP1 and BARD1 to the HPV ori with BRD4-S selectively required for BARD1 but not 53BP1 recruitment. (D) Time- and BRD4-L/S-dependent recruitment of 53BP1 and BARD1 to the HPV ori. (E) Model for BRD4-L/S-selective recruitment of 53BP1 and BARD1 to the HPV ori. Data represent mean ± SEM. Significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using unpaired two-tailed Student’s t-test.