Small Compound Inhibitors Targeting HPV Genome Replication

Human papillomavirus (HPV) is the causative agent for cervical cancer and is etiologically linked to penile cancer, anal cancer, and oral cancers induced through sexual contact with HPV-infected individuals. Although three prophylactic vaccines that prevent up to nine types of HPV infection (Gardasil 9 for types 6, 11, 16, 18, 31, 33, 45, 52 and 58) are available and recommended for vaccination of adolescent girls/boys and adults up to age 45, vaccine hesitance and lack of therapeutics have made it difficult to eradicate this highly infectious human pathogen. As such, there is an urgent need to develop therapeutic agents able to block HPV propagation during early-stage viral episomal replication and late-stage HPV integration-perturbed host transcription programs. In our effort to identify cellular targets potentiating HPV-associated cervical and head-and-neck cancers, we found a chromatin-binding factor, bromodomain-containing protein 4 (BRD4), plays a crucial role in HPV DNA replication, genome segregation, viral gene regulation, and induction of cellular apoptosis and senescence through physical and functional association with HPV-encoded early protein 2 (E2). Besides being an emerging cancer therapeutic target, BRD4 is also a promising target for antiviral development. To understand the molecular basis of BRD4-dependent HPV replication, we knocked down BRD4 in cultured human C-33A cervical cancer cells and found that replication of high-risk HPV16 was completely abolished with no detectable origin of replication activity. Intriguingly, loss of HPV16 origin replication could be efficiently rescued by introducing wildtype, but not a phosphorylation-defective mutant, BRD4 back into the BRD4-knockdown cells. This finding suggests pharmacological inhibitors and stimulators regulating the extent of BRD4 phosphorylation and small compounds that target this phospho region of BRD4 are promising agents capable of blocking HPV replication and thus inhibiting HPV propagation in infected host cells. In this proposal, we will test the hypothesis that compounds targeting the phospho sites in a low-complexity intrinsically disordered region of BRD4 are potent inhibitors of HPV genome replication. The aims are: 1. Identify compounds effectively inhibiting HPV genome replication 2. Define the mechanistic action of phospho-BRD4-targeting compounds Three dozen or so compounds that we identified from a 200,000-chemical library screening, along with their newly derived analogs, will be analyzed for their ability to inhibit the three stages (establishment, maintenance, and amplification) of HPV DNA replication with promising compounds further examined for their pharmacokinetics, in vivo toxicology, and mechanistic action in inhibiting HPV life cycle. This new class of phospho-BRD4-binding compounds as target-selective protein-protein interaction inhibitors, unlike panbromodomain inhibitors (e.g., JQ1 and I-BET) that non-selectively inhibit BRD4 chromatin association, provides a new line of research for developing anti-HPV agents for treating populations already infected by HPVs.