Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay

Abstract

Human papillomaviruses (HPVs) are oncogenic viruses that cause numerous different cancers as well as benign lesions in the epithelia. To date, there is no effective cure for an ongoing HPV infection. Here, we describe the generation process of a platform for the development of anti-HPV drugs. This system consists of engineered full-length HPV genomes that express reporter genes for evaluation of the viral copy number in all three HPV replication stages. We demonstrate the usefulness of this system by conducting high-throughput screens to identify novel high-risk HPV-specific inhibitors. At least five of the inhibitors block the function of Tdp1 and PARP1, which have been identified as essential cellular proteins for HPV replication and promising candidates for the development of antivirals against HPV and possibly against HPV-related cancers.

The molecular biology and HPV drug responsiveness of cynomolgus macaque papillomaviruses support their use in the development of a relevant in vivo model for antiviral drug testing

“…The DpnI-resistant replication signal strengths were quantified and expressed relative to the vehicle-only control (DMSO), and the inhibition curves and IC50 values for each compound are provided in Fig 4. The results indicate that all five compounds inhibited the replication intensity of the MfPV8 genomes in a concentration-dependent manner, with IC50 values ranging from 5.7 to 58.9 μM (Fig 4), which are very similar to what is seen for HPV18, for example [32]. Thus, MfPV genome replication in U2OS cells can potentially be used for screening or validation of anti-HPV drugs.…”

“…We demonstrated that the human osteosarcoma cell line U2OS supports the genome replication of alpha and beta HPVs and is suitable for studying HPV genome replication and transcription [26–32,42]. To identify whether the same cell line also supports the replication of the MfPV genomes, a transient replication assay mimicking the phase of the establishment of the viral infection was performed.…”

“…Thus, we decided to analyze whether the previously described high-risk HPV-specific drug candidates can also inhibit MfPV8 replication. The five compounds used (NSC 9782, NSC 88915, NSC 82269, NSC 109128 and NSC 305831) were identified in high-throughput screening of NCI Diversity Set IV chemical library as specific inhibitors of high-risk mucosal HPV replication [32]. The MfPV8 minicircle genomes were transfected into U2OS cells, and genomic DNA was harvested after cultivating the cells in the presence of each compound at the concentrations for 5 days.…”

“…Interestingly, we found that the human U2OS cell line is capable of supporting the replication of not only HPV [26–32,36,42] but also cynomolgus macaque PV genomes (Fig 1) and therefore provides an opportunity to characterize these viruses, which are closely related to HPVs, at the molecular level. Hence, we used the U2OS cell line-based assay system for experimental description of the high-risk alpha and beta MfPV transcriptomes.…”

“…The U2OS cell line derived from a moderately differentiated osteosarcoma has a unique capability to support the transient, stable, and late amplificational replication of both cutaneous and mucosal HPV genomes efficiently [26]. The replication and transcriptome studies suggest that U2OS cells provide an adequate cellular environment to study HPV [26–32]. It has been shown that the same HPV DNA replication intermediates observed in U2OS cells are also present in keratinocyte cell lines [26,28,33].…”