Abstract

Expression and function of the human papillomavirus (HPV) early protein 6 (E6) is necessary for viral replication and oncogenesis in cervical cancers. HPV E6 targets the tumor suppressor protein p53 for degradation. To achieve this, “high-risk” HPV E6 proteins bind to and modify the target specificity of the ubiquitin ligase E6AP (E6 associated protein). This E6-dependent loss of p53 enables the virus to bypass host cell defenses and facilitates virally induced activation of the cell cycle progression during viral replication. Disruption of the interaction between E6 and E6AP and stabilization of p53 should decrease viability and proliferation of HPV positive cells. A new in vitro high-throughput binding assay was developed to assay binding between HPV-16 E6 and E6AP and to identify compounds that inhibit this interaction. The compound luteolin emerged from the screen and a library of novel flavones based on its structure was synthesized and characterized using this in vitro binding assay. The compounds identified in this study disrupt the E6/E6AP interaction, increase the levels of p53 and p21Cip1/Waf1, and decrease proliferation of HPV positive cell lines. The new class of flavonoid E6 inhibitors displays a high degree of specificity for HPV positive cells. Docking analyses suggest that these compounds bind in a hydrophobic pocket at the interface between E6 and E6AP and mimic the leucines in the conserved α-helical motif of E6AP. The activity and specificity of these compounds represent a promising new lead for development as an antiviral therapy in the treatment of HPV infection and cervical cancer.

Highlights

  • human papillomavirus (HPV) causes common cutaneous, mucosal, anogenital, and oropharyngeal epithelial growths

  • Filter plate based HPV-16 early protein 6 (E6)/E6AP interaction assay for lead conformation

  • Glutathione-bead bound GST-16 E6 was immobilized on 96-well filter plates, incubated with free E6APBAP fusion protein, and unbound E6AP-bacterial alkaline phosphatase (BAP) fusion was washed from the plates

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Summary

Introduction

HPV causes common cutaneous, mucosal, anogenital, and oropharyngeal epithelial growths. Genital warts are highly transmissible and affect all socioeconomic groups. The CDC estimated there are ~750,000 new cases of genital warts each year and 1.5 million persons under treatment in the USA. Three million new cases of abnormal Pap smears are detected in the USA, indicating active HPV infection. A minority of these lesions progress to pre-cancerous dysplasia and to invasive malignancy. On a worldwide basis, ~500,000 new cases of cervical cancer are diagnosed and nearly 250,000 deaths occur each year. HPV type 16 is found in approximately 50% of all cervical cancers [1] and is the most frequent isolate from oropharyngeal cancers, of which 25-50% are attributed to HPV [2,3,4]

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