Human papillomaviruses (HPVs) are the most common sexually transmitted pathogens known today, infecting approximately 75% of sexually active individuals (Koutsky, 1997
). In the US over 20 million people are actively infected at any one time with over 5 million new infections arising each year (Cates, 1999
). Cervical cancer, a leading cause of death by cancer among women worldwide, with approximately a half million new cases and nearly a quarter of a million deaths each year (Arbyn et al., 2011
), is caused by a subset of “high-risk” HPVs, including HPV16, 18, 31, 33, and 45 that are classified as carcinogens by the World Health Organization (Cogliano et al., 2005
). Among the “high risk” HPVs, HPV16 is most notable, whose DNA can be detected virtually in over half of cervical cancers. These high-risk HPVs are also causally associated with other anogenital cancers of the vulva, vagina, penis, anus and periungal region, and approximately 20% of head and neck cancers, particularly of the tongue, tonsil and oropharynx (zur Hausen, 2009
Prevention of HPV infections remains the primary means by which we can reduce the incidence of HPV-associated cancers. Vaccines against human papillomaviruses, Cervarix and Gardasil, have made this goal achievable; they possess remarkable efficiency at inhibiting infection in vaccinated populations by those HPV genotypes they are designed to protect against(Frazer et al., 2011
). These vaccines reduced by at least 90% the infection rate and early neoplasia by those genotypes targeted by the vaccines (HPVs 16 and 18 in the case of Cervarix, HPVs 6, 11, 16 and 18 in the case of Gardasil) (Garland et al., 2007
; Paavonen et al., 2007
; Villa et al., 2005
). However, the long-term effectiveness of these prophylactic vaccines in those vaccinated remains unclear. The current vaccines, even if 100% effective will only prevent cervical cancers arising from infection by two of the high-risk HPV genotypes, HPV16 and HPV18. As cancers caused by other high-risk HPVs account for approximately 25 – 30% of all cervical cancers, second-generation vaccines with broader range effectiveness for more high-risk HPV genotypes or alternative means to prevent infection by these high-risk HPVs are needed. Another critical issue is accessibility of the vaccines. These vaccines are unlikely to be widely disseminated in most developing countries where cervical cancer is more frequent because of the high cost of whole scale vaccinations of young women in these populations. Clearly, there is need for identifying other less expensive modalities for preventing HPV infections.
Ready access to effective antivirals represents one potentially valuable approach to the prevention of sexually transmitted diseases (STDs) including genital HPV infections (Howett and Kuhl, 2005
). To make an effective topical HPV antiviral commercially available, it must fulfill several criteria. It should efficiently target an early step in HPV infection, possess minimal side-effects/cytotoxicity, it should be easy to formulate and chemically stable in the formulation, and inexpensive to manufacture.
Recent advances now make it possible to pursue HPV antiviral development. Specifically, methodologies to efficiently produce HPV pseudoviruses carrying reporter genes (Buck et al., 2004
) or infectious papillomavirus (Pyeon et al., 2005
) at very high yields have been developed. These advances overcame the major limitation in the identification of HPV-antivirals, by providing access to useful amounts of virus particles for high throughput assays to identify small molecules that inhibit early steps in infection.
In this study, we identified small molecules available in the University of Wisconsin Small Molecule Screening Facility (UW-SMSF) that fulfill the above stated criteria for an effective HPV-antiviral using a well-validated cell-based high throughput screening (HTS) assay using HPV16 pseudoviruses expressing a reporter gene. Candidate lead compounds were screened in multiple secondary and tertiary screens using HPV11, 16 and 31 pseudoviruses containing alternative reporter genes as well as infection with bona fide HPV16 particles. Lead compounds were chosen for further analyses based upon the pharmacological property, scaffold diversity, strength of the inhibitory activity and low cytotoxicity. Analogs of the best two lead compounds were screened to inform on the pharmacophore for each compound. Initial studies indicate these compounds act at early steps of infection, in the first 12 hours post exposure of cells to virus like particles, but do not prevent internalization of the bulk of virus particles based upon in situ microscopy studies. Potential mechanisms of action of these compounds are discussed.