HPV episome levels are potently decreased by pyrrole–imidazole polyamides

Abstract

Human papillomavirus (HPV) causes cervical cancer and other hyperproliferative diseases. There currently are no approved antiviral drugs for HPV that directly decrease viral DNA load and that have low toxicity. We report the potent anti-HPV activity of two N-methylpyrrole-imidazole polyamides of the hairpin type, polyamide 1 (PA1) and polyamide 25 (PA25). Both polyamides have potent anti-HPV activity against 3 different genotypes when tested on cells maintaining HPV episomes. The compounds were tested against HPV16 (in W12 cells), HPV18 (in Ker4-18 cells), and HPV31 (tested in HPV31 maintaining cells). From a library of polyamides designed to recognize AT-rich DNA sequences such as those in or near E1 or E2 binding sites of the HPV16 origin of replication (ori), 4 polyamides were identified that possessed apparent IC50s ≤ 150 nM with no evidence of cytotoxicity and we report two highly-active compounds here. Treatment of epithelia engineered in organotypic cultures with these compounds also causes a dose-dependent loss of HPV episomal DNA that correlates with accumulation of compounds in the nucleus. Bromodeoxyuridine (BrdU) incorporation demonstrates that DNA synthesis in organotypic cultures is suppressed upon compound treatment, correlating with a loss of HPV16 and HPV18 episomes. PA1 and PA25 are currently in preclinical development as an antiviral compound for treatment of HPV-related disease, including cervical dysplasia. PA1 and related polyamides offer promise as antiviral agents and the ability to regulate HPV episomal levels in cells for the study of HPV biology. We also report that anti-HPV16 activity for Distamycin A, a natural product related to PA1, is accompanied by significant cellular toxicity.

Human Papillomavirus Episome Stability Is Reduced by Aphidicolin and Controlled by DNA Damage Response Pathways

“…Tissue culture of normal foreskin keratinocytes (Ker-4) and keratinocytes maintaining HPV16 (W12E cells), HPV18 (Ker4-18 cells), or HPV31 was previously described (15,19). Cells were cultured on mitomycin C-treated J2 3T3 cells in media containing three parts Dulbecco's modified Eagle medium (DMEM) and one part F12 medium.…”

“…However, little is known of the factors that contribute to episome stability over short periods of time in culture. We employ a quantitative PCR (Q-PCR) assay for HPV episomes that permits the rapid and accurate examination of changes in episome levels over short periods of time in culture (15,19). Two DNA binding compounds, polyamide 1 (PA1) and polyamide 25 (PA25), targeting HPV were recently reported to cause a large, rapid loss of episomes in monolayer and organotypic cultures (19).…”

“…Keratinocytes maintaining high-risk HPV genomes in vitro, whether derived from clinical samples or engineered in the laboratory, generally maintain a constant number of episomes per cell (15,(18)(19)(20). The E2 protein and E8Ê2C have been implicated as playing an important role in HPV episome copy number control via their role in modulating both E1-dependent replication and transcriptional repression (21).…”

“…We employ a quantitative PCR (Q-PCR) assay for HPV episomes that permits the rapid and accurate examination of changes in episome levels over short periods of time in culture (15,19). Two DNA binding compounds, polyamide 1 (PA1) and polyamide 25 (PA25), targeting HPV were recently reported to cause a large, rapid loss of episomes in monolayer and organotypic cultures (19). In investigating the mechanism by which PA25 acts to destabilize episomes, we discovered that aphidicolin by itself also elicits episome loss from cells.…”

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

“…4,5,[7][8][9][10][11] Polyamides have been employed to both repress and activate the expression of specific genes. 4,[12][13][14][15][16][17][18][19] They also have interesting antiviral [20][21][22][23][24] and anticancer 12,13,[25][26][27][28][29][30] properties. One attractive feature of polyamides is their ability to access DNA sequences that are methylated 31,32 and wrapped around histone proteins 9,10,33 .…”