Interactions of two large antiviral polyamides with the long control region of HPV16

Abstract

PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes (DNA) in cell culture, and PA25 has broad spectrum activity against three cancer-causing forms of HPV (Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok, K., Helmus, M., Bashkin, J. K., Fisher, C., Antiviral Res. 91 (2011) 177-186). Described here are the interactions of these PAs with sequences in the long control region (LCR) of HPV16 (7348-122). Using an FeEDTA conjugate of PA1 (designed to recognize 5’-W2GW7-3’; W = A or T), 34 affinity cleavage (AC) patterns were detected for this fragment. These sites can be rationalized with sequences featuring perfect, single, double, triple and quadruple mismatches. Quantitative DNase I footprinting analysis indicates that perfect sites bind PA1 with Kds between 0.7-2.2 nM. Kds for single, double, triple and quadruple mismatch sites range from 1-3 nM to 20 nM. Using AC and EDTA conjugates, we report that unlike smaller 8-ring hairpin PAs, introduction of a chiral turn in this large polyamide has no effect on binding orientation (forward vs. reverse). Despite its design to recognize 5’-W2GW5GW4-3’ via two Im residues, a motif not represented in this HPV sequence, a PA25-EDTA conjugate yielded 31 affinity cleavage sites on the region. Low nM Kds for PA25 without EDTA indicate a high tolerance for triple and quadruple mismatches. While there is extensive coverage of the sequence examined, AC cleavage patterns for the two PAs show discrete binding events and do not overlap significantly. This indicates that within the context of A/T rich sequences, these PAs do not recognize a simple shared sequence-related feature of the DNA. These insights continue to inform the complex nature of large hairpin PA-DNA interactions and antiviral behavior.

Improved antiviral activity of a polyamide against high-risk human papillomavirus via N-terminal guanidinium substitution

“…9,26 The first biophysical studies of PA25 were reported recently. 18b However, PA25 is a more difficult synthetic and purification challenge than PA1 and its derivatives, though we have scaled up both PA1 and PA25 to 10 g, and the cost-of-goods is much higher for the 20-ring PA25 than the 14-ring PA1 , so we were interested to see if modifications to PA1 could increase its activity significantly. That goal was successful with PA31 .…”

“…18 In fact, in that prior work we found for 14-ring PA1 that numerous “mismatch” sites were bound with higher affinity than so-called canonical sites. Also, up to four mismatches were tolerated with 2 nM dissociation constants identical to numerous “perfect-match” sites.…”

“…Since we measured polyamide-DNA binding properties in the context of a large, natural DNA sequences instead of small artificial, DNA sequences designed to examine sequence preferences at a single location, our results are much more complex than those found in the literature. Published binding rules are not obeyed in our system with significant fidelity, and “mismatches” are very well tolerated (see also He et al 18 and Qiao et al 20 ).…”

“…K d was determined by the Hill or Langmuir equation, based on the magnitude of the derived Hill coefficient and overall fit (see Table S 1 and 2). 18 …”

“…For a larger, 20-ring antiviral polyamide ( PA25 ), four mismatches were even more common than with PA1 , leading to a complete blanketing of the HPV16 long control region when only sparse binding, at best, was predicted. 18b …”