Antiviral compounds and methods for treating infections caused by double-stranded DNA viruses

The present invention relates to polyamide compounds and their use in pharmaceutical compositions and in medical applications for the treatment of human papillomavirus infections and/or polyomavirus infections. For the most up-to-date information about these patents, including the availability of Certificates of Correction, be sure to check the United States Patent and Trademark Office\u27s free, publicly accessible database: Patent Public Search https://ppubs.uspto.gov/pubwebapp/static/pages/landing.htmlhttps://irl.umsl.edu/patents/1001/thumbnail.jp

DNA Binding Polyamides and the Importance of DNA Recognition in their use as Gene-Specific and Antiviral Agents

There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyamides (PAs) that are higher homologs of natural products such as distamycin A and netropsin. This work has been pursued by many groups, with the Dervan and Sugiyama groups responsible for many breakthroughs. We have studied PAs since about 1999, partly in industry and partly in academia. Early in this program, we reported methods to control cellular uptake of polyamides in cancer cell lines and other cells likely to have multidrug resistance efflux pumps induced. We went on to discover antiviral polyamides active against HPV31, where SAR showed that a minimum binding size of about 10 bp of DNA was necessary for activity. Subsequently we discovered polyamides active against two additional high-risk HPVs, HPV16 and 18, a subset of which showed broad spectrum activity against HPV16, 18 and 31. Aspects of our results presented here are incompatible with reported DNA recognition rules. For example, molecules with the same cognate DNA recognition properties varied from active to inactive against HPVs. We have since pursued the mechanism of action of antiviral polyamides, and polyamides in general, with collaborators at NanoVir, the University of Missouri-St. Louis, and Georgia State University. We describe dramatic consequences of β-alanine positioning even in relatively small, 8-ring polyamides; these results contrast sharply with prior reports. This paper was originally presented by JKB as a Keynote Lecture in the 2nd International Conference on Medicinal Chemistry and Computer Aided Drug Design Conference in Las Vegas, NV, October 2013

Employing the Metabolic “Branch Point Effect” to Generate an All-or-None, Digital-like Response in Enzymatic Outputs and Enzyme-Based Sensors

Here, we demonstrate a strategy to convert the graded Michaelis−Menten response typical of unregulated enzymes into a sharp, effectively all-or-none response. We do so using an approach analogous to the “branch point effect”, a mechanism observed in naturally occurring metabolic networks in which two or more enzymes compete for the same substrate. As a model system, we used the enzymatic reaction of glucose oxidase (GOx) and coupled it to a second, nonsignaling reaction catalyzed by the higher affinity enzyme hexokinase (HK) such that, at low substrate concentrations, the second enzyme outcompetes the first, turning off the latter’s response. Above an arbitrarily selected “threshold” substrate concentration, the nonsignaling HK enzyme saturates leading to a “sudden” activation of the first signaling GOx enzyme and a far steeper dose−response curve than that observed for simple Michaelis−Menten kinetics. Using the well-known GOx-based amperometric glucose sensor to validate our strategy, we have steepen the normally graded response of this enzymatic sensor into a discrete yes/no output similar to that of a multimeric cooperative enzyme with a Hill coefficient above 13. We have also shown that, by controlling the HK reaction we can precisely tune the threshold target concentration at which we observe the enzyme output. Finally, we demonstrate the utility of this strategy for achieving effective noise attenuation in enzyme logic gates. In addition to supporting the development of biosensors with digital-like output, we envisage that the use of all-or-none enzymatic responses will also improve our ability to engineer efficient enzyme-based catalysis reactions in synthetic biology applications

Synthesis and characterization of a novel series of bis-linked diaza-18-crown-6 porphyrins

The synthesis and physicochemical characterization of a novel family of \u27crowned-porphyrins\u27 is presented. The compounds uniquely possess a 5,15-di(2-alkylamidophenyl)etioporphyrin bis-linked to the nitrogens on opposite sides of a 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane moiety. The size of the cavity in between the porphyrin and the diaza-18-crown-6 ring has been regulated by varying the length of the alkylamido linking units. The structures of 5 and 7 bear diacetamido linkers whereas 6 is linked via dipropionamido groups. The synthetic pathway presented is generalized such that a wide variety of useful porphyrin-based bis-macrocycles can be prepared. Products have been fully characterized by elemental analysis, mass spectrometry, spectrophotometry, 1H NMR, 13C NMR and 1H-13C heteronuclear correlation NMR spectroscopies. X-Ray structural data are presented for the free-base porphyrin 6 and the ZnIIporphyrin 7. The structural data confirm that the porphyrins and their diaza-18-crown-6 substituents adopt cofacial orientations. The size of the cavity between the macrocycles, while demonstrated to accommodate small molecules as shown by the binding of H2O as an axial ligand to the central ZnII ion in the crystal structure of 7, will probably not accommodate large ligands or substrates. Moreover, these compounds should allow sequential metallations to yield heterobimetallic species. Therefore, they are ideally suited for study as model systems for biologically important heme-dependent phenomena such as xenon-129 binding to myoglobin or the mimicking of processes related to the structure and function of cytochrome c oxidase

DNA Damage Repair Genes Controlling Human Papillomavirus (HPV) Episome Levels under Conditions of Stability and Extreme Instability

<div><p>DNA damage response (DDR) genes and pathways controlling the stability of HPV episomal DNA are reported here. We set out to understand the mechanism by which a DNA-binding, N-methylpyrrole-imidazole hairpin polyamide (PA25) acts to cause the dramatic loss of HPV DNA from cells. Southern blots revealed that PA25 alters HPV episomes within 5 hours of treatment. Gene expression arrays identified numerous DDR genes that were specifically altered in HPV16 episome-containing cells (W12E) by PA25, but not in HPV-negative (C33A) cells or in cells with integrated HPV16 (SiHa). A siRNA screen of 240 DDR genes was then conducted to identify enhancers and repressors of PA25 activity. Serendipitously, the screen also identified many novel genes, such as TDP1 and TDP2, regulating normal HPV episome stability. MRN and 9-1-1 complexes emerged as important for PA25-mediated episome destruction and were selected for follow-up studies. Mre11, along with other homologous recombination and dsDNA break repair genes, was among the highly significant PA25 repressors. The Mre11 inhibitor Mirin was found to sensitize HPV episomes to PA25 resulting in a ∼5-fold reduction of the PA25 IC50. A novel assay that couples end-labeling of DNA to Q-PCR showed that PA25 causes strand breaks within HPV DNA, and that Mirin greatly enhances this activity. The 9-1-1 complex member Rad9, a representative PA25 enhancer, was transiently phosphorylated in response to PA25 treatment suggesting that it has a role in detecting and signaling episome damage by PA25 to the cell. These results establish that DNA-targeted compounds enter cells and specifically target the HPV episome. This action leads to the activation of numerous DDR pathways and the massive elimination of episomal DNA from cells. Our findings demonstrate that viral episomes can be targeted for elimination from cells by minor groove binding agents, and implicate DDR pathways as important mediators of this process.</p></div

Genes whose expression is significantly altered by PA25 in W12E cells.

<p><b>DSB:</b> double-strand break; <b>BER:</b> base excision repair; <b>MMR:</b> mismatch repair; <b>HR:</b> homologous recombination; <b>TLS:</b> translesion repair; <b>MMR:</b> mismatch repair; <b>NHEJ</b>: non-homologous end-joining.</p

Antiviral activity and structure of anti-HPV N-methylpyrrole-imidazole polyamides following 48 hours of treatment in W12E cells.

<p>A. PA1 and PA25 dramatically decrease HPV16 episome levels in W12E cells while the related PA11 has no effect. B. Structure of PA11. C. Structure of PA25.</p

Southern blots of PA25 treated HPV16 episomes from W12E cells.

<p>A. Southern blot of intact HPV episomes following treatment over 5 µM PA25. Migration of linearized HPV16 is shown (BamH1). Note retardation of migration of HPV16 Form 1 DNA (arrow) over time in the presence of PA25, and the appearance of Form 3 viral DNA (linear) at 5 hours of treatment (arrowhead). Open circle form of HPV is indicated with asterisk. B. Southern blot of episomes following treatment with 10 µM PA25. Migration of linearized HPV16 is shown (BamH1). Note the pattern of migration of HPV16 Form 1 DNA (arrow) over time in presence of PA25 resulting in a step-like appearance of HPV topoisomers. Open circle form of HPV is indicated with asterisk.</p

Southern blots of linearized (left) and intact (right) HPV16 episomes over time following treatment with 1 µM PA25 for 48 hours.

<p>The blots are loaded identically except HPV16 was linearized by BamH1 in one set of samples (left) or digested with HindIII, which does not restrict viral DNA (right). An additional, over-exposed HindIII blot is also provided. OC: open circle; SC: super-coiled.</p