Human cytomegalovirus UL97 kinase is involved in the mechanism of action of methylenecyclopropane analogs with 6-ether and -thioether substitutions.

Methylenecyclopropane nucleoside (MCPN) analogs are being investigated for treatment of human cytomegalovirus (HCMV) infection because of favorable preclinical data and limited ganciclovir cross-resistance. Monohydroxymethyl MCPNs bearing ether and thioether functionalities at the purine 6 position have antiviral activity against herpes simplex virus (HSV) and varicella-zoster virus (VZV) in addition to HCMV. The role of the HCMV UL97 kinase in the mechanism of action of these derivatives was examined. When tested against a kinase-inactive UL97 K355M virus, a moderate 5- to 7-fold increase in 50% effective concentration (EC50) was observed, in comparison to a 13- to 25-fold increase for either cyclopropavir or ganciclovir. Serial propagation of HCMV under two of these compounds selected for three novel UL97 mutations encoding amino acid substitutions D456N, C480R,and Y617del. When transferred to baseline laboratory HCMV strains, these mutations individually conferred resistance to all of the tested MCPNs, ganciclovir, and maribavir. However, the engineered strains also demonstrated severe growth defects and abnormal cytopathic effects similar to the kinase-inactive mutant. Expressed and purified UL97 kinase showed in vitro phosphorylation of the newly tested MCPNs. Thus, HCMV UL97 kinase is involved in the antiviral action of these MCPNs, but the in vitro selection of UL97-defective viruses suggests that their activity against more typical ganciclovir-resistant growth-competent UL97 mutants may be relatively preserved.

Small molecule inhibitors of anthrax lethal factor toxin.

This manuscript describes the preparation of new small molecule inhibitors of Bacillus anthracis lethal factor. Our starting point was the symmetrical, bis-quinolinyl compound 1 (NSC 12155). Optimization of one half of this molecule led to new LF inhibitors that were desymmetrized to afford more drug-like compounds.

Synthesis and antiviral activities of methylenecyclopropane analogs with 6-alkoxy and 6-alkylthio substitutions that exhibit broad-spectrum antiviral activity against human herpesviruses.

Methylenecyclopropane nucleosides have been reported to be active against many of the human herpesviruses. The most active compound of this class is cyclopropavir (CPV), which exhibits good antiviral activity against human cytomegalovirus (HCMV), Epstein-Barr virus, both variants of human herpesvirus 6, and human herpesvirus 8. CPV has two hydroxymethyl groups on the methylenecyclopropane ring, but analogs with a single hydroxymethyl group, such as the prototypical (S)-synguanol, are also active and exhibit a broader spectrum of antiviral activity that also includes hepatitis B virus and human immunodeficiency virus. Here, a large set of monohydroxymethyl compounds with ether and thioether substituents at the 6 position of the purine was synthesized and evaluated for antiviral activity against a range of human herpesviruses. Some of these analogs had a broader spectrum of antiviral activity than CPV, in that they also inhibited the replication of herpes simplex viruses 1 and 2 and varicella-zoster virus. Interestingly, the antiviral activity of these compounds appeared to be dependent on the activity of the HCMV UL97 kinase but was relatively unaffected by the absence of thymidine kinase activity in HSV. These data taken together indicate that the mechanism of action of these analogs is distinct from that of CPV. They also suggest that they might be useful as broad-spectrum antiherpesvirus agents and may be effective in the treatment of resistant virus infections.

Discovery of N-(4'-(indol-2-yl)phenyl)sulfonamides as novel inhibitors of HCV replication.

A series of novel 2-phenylindole analogs were synthesized and evaluated for activity in subgenomic HCV replicon inhibition assays. Several compounds containing small alkyl sulfonamides on the phenyl ring exhibiting submicromolar EC50 values against the genotype 1b replicon were identified. Among these, compound 25d potently inhibited the 1b replicon (EC50=0.17 µM) with 147-fold selectivity with respect to cytotoxicity. Compound 25d was stable in the presence of human liver microsomes and had a good pharmacokinetic profile in rats with an IV half-life of 4.3h and oral bioavailability (F) of 58%.

PTC124 targets genetic disorders caused by nonsense mutations.

Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.

Synthesis and antiviral activity of certain second generation methylenecyclopropane nucleosides.

A second-generation series of substituted methylenecyclopropane nucleosides (MCPNs) has been synthesized and evaluated for antiviral activity against a panel of human herpesviruses, and for cytotoxicity. Although alkylated 2,6-diaminopurine analogs showed little antiviral activity, the compounds containing ether and thioether substituents at the 6-position of the purine did demonstrate potent and selective antiviral activity against several different human herpesviruses. In the 6-alkoxy series, antiviral activity depended on the length of the ether carbon chain, with the optimum chain length being about four carbon units long. For the corresponding thioethers, compounds containing secondary thioethers were more potent than those with primary thioethers.

Aryl rhodanines specifically inhibit staphylococcal and enterococcal biofilm formation.

Staphylococcus epidermidis and Staphylococcus aureus are the leading causative agents of indwelling medical device infections because of their ability to form biofilms on artificial surfaces. Here we describe the antibiofilm activity of a class of small molecules, the aryl rhodanines, which specifically inhibit biofilm formation of S. aureus, S. epidermidis, Enterococcus faecalis, E. faecium, and E. gallinarum but not the gram-negative species Pseudomonas aeruginosa or Escherichia coli. The aryl rhodanines do not exhibit antibacterial activity against any of the bacterial strains tested and are not cytotoxic against HeLa cells. Preliminary mechanism-of-action studies revealed that the aryl rhodanines specifically inhibit the early stages of biofilm development by preventing attachment of the bacteria to surfaces.