The purpose of this study was to characterize the antiviral activity, cytotoxicity, and mechanism of action of TMC114, a novel human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI). TMC114 exhibited potent anti-HIV activity with a 50% effective concentration (EC 50 ) of 1 to 5 nM and a 90% effective concentration of 2.7 to 13 nM. TMC114 exhibited no cytotoxicity at concentrations up to 100 M (selectivity index, >20,000). All viruses in a panel of 19 recombinant clinical isolates carrying multiple protease mutations and demonstrating resistance to an average of five other PIs, were susceptible to TMC114, defined as a fold change in EC 50 of <4. TMC114 was also effective against the majority of 1,501 PI-resistant recombinant viruses derived from recent clinical samples, with EC 50 s of <10 nM for 75% of the samples. In sequential passage experiments using HIV-1 LAI, two mutations (R41T and K70E) were selected. One selected virus showed a 10-fold reduction in susceptibility to TMC114, but <10-fold reductions in susceptibility to the current PIs (atazanavir was not assessed), except saquinavir. However, when the selected mutations were introduced into a laboratory strain by site-directed mutagenesis, they had no effect on susceptibility to TMC114 or other PIs. There was no evidence of antagonism between TMC114 and any currently available PIs or reverse transcriptase inhibitors. Combinations with ritonavir, nelfinavir, and amprenavir showed some evidence of synergy. These results suggest that TMC114 is a potential candidate for the treatment of both naïve and PI-experienced patients with HIV.

The screening of known HIV-1 protease inhibitors against a panel of multi-drug-resistant viruses revealed the potent activity of TMC126 on drug-resistant mutants. In comparison to amprenavir, the improved affinity of TMC126 is largely the result of one extra hydrogen bond to the backbone of the protein in the P2 pocket. Modification of the substitution pattern on the phenylsulfonamide P2' substituent of TMC126 created an interesting SAR, with the close analogue TMC114 being found to have a similar antiviral activity against the mutant and the wild-type viruses. X-ray and thermodynamic studies on both wild-type and mutant enzymes showed an extremely high enthalpy driven affinity of TMC114 for HIV-1 protease. In vitro selection of mutants resistant to TMC114 starting from wild-type virus proved to be extremely difficult; this was not the case for other close analogues. Therefore, the extra H-bond to the backbone in the P2 pocket cannot be the only explanation for the interesting antiviral profile of TMC114. Absorption studies in animals indicated that TMC114 has pharmacokinetic properties comparable to currently approved HIV-1 protease inhibitors.

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HCV NS5B polymerase, an essential and virus-specific enzyme, is an important target for drug discovery. Using structure-based design, we optimized a 1,5-benzodiazepine NS5B polymerase inhibitor chemotype into a new sulfone-containing scaffold. The design yielded potent inhibitor (S)-4c (K(D) = 0.79 nM), which has approximately 20-fold greater affinity for NS5B than its carbonyl analogue (R)-2c.

The synthesis and the spectral and cation binding properties of new fluorescent indicators for Na ؉ , dicaesium 5-[5-(2,3,5,6,8,9,11,12-octahydro-1,4,7,10,13-benzopentaoxacyclopentadecin-15-yl)-2-thienyl]-isophthalate (Benzos), and for K ؉ , dicaesium 5-[5-(2,3,5,6,8,9,11,12,14,15-decahydro-1,4,7,10,13,16-benzohexaoxacyclooctadecin-18-yl)-2-thienyl]isophthalate (Benzop), are reported. Both indicators consist of a benzocrown ether with an appropriate cavity for the sodium and the potassium cation, respectively, linked to an aryl thiophene fluorophore. In aqueous solution, the indicators have an absorption maximum at 340 nm and an emission maximum at 430 nm. The chelating abilities of Benzos and Benzop with Na ؉ , K ؉ , Li ؉ , Cs ؉ , Mg 2؉ and Ca 2؉ were studied by means of their fluorescence properties. Upon cation binding, a change in the fluorescence intensity is observed, whereas the spectral distribution remains unaltered. While Benzos cannot discriminate between Na ؉ and other monovalent cations, Benzop shows a three-fold selectivity for K ؉ over Na ؉ .