4-Hydroxy-5,6-dihydropyrones as inhibitors of HIV protease: the effect of heterocyclic substituents at C-6 on antiviral potency and pharmacokinetic parameters.

Due largely to the emergence of multi-drug-resistant HIV strains, the development of new HIV protease inhibitors remains a high priority for the pharmaceutical industry. Toward this end, we previously identified a 4-hydroxy-5,6-dihydropyrone lead compound (CI-1029, 1) which possesses excellent activity against the protease enzyme, good antiviral efficacy in cellular assays, and promising bioavailability in several animal species. The search for a suitable back-up candidate centered on the replacement of the aniline moiety at C-6 with an appropriately substituted heterocyle. In general, this series of heterocyclic inhibitors displayed good activity (in both enzymatic and cellular tests) and low cellular toxicity; furthermore, several analogues exhibited improved pharmacokinetic parameters in animal models. The compound with the best combination of high potency, low toxicity, and favorable bioavailabilty was (S)-3-(2-tert-butyl-4-hydroxymethyl-5-methyl-phenylsulfanyl)-4-hydroxy-6-isopropyl-6-(2-thiophen-3-yl-ethyl)-5,6-dihydro-pyran-2-one (13-(S)). This thiophene derivative also exhibited excellent antiviral efficacy against mutant HIV protease and resistant HIV strains. For these reasons, compound 13-(S) was chosen for further preclinical evaluation.

Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability.

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of > 80 degrees C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.

5,6-Dihydropyran-2-ones possessing various sulfonyl functionalities: potent nonpeptidic inhibitors of HIV protease.

On the basis of previous SAR findings and molecular modeling studies, a series of compounds were synthesized which possessed various sulfonyl moieties substituted at the 4-position of the C-3 phenyl ring substituent of the dihydropyran-2-one ring system. The sulfonyl substituents were added in an attempt to fill the additional S(3)' pocket and thereby produce increasingly potent inhibitors of the target enzyme. Racemic and enantiomerically resolved varieties of selected compounds were synthesized. All analogues in the study displayed decent binding affinity to HIV protease, and several compounds were shown to possess very good antiviral efficacy and safety margins. X-ray crystallographic structures confirmed that the sulfonamide and sulfonate moieties were filling the S(3)' pocket of the enzyme. However, the additional substituent did not provide improved enzymatic inhibitory or antiviral activity as compared to the resolved unsubstituted aniline. The addition of the sulfonyl moiety substitution does not appear to provide favorable pharamacokinectic parameters. Selected inhibitors were tested for antiviral activity in clinical isolates and exhibited similar antiviral activity against all of the HIV-1 strains tested as they did against the wild-type HIV-1. In addition, the inhibitors exhibited good antiviral efficacies against HIV-1 strains that displayed resistance to the currently marketed protease inhibitors.

4-hydroxy-5,6-dihydro-2H-pyran-2-ones.3. Bicyclic and hetero-aromatic ring systems as 3-position scaffolds to bind to S1' and S2' of the HIV-1 protease enzyme.

5,6-Dihydro-2H-pyran-2-ones are potent inhibitors of HIV-1 protease, which bind to the S1, S2, S1', and S2' pockets and have a unique binding mode with the catalytic aspartyl groups and the flap region of the enzyme. Efforts to explore 3-position heterocyclic scaffolds that bind to the S1' and S2' pockets have provided a number of selected analogs that display high HIV-1 protease inhibitory activity. reserved.

Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor.

With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfa nyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC50 of 200 nM with a therapeutic index of > 1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. Cmax and absolute bioavailability of 34S were higher and half-life and time above EC95 were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.

Identification of glu-277 as the catalytic nucleophile of Thermoanaerobacterium saccharolyticum beta-xylosidase using electrospray MS.

Thermoanaerobacterium saccharolyticum beta-xylosidase is a member of family 39 of the glycosyl hydrolases. This grouping comprises both retaining beta-d-xylosidases and alpha-l-iduronidases. T. saccharolyticum beta-xylosidase catalyses the hydrolysis of short xylo-oligosaccharides into free xylose via a covalent xylosyl-enzyme intermediate. Incubation of T. saccharolyticum beta-xylosidase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-xyloside resulted in time-dependent inactivation of the enzyme (inactivation rate constant ki=0.089 min-1, dissociation constant for the inactivator Ki=65 microM) through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-d-xylosyl-enzyme, as observed by electrospray MS. Removal of excess inactivator and regeneration of the free enzyme through transglycosylation with either xylobiose or thiobenzyl xyloside demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-alpha-d-xylosyl-enzyme intermediate and subsequent analysis by electrospray ionization triple-quadrupole MS in the neutral-loss mode indicated the presence of a 2-deoxy-2-fluoro-alpha-d-xylosyl peptide. Sequence determination of the labelled peptide by tandem MS in the daughter-ion scan mode permitted the identification of Glu-277 (bold and underlined) as the catalytic nucleophile within the sequence IILNSHFPNLPFHITEY.