Discovery of GS-9131: Design, synthesis and optimization of amidate prodrugs of the novel nucleoside phosphonate HIV reverse transcriptase (RT) inhibitor GS-9148.

GS-9148 [(5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonic acid] 4 is a novel nucleoside phosphonate HIV-1 reverse transcriptase (RT) inhibitor with a unique resistance profile toward N(t)RTI resistance mutations. To effectively deliver 4 and its active phosphorylated metabolite 15 into target cells, a series of amidate prodrugs were designed as substrates of cathepsin A, an intracellular lysosomal carboxypeptidase highly expressed in peripheral blood mononuclear cells (PBMCs). The ethylalaninyl phosphonamidate prodrug 5 (GS-9131) demonstrated favorable cathepsin A substrate properties, in addition to favorable in vitro intestinal and hepatic stabilities. Following oral dosing (3mg/kg) in Beagle dogs, high levels (>9.0microM) of active metabolite 15 were observed in PBMCs, validating the prodrug design process and leading to the nomination of 5 as a clinical candidate.

Design, synthesis, and anti-HIV activity of 4'-modified carbocyclic nucleoside phosphonate reverse transcriptase inhibitors.

A diphosphate of a novel cyclopentyl based nucleoside phosphonate with potent inhibition of HIV reverse transcriptase (RT) (20, IC(50)=0.13 microM) has been discovered. In cell culture the parent phosphonate diacid 9 demonstrated antiviral activity EC(50)=16 microM, within two-fold of GS-9148, a prodrug of which is currently under clinical investigation, and within 5-fold of tenofovir (PMPA). In vitro cellular metabolism studies using 9 confirmed that the active diphosphate metabolite is produced albeit at a lower efficiency relative to GS-9148.

Chalcomycin biosynthesis gene cluster from Streptomyces bikiniensis: novel features of an unusual ketolide produced through expression of the chm polyketide synthase in Streptomyces fradiae.

Chalcomycin, a 16-membered macrolide antibiotic made by the bacterium Streptomyces bikiniensis, contains a 2,3-trans double bond and the neutral sugar D-chalcose in place of the amino sugar mycaminose found in most other 16-membered macrolides. Degenerate polyketide synthase (PKS)-specific primers were used to amplify DNA fragments from S. bikiniensis with very high identity to a unique ketosynthase domain of the tylosin PKS. The resulting amplimers were used to identify two overlapping cosmids encompassing the chm PKS. Sequencing revealed a contiguous segment of >60 kb carrying 25 putative genes for biosynthesis of the polyketide backbone, the two deoxysugars, and enzymes involved in modification of precursors of chalcomycin or resistance to it. The chm PKS lacks the ketoreductase and dehydratase domains in the seventh module expected to produce the 2,3-double bond in chalcomycin. Expression of PKS in the heterologous host Streptomyces fradiae, from which the tyl genes encoding the PKS had been removed, resulted in production of at least one novel compound, characterized as a 3-keto 16-membered macrolactone in equilibrium with its 3-trans enol tautomer and containing the sugar mycaminose at the C-5 position, in agreement with the structure predicted on the basis of the domain organization of the chm PKS. The production of a 3-keto macrolide from the chm PKS indicates that a discrete set of enzymes is responsible for the introduction of the 2,3-trans double bond in chalcomycin. From comparisons of the open reading frames to sequences in databases, a pathway for the synthesis of nucleoside diphosphate-D-chalcose was proposed.

Production of hybrid 16-membered macrolides by expressing combinations of polyketide synthase genes in engineered Streptomyces fradiae hosts.

Combinations of the five polyketide synthase (PKS) genes for biosynthesis of tylosin in Streptomyces fradiae (tylG), spiramycin in Streptomyces ambofaciens (srmG), or chalcomycin in Streptomyces bikiniensis (chmG) were expressed in engineered hosts derived from a tylosin-producing strain of S. fradiae. Surprisingly efficient synthesis of compounds predicted from the expressed hybrid PKS was obtained. The post-PKS tailoring enzymes of tylosin biosynthesis acted efficiently on the hybrid intermediates with the exception of TylH-catalyzed hydroxylation of the methyl group at C14, which was efficient if C4 bore a methyl group, but inefficient if a methoxyl was present. Moreover, for some compounds, oxidation of the C6 ethyl side chain to an unprecedented carboxylic acid was observed. By also expressing chmH, a homolog of tylH from the chalcomycin gene cluster, efficient hydroxylation of the 14-methyl group was restored.