A-33853

The first synthesis and biological evaluation of antibiotic 31 (A-33853) and its analogues are reported. Initial screening for inhibition of L. donovani, T. b. rhodesiense, T. cruzi, and P. falciparum cultures followed by determination of IC(50) in L. donovani and cytotoxicity on L6 cells revealed 31 to be 3-fold more active than miltefosine, a known antileishmanial drug. Compounds 14, 15, and 25 selectively inhibited L. donovani at nanomolar concentrations and showed much lower cytotoxicity.

The structure of antibiotic A33853, isolated from the culture broth of Streptomyces sp., NRRL 12068, is reported. The structure was deduced from an X-ray crystallographic study of its tetraacetyl derivative. Tetraacetyl A33853 is unique because it contains an anhydride moiety, an unexpected product from the reaction of A33853 with acetic anhydride and pyridine.

A33853, which shows excellent bioactivity against Leishmania, is a benzoxazole-family compound formed from two moieties of 3-hydroxyanthranilic acid and one 3-hydroxypicolinic acid. In this study, we have identified the gene cluster responsible for the biosynthesis of A33853 in Streptomyces sp. NRRL12068 through genome mining and heterologous expression. Bioinformatics analysis and functional characterization of the orfs contained in the gene cluster revealed that the biosynthesis of A33853 is directed by a group of unusual enzymes. In particular, BomK, annotated as a ketosynthase, was found to catalyze the amide bond formation between 3-hydroxypicolinic and 3-hydroxyanthranilic acid during the assembly of A33853. BomJ, a putative ATP-dependent coenzyme A ligase, and BomN, a putative amidohydrolase, were further proposed to be involved in the benzoxazole formation in A33853 according to gene deletion experiments. Finally, we have successfully utilized mutasynthesis to generate two analogs of A33853, which were reported previously to possess excellent anti-leishmanial activity.