Novel approaches to antifungal drug delivery

This study describes the development and characterization of novel antifungal agents directed towards the opportunistic pathogen Candida albicans. Six aromatic dipetidyl derivatives of uracil polyoxin C were sjmthesized. With the exception of (D)-Trp-UPOC these compounds strongly inhibited chitin synthetase from C. albicans with inhibition constants in the micromolar range. Seven nonaromatic dipeptdyl polyoxins were synthesized, several of which incorporated peptidase resistant chemical modifications. None of these compounds effectively competed with the entry of labelled trimethionine into C. albicans. N-alkyaltion of Nle-UPOC with either N-methyl or N-npropyl groups, or amidation of the C₅’ carboxyl group resulted in a significant reduction in chitin synthetase inhibitory activity. Two novel polyoxins, Oct-Lys-UPOC and Oct-Gln-UPOC, incorporating hydrophobic amino acid side chains were strong inhibitors of C. albicans chitin synthetase with Ki's equal to 1.7 x 10^-6 M. Both compounds were resistant to hydrolysis by a cell extract of C. albicans, however, Oct-Gln-UPOC was hydrolyzed by a permeabilized cell preparation with a half-life equal to 23 min. Oct-Lys-UPOC and Oct-Gln-UPOC were ineffective in the inhibition of cell growth or reduction of cell viability at millimolar concentrations, but they induced aberrant morphologies at concentrations of 0.25 mM. Five tripeptidyl polyoxins were developed, two of which incorporated peptidase resistant chemical modifications. Leu-Polyoxin D, Nle-UPOC-Leu, and Leu-Nle-UPOC did not inhibit a C. albicans chitin synthetase membrane preparation. In whole cells of C. albicans, these compounds inhibited growth and affected cell morphology similarly to Polyoxin D. Leu-Nle-UPOC competitively inhibited the transport of labelled trimethionine with a Ki equal to 1.95 x 10^-4 M. Leu-Polyoxin D and Leu-Nle-UPOC represent prodrugs which are hydrolyzed within cells of C. albleans to toxic compounds. Phe-Δ-Phe-UPOC and Oct-Phe-Polyoxin D were strong inhibitors of chitin synthetase present in either a mixed-membrane preparation or in permeabilized cells. Both compounds were resistant to hydrolysis by C. albicans peptldases. Many of the analogues caused morphological alterations of the yeast in culture, and a number of the analogues killed C albicans at millimolar concentrations. The presented Insights into the structure function relationship of the polyoxins to chitin synthetase will aid in the future development of polyoxin derived antifungal agents.