Inhibition of nucleotide biosynthesis potentiates the antifungal activity of amphotericin B.

The polyene antifungal agent Amphotericin B exhibits potent and broad spectrum fungicidal activity. However, high nephrotoxicity can hinder its administration in resource poor settings. Quantification of early fungicidal activity in studies of HIV patients with cryptococcosis demonstrate that 5-Fluorocytosine therapy in combination with Amphotericin B results in faster clearance than with Amphotericin B alone. In vitro synergy between the two drugs has also been reported but the mechanism by which 5-Fluorocytosine synergizes with Amphotericin B has not been delineated. In this study we set out to investigate the effect of genetic mutation or pharmacologic repression of de novo pyrimidine and purine biosynthesis pathways on the Amphotericin B susceptibility of Cryptococcus neoformans. We demonstrate that a ura- derivative of wild type Cryptococcus neoformans strain H99 is hypersensitive to Amphotericin B. This sensitivity is remediated by re-introduction of a wild type URA5 gene, but not by addition of exogenous uracil to supplement the auxotrophy. Repression of guanine biosynthesis by treatment with the inosine monophosphate dehydrogenase inhibitor, mycophenolic acid, was synergistic with Amphotericin B as determined by checkerboard analysis. As in Cryptococcus neoformans, a ura(-) derivative of Candida albicans was also hypersensitive to Amphotericin B, and treatment of Candida albicans with mycophenolic acid was likewise synergistic with Amphotericin B. In contrast, neither mycophenolic acid nor 5-FC had an effect on the Amphotericin B susceptibility of Aspergillus fumigatus. These studies suggest that pharmacological targeting of nucleotide biosynthesis pathways has potential to lower the effective dose of Amphotericin B for both C. neoformans and C. albicans. Given the requirement of nucleotide and nucleotide sugars for growth and pathogenesis of Cryptococcus neoformans, disrupting nucleotide metabolic pathways might thus be an effective mechanism for the development of novel antifungal drugs

Drug interaction between AmB and 5-FC against <i>A. fumigatus.</i>

<p>AmB: Amphotericin B, 5-FC: 5-Flourocytosine.</p><p>MICs of AmB and 5-FC alone and in combination against <i>A. fumigatus</i> in 3 separate experiments and calculated FIC values.</p

Perturbations in nucleotide biosynthesis sensitize <i>Candida albicans</i> to AmB.

<p>E-Test analysis for AmB susceptibility of (A) <i>C. albicans</i> wt and <i>ura</i>⁻ mutant on AM3 medium (B) <i>C. albicans</i> wt on AM3 medium alone or supplemented with 3.25 µg/mL MPA (C) <i>C. albicans</i> wt and <i>ura</i>⁻ mutant supplemented with 20 µM uracil or 20 µM uridine.</p

Perturbations in nucleotide metabolism do not alter the AmB susceptibility of <i>Aspergillus fumigatus</i>.

<p>E-Test analysis of <i>A. fumigatus</i> wt on AM3 medium alone or supplemented with 3.25 µg/mL MPA. (B) Checkerboard analysis to assess drug interaction between AmB and 5-FC against <i>A. fumigatus</i> using the following drug ranges; AmB 0.097 µg/mL to 50 µg/mL and 5-FC from 1.56 µg/mL to 100 µg/mL.</p

Drug interaction between AmB and MPA in <i>C. albicans.</i>

<p>AmB: Amphotericin B, MPA: Mycophenolic acid.</p><p>MICs of AmB and MPA alone and in combination against <i>C. albicans</i> in 3 separate experiments and calculated FIC values.</p

MPA potentiates the killing of <i>C. neoformans</i> by AmB.

<p>Time kill assay of <i>C. neoformans</i> in the presence of AmB at either 0.03 µg/mL or 0.06 µg/mL of AmB alone or in combination with 30 µg/mL MPA. CFU were enumerated from triplicate plates. The graph represents mean CFU +/− SEM (****<i>p</i><0.0001, ***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05).</p

A <i>C. neoformans ura</i>⁻ strain is hypersensitive to AmB.

<p>(A) E-Test for AmB susceptibility of <i>C. neoformans</i> wt, <i>ura</i>⁻ mutant and <i>ura</i>⁻ strain complemented with the wild type <i>URA5</i> gene on AM3 medium (B) E-Test for AmB susceptibility of <i>C. neoformans</i> wt and <i>ura</i>⁻ mutant on AM3 medium supplemented with 20 µM uracil and 20µM uridine.</p

AmB and MPA or 5-FC exhibit positive interactions against <i>Candida albicans</i>.

<p>(A) Checkerboard analysis of drug interaction between AmB and MPA against <i>C. albicans.</i> Assay range of concentrations for AmB was 0.0009 µg/mL to 0.5 µg/mL and MPA was 0.45 µg/mL to 30 µg/mL. (B) Checkerboard analysis of drug interaction between AmB and 5-FC against <i>C. albicans</i>. The range of drug concentration used for AmB was the same as in previous experiments, for 5-FC the range was 0.015 µg/mL to 0.96 µg/mL.</p

AmB and MPA synergize against <i>C. neoformans</i>.

<p>Assay range of concentrations for AmB is 0.0009 µg/mL to 0.5 µg/mL and for MPA is 0.45 µg/mL to 30 µg/mL. Well A12 is the sterility control (SC) and wells H1 and H12 are growth controls (GC) for MPA and AmB respectively.</p

Inhibition of guanosine biosynthesis sensitizes <i>C. neoformans</i> to AmB.

<p>(A) E-Test of <i>C. neoformans</i> wt on AM3 medium (B) AM3 medium supplemented with 3.25 µg/mL MPA and (C) E-test analysis for AmB susceptibility of <i>C. neoformans</i> wt on AM3 supplemented with 3.25 µg/mL MPA and 20 µg/mL exogenous guanine.</p