The aminoalkylindole, BML-190, negatively regulates chitosan synthesis via the cAMP/PKA1 pathway in Cryptococcus neoformans

Cryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immune-compromising conditions. Current antifungals are suboptimal to treat this disease, therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall, is required for survival in the mammalian host, and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis. To identify these compounds we developed a robust and novel cell-based flow cytometry screening method to identify small molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in C. neoformans. We used flow cytometry-based counter and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits. One of the confirmed hits that reduced chitosan content was the aminoalkylindole, BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the C. neoformans G-protein coupled receptor, Gpr4, and via the cAMP/PKA signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis, and could lead to potential novel therapeutics against C. neoformans.