Exploring therapeutic targets for cryptococcosis: in silico and in vitro testing for isocitrate lyase (ICL1) potential inhibitors.

Cryptococcosis, caused by Cryptococcus neoformans and Cryptococcus gattii, remains a severe fungal infection, with current antifungal treatments facing challenges such as toxicity, prolonged therapy, and resistance. Isocitrate lyase (ICL1), a key enzyme in the glyoxylate cycle, is a potential antifungal target. This study combined in silico and in vitro approaches to identify ICL1 inhibitors. Virtual screening of FDA-approved drugs selected five candidates: bufexamac, isoniazid, nifuraldezone, nifuroxazide, and ribavirin. Molecular dynamics simulations and binding free energy calculations highlighted π-interactions with Trp97 as crucial for ligand stabilization, with isoniazid emerging as a top candidate due to strong binding and structural stability. In vitro testing confirmed isoniazid's antifungal activity against Cryptococcus spp., but MIC values were high, indicating variable susceptibility. For C. neoformans, ATCC 499 showed the highest MIC (70 mg/mL), while IEC-Crypto01 exhibited 35 mg/mL. For C. gattii, ATCC R265 displayed 2.19 mg/mL, and IEC-Crypto04 was inhibited at 8.75 mg/mL. These results suggest a strain-dependent response and a limited direct antifungal effect at high concentrations. However, previous reports showed that isoniazid also inhibits cryptococcal biofilm formation, reinforcing its potential role in combination therapies. Additionally, the data suggests a dual mechanism of action, targeting both metabolism and membrane integrity. This study provides novel insights into ICL1 inhibition and contributes to drug repurposing efforts for cryptococcosis. Despite high MIC values, isoniazid's antifungal activity warrants further investigation, particularly in synergistic combinations with existing antifungals.