Inositol polyphosphate kinases: Investigating their function and targeting potential for new antifungal drug development

Abstract

Invasive fungal infections kill ~1.5 million people annually. Existing drugs target the cell wall and ergosterol but have drawbacks including toxicity and a narrow efficacy range. Resistance is also emerging. Our aim is to target inositol polyphosphate (IP) synthesis to develop a novel class of antifungal with a different mode of action. Genetically ablating the kinase that produces IP4/5 (IP3-4K) in the human fungal pathogens Cryptococcus neoformans and Candida albicans leads to cellular defects and loss of virulence in mouse models, suggesting that IP3-4K inhibition would be effective. We used rational design on a dibenzylpurine compound, TNP, to produce 38 analogues. Compounds 13, 30, and 42 inhibited CnArg1 at low µM IC50 (10-30 µM) and up to 6-fold more selective than HsIPMK but did not inhibit CaIpk2. However, their solubility was insufficient for antifungal testing. Further efforts created four compounds with improved potency and solubility. Lead compound DT-23 inhibited CaIpk2 (IC50 = 41 µM) and CnArg1 (IC50 = 0.6 µM), a 17-fold improvement, and inhibited IP synthesis and growth of both pathogens, especially with amphotericin B. DT-23 represents a new chemical class without dibenzyl extensions. Two other IPK inhibitor scaffolds were also explored: spiro-indoles and benzoisoxazoles. Of 11 spiro-indoles, one inhibited CnArg1 (IC50 ~2 µM). Sixteen benzoisoxazoles showed strong inhibition of CnArg1 and CaIpk2 (low nM IC50s) but were not antifungal. We also attempted to crystallize CnArg1, creating five deletion constructs. Three expressed well and were active, confirming the proline-rich region's importance for stability, although crystals did not form. We confirmed IP3-4K catalytic activity's role in fungal virulence, supporting Arg1's function through its catalytic product, IP7. Unlike ScIpk2 and HsIPMK, CnArg1 and CaIpk2 did not display PI3K activity, highlighting functional differences and supporting compromised PLC activity when Arg1 is blocked.