Abstract：
OBJECTIVE: This study aimed to discover novel antifungals targeting Candida albicans glyceraldehyde-3-phosphate dehydrogenase (CaGAPDH), have an insight into inhibitory mode, and provide evidence supporting CaGAPDH as a target for new antifungals.
METHODS: Virtual screening was utilized to discover inhibitors of CaGAPDH. The inhibitory effect on cellular GAPDH was evaluated by determining the levels of ATP, NAD, NADH, etc., as well as examining GAPDH mRNA and protein expression. The role of GAPDH inhibition in C. albicans was supported by drug affinity responsive target stability and overexpression experiments. The mechanism of CaGAPDH inhibition was elucidated by Michaelis-Menten enzyme kinetics and site-specific mutagenesis based on docking. Chemical synthesis was used to produce an improved candidate. Different sources of GAPDH were used to evaluate inhibitory selectivity across species. In vitro and in vivo antifungal tests, along with anti-biofilm activity, were carried out to evaluate antifungal potential of GAPDH inhibitors.
RESULTS: A natural xanthone was identified as the first competitive inhibitor of CaGAPDH. It demonstrated in vitro anti-C. albicans potential but also caused hemolysis. XP-W, a synthetic side-chain-optimized xanthone, demonstrated a better safety profile, exhibiting a 50-fold selectivity for CaGAPDH over human GAPDH. XP-W also exhibited potent anti-biofilm activity and displayed broad-spectrum anti-Candida activities in vitro and in vivo, including multi-azole-resistant C. albicans.
CONCLUSIONS: These results demonstrate for the first time that CaGAPDH is a valuable target for antifungal drug discovery, and XP-W provides a promising lead.
METHODS: Virtual screening was utilized to discover inhibitors of CaGAPDH. The inhibitory effect on cellular GAPDH was evaluated by determining the levels of ATP, NAD, NADH, etc., as well as examining GAPDH mRNA and protein expression. The role of GAPDH inhibition in C. albicans was supported by drug affinity responsive target stability and overexpression experiments. The mechanism of CaGAPDH inhibition was elucidated by Michaelis-Menten enzyme kinetics and site-specific mutagenesis based on docking. Chemical synthesis was used to produce an improved candidate. Different sources of GAPDH were used to evaluate inhibitory selectivity across species. In vitro and in vivo antifungal tests, along with anti-biofilm activity, were carried out to evaluate antifungal potential of GAPDH inhibitors.
RESULTS: A natural xanthone was identified as the first competitive inhibitor of CaGAPDH. It demonstrated in vitro anti-C. albicans potential but also caused hemolysis. XP-W, a synthetic side-chain-optimized xanthone, demonstrated a better safety profile, exhibiting a 50-fold selectivity for CaGAPDH over human GAPDH. XP-W also exhibited potent anti-biofilm activity and displayed broad-spectrum anti-Candida activities in vitro and in vivo, including multi-azole-resistant C. albicans.
CONCLUSIONS: These results demonstrate for the first time that CaGAPDH is a valuable target for antifungal drug discovery, and XP-W provides a promising lead.
摘要：
目的：本研究旨在发现针对白色念珠菌甘油醛-3-磷酸脱氢酶（CaGAPDH）的新型抗真菌药物，对抑制模式有深入的了解，并提供证据支持CaGAPDH作为新的抗真菌药物的目标。
方法：利用虚拟筛选发现CaGAPDH的抑制剂。对细胞GAPDH的抑制作用通过测定ATP的水平来评估，NAD,NADH,等。,以及检测GAPDHmRNA和蛋白质表达。GAPDH抑制在白色念珠菌中的作用得到药物亲和响应靶标稳定性和过表达实验的支持。通过Michaelis-Menten酶动力学和基于对接的位点特异性诱变阐明了CaGAPDH抑制的机制。使用化学合成来产生改进的候选物。GAPDH的不同来源用于评估物种间的抑制选择性。体外和体内抗真菌试验，以及抗生物膜活性，进行了评估GAPDH抑制剂的抗真菌潜力。
结果：天然黄吨酮被鉴定为CaGAPDH的第一个竞争性抑制剂。它在体外证明了抗C。白色念珠菌的潜力，但也引起溶血。XP-W,合成侧链优化的黄原酮，表现出更好的安全性，对CaGAPDH的选择性是人GAPDH的50倍。XP-W还表现出有效的抗生物膜活性，并在体外和体内表现出广谱抗念珠菌活性，包括耐多唑的白色念珠菌。
结论：这些结果首次表明CaGAPDH是发现抗真菌药物的有价值的靶标，和XP-W提供了一个有前途的领先优势。
方法：利用虚拟筛选发现CaGAPDH的抑制剂。对细胞GAPDH的抑制作用通过测定ATP的水平来评估，NAD,NADH,等。,以及检测GAPDHmRNA和蛋白质表达。GAPDH抑制在白色念珠菌中的作用得到药物亲和响应靶标稳定性和过表达实验的支持。通过Michaelis-Menten酶动力学和基于对接的位点特异性诱变阐明了CaGAPDH抑制的机制。使用化学合成来产生改进的候选物。GAPDH的不同来源用于评估物种间的抑制选择性。体外和体内抗真菌试验，以及抗生物膜活性，进行了评估GAPDH抑制剂的抗真菌潜力。
结果：天然黄吨酮被鉴定为CaGAPDH的第一个竞争性抑制剂。它在体外证明了抗C。白色念珠菌的潜力，但也引起溶血。XP-W,合成侧链优化的黄原酮，表现出更好的安全性，对CaGAPDH的选择性是人GAPDH的50倍。XP-W还表现出有效的抗生物膜活性，并在体外和体内表现出广谱抗念珠菌活性，包括耐多唑的白色念珠菌。
结论：这些结果首次表明CaGAPDH是发现抗真菌药物的有价值的靶标，和XP-W提供了一个有前途的领先优势。
