Abstract：
Clostridioides difficile, a gram-positive anaerobic bacterium, is one of the most frequent causes of nosocomial infections. C. difficile infection (CDI) results in almost a half a million infections and approximately 30,000 deaths in the U.S. each year. Broad-spectrum antibacterial use is a strong risk factor for development of recurring CDI. There is a critical need for narrow-spectrum antibacterials with activity limited to C. difficile. The C. difficile enoyl-acyl carrier protein (ACP) reductase II enzyme (CdFabK), an essential and rate-limiting enzyme in the organism\'s fatty acid biosynthesis pathway (FAS-2), is an attractive target for narrow-spectrum CDI therapeutics as it is not present in many of the non-pathogenic gut organisms. We have previously characterized inhibitors of the CdFabK enzyme with narrow-spectrum anti-difficile activity and favorable in vivo efficacy, ADME, and low dysbiosis. To expand our knowledge of the structural requirements for CdFabK inhibition, we seek to identify new inhibitors with novel chemical scaffolds. Herein we present the optimization of a thermo-FMN biophysical assay based on the principles of differential scanning fluorimetry, or thermal shift, which leverages the fluorescence signal of the FabK enzyme\'s FMN prosthetic group. The optimized assay was validated by pilot testing a 10K diversity-based chemical library and novel scaffold hit compounds were identified and biochemically characterized. Additionally, we show that the thermo-FMN assay can be used to determine the thermodynamic dissociation constant, Kd, of CdFabK inhibitors.
摘要：
艰难梭菌，革兰氏阳性厌氧菌,是医院感染的最常见原因之一。艰难梭菌感染(CDI)在美国每年导致近50万例感染和大约30,000例死亡。广谱抗细菌的使用是复发性CDI发展的强烈风险因素。对于具有限于艰难梭菌的活性的窄谱抗菌剂存在迫切需求。艰难梭菌烯酰-酰基载体蛋白（ACP）还原酶II酶（CdFabK），生物体脂肪酸生物合成途径（FAS-2）中的一种必需和限速酶，是窄谱CDI治疗剂的有吸引力的靶标,因为它不存在于许多非致病性肠道生物体中。我们以前已经表征了CdFabK酶的抑制剂具有窄谱抗艰难菌活性和良好的体内功效，ADME,和低生态失调。为了扩大我们对CdFabK抑制的结构要求的认识，我们寻求用新型化学支架鉴定新的抑制剂。在这里，我们提出了基于差示扫描荧光分析原理的热FMN生物物理测定的优化，或热移位，利用FabK酶的FMN辅基的荧光信号。通过对基于10K多样性的化学文库进行初步测试来验证优化的测定,并鉴定和生化表征新型支架命中化合物。此外,我们表明，热-FMN测定可用于确定热力学解离常数，Kd,CdFabK抑制剂。
