Gowri Meiyazhagan, Sofi Beaula Winfred, Biswal Jayashree, Dhamodharan Prabhu, Saiharish Raghavan, Rohan prasad Surabi, Pitani Ravishankar, Kandaswamy Deivanayagam, Raghunathan Ragavachary, Jeyakanthan Jeyaraman, Rayala Suresh K, Venkatraman Ganesh
Department of Human Genetics, College of Biomedical Sciences, Technology & Research, Sri Ramachandra University, Porur, Chennai- 600 116, India.
Department of Bioinformatics, Alagappa University, Karaikudi- 630003, India.
Biochim Biophys Acta. 2016 Apr;1860(4):636-47. doi: 10.1016/j.bbagen.2015.12.020. Epub 2015 Dec 23.
Further quest for new anti-fungal compounds with proven mechanisms of action arises due to resistance and dose limiting toxicity of existing agents. Among the human fungal pathogens C. albicans predominate by infecting several sites in the body and in particular oral cavity and root canals of human tooth.
In the present study, we screened a library of β-lactam substituted polycyclic fused pyrrolidine/pyrrolizidine compounds against Candida sp. Detailed molecular studies were carried out with the active compound 3 on C. albicans. Morphological damage and antibiofilm activity of compound 3 on C. albicans was studied using scanning electron microscopy (SEM). Biochemical evidence for membrane damage was studied using flow cytometry. In silico docking studies were carried out to elucidate the mechanism of action of compound 3. Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model.
Screening data showed that several new compounds were active against Candida sp. Among them, Compound 3 was most potent and exerted time kill effect at 4h, post antifungal effect up to 6h. When used in combination with fluconazole or nystatin, compound 3 revealed an minimum inhibitory concentration (MIC) decrease by 4 fold for both drugs used. In-depth molecular studies with compound 3 on C. albicans showed that this compound inhibited yeast to hyphae (Y-H) conversion and this involved the cAMP pathway. Further, SEM images of C. albicans showed that compound 3 caused membrane damage and inhibited biofilm formation. Biochemical evidence for membrane damage was confirmed by increased propidium iodide (PI) uptake in flow cytometry. Further, in silico studies revealed that compound 3 docks with the active site of the key enzyme 14-α-demethylase and this might inhibit ergosterol synthesis. In support of this, ergosterol levels were found to be decreased by 32 fold in compound 3 treated samples as analyzed by high performance liquid chromatography (HPLC). Further, the antifungal activity of compound 3 was evaluated in an ex vivo dentinal tubule infection model, which mimics human tooth root canal infection. Confocal laser scanning microscopy studies showed 83% eradication of C. albicans and a 6 log reduction in colony forming unit (CFU) after 24h treatment in the infected tooth samples in this model.
Compound 3 was found to be very effective in eradicating C. albicans by inhibiting cAMP pathway and ergosterol biosynthesis.
The results of this study can pave the way for developing new antifungal agents with well deciphered mechanisms of action and can be a promising antifungal agent or medicament against root canal infection.
由于现有抗真菌药物的耐药性和剂量限制性毒性，人们进一步寻求具有已证实作用机制的新型抗真菌化合物。在人类真菌病原体中，白色念珠菌占主导地位，它可感染人体多个部位，尤其是口腔和人类牙齿的根管。
在本研究中，我们针对念珠菌属筛选了一个β-内酰胺取代的多环稠合吡咯烷/吡咯里西啶化合物库。对活性化合物3进行了针对白色念珠菌的详细分子研究。使用扫描电子显微镜（SEM）研究了化合物3对白色念珠菌的形态损伤和抗生物膜活性。使用流式细胞术研究了膜损伤的生化证据。进行了计算机对接研究以阐明化合物3的作用机制。此外，在体外牙本质小管感染模型中评估了化合物3的抗真菌活性。
筛选数据表明，几种新化合物对念珠菌属具有活性。其中，化合物3最有效，在抗真菌作用4小时后发挥时间杀灭作用，后效应长达6小时。当与氟康唑或制霉菌素联合使用时，化合物3使两种药物的最低抑菌浓度（MIC）降低了4倍。对化合物3针对白色念珠菌进行的深入分子研究表明，该化合物抑制酵母向菌丝（Y-H）的转化，这涉及cAMP途径。此外，白色念珠菌的SEM图像显示化合物3导致膜损伤并抑制生物膜形成。流式细胞术中碘化丙啶（PI）摄取增加证实了膜损伤的生化证据。此外，计算机研究表明化合物3与关键酶14-α-脱甲基酶的活性位点对接，这可能抑制麦角甾醇的合成。支持这一点的是，通过高效液相色谱（HPLC）分析发现，在化合物3处理的样品中麦角甾醇水平降低了32倍。此外，在模拟人类牙根管感染的体外牙本质小管感染模型中评估了化合物3的抗真菌活性。共聚焦激光扫描显微镜研究表明，在该模型中，感染牙齿样品在处理24小时后，白色念珠菌的根除率为83%，菌落形成单位（CFU）减少了6个对数。
发现化合物3通过抑制cAMP途径和麦角甾醇生物合成，在根除白色念珠菌方面非常有效。
本研究结果可为开发具有明确作用机制的新型抗真菌药物铺平道路，并且可能成为一种有前景的抗根管感染的抗真菌剂或药物。
