几丁质与葡聚糖是真菌细胞壁特有的主要组分，但其生物合成途径中单一调控基因缺陷不能使菌体死亡，而二者合成途径中的调控基因双缺陷是致死的。前期合作研究以真菌细胞壁合成通路为靶标，创新性地利用几丁质与葡聚糖合成双阻断致死这一崭新的遗传学思路进行抗真菌药物筛选, 得到多个阳性化合物。本研究拟以得到的苗头化合物为探针进行深入抗真菌机制与靶标确认研究，以期发现新型抗真菌先导物和新的细胞壁药靶；前期合作中，在申请者指导下成功应用酵母双杂交进行了以L12-L10蛋白相互作用为靶标的新型抗结核药物的筛选及研究, 本研究将进一步拓展酵母双杂交技术在新药发现中的应用，以革兰氏阴性菌脂多糖转运过程中的转运蛋白相互作用为靶标，通过酵母双杂交系统筛选LptA-LptD和LptA-LptE相互作用抑制剂，深入研究阳性化合物阻断相互作用机制，探索其作为药物靶标的可行性，从而发现新型高效低毒的抗耐药革兰氏阴性菌先导化合物。

Yeast mutants lacking genes involved in the synthesis of either glucan or chitin, the two major components of fungal cell wall, are viable, but the combination of both defects leads to synthetic lethality. Based on this new concept, we performed high throughput screen for small molecules that inhibit the synthesis of glucan or chitin, and isolated dozens candidate compounds. This proposal will further elucidate the molecular mechanism for these candidates, which will not only find new leading compounds targeting fungal cell wall but also identify new targets for antifungal agents. Our collaboration has also established the methodology for the screen of anti-TB drugs that disrupt the interaction of two ribosome proteins L10 and L12 using yeast two-hybrid assay, and this technology enables us to isolate compounds that disrupts a specific protein-protein interaction. This proposal will apply the established two-hybrid assay system to identify new antibacterial drugs that block transport of lipopolysaccharide (LPS) to outer membrane in gram-negative bacteria. We will first use the yeast two-hybrid assay to confirm the interactions between E. coli proteins LptA-LptD and LptA-LptE involved in LPS transport and further isolate compounds that inhibit these interactions. We expect to isolate new leading compounds that inhibit the cell growth of gram-negative bacteria with low toxicity.