靶向单基因模型构建与新型小分子化合物CB抗衣原体分子作用机制研究

Chlamydiae are widely distributed and have high prevalence in human populations. Although chlamydiae remain sensitive to several classes of antibiotics, antibiotic-resistant variants are reported at times. The identification of chlamydia-specific inhibitory compounds might be clinically significant. Recently, we identified a novel family of small compounds exemplified by N'-(3,5-dibromo-4-hydroxybenzylidene)-3-nitrobenzohydrazide (CB) that inhibited all chlamydia species tested without detectable effects on the host cells. We isolated a CB-resistant Chlamydia muridarum variant, designated MCR. Whole genome sequencing revealed four point mutations in MCR. Three mutations were in the coding region of MOMP, TC0412 and GrgA respectively, and the last one was in the 5'-noncoding region of the ADP/ATP translocase gene. In this study, a chlamydia-derived plasmid pGFP:SW2 that was reconstructed to express exogenous gene or antisense RNA will be used as shuttle vector to transform the plasmid-free chlamydia muridarum (Nigg). Stable transformants will be isolated and used as research models to evaluate single-gene based functional changes in chlamydia. Relying on this model, we can explore the relationship between CB-resistant and these mutations-assocaited genes one by one. In this project, we aim at clarifying the molecular mechanism underlying the inhibitory effect of CB and identifying novel genes that can be used as targets for anti-chlamydial drugs development.

衣原体感染容易引起包括性传播疾病和肺炎在内的多种疾病的高发。抗生素一直是治疗衣原体感染的首选药物，然而抗生素耐受的衣原体菌株时有发现，给抗生素治疗衣原体感染带来了潜在的风险，亟需寻找并发现新型抗衣原体特效药物。N'-(3,5-二溴-4-羟基苯亚甲基)-3-硝基苯酰肼（CB）是一种新型抗衣原体小分子化合物，能够有效抑制多种衣原体的增殖。通过筛选出具有CB抗性的衣原体克隆株MCR，对其进行全基因组测序，发现了4个可能是CB潜在分子靶标的突变位点。本项目借助衣原体质粒作为模板，构建可表达外源基因和反义RNA的载体；通过筛选稳定表达外源基因或者反义RNA的转化衣原体，在衣原体上建立针对单个基因的研究模型，逐个探讨这些突变位点与CB药物抗性之间的关系。本研究有助于阐明CB抗衣原体作用的分子机制，在此基础上找到新的可作为抗衣原体药物开发的分子靶标，为抗衣原体药物的研发提供新的思路。

传统的抗生素治疗衣原体感染存在潜在的风险，而抗衣原体疫苗的研制也一直未获成功，寻找新型抗衣原体化合物意义重大。CF0001是一类新型抗衣原体小分子化合物，本研究通过逐次提高药物浓度的方式，筛选出具有CF0001抗性的衣原体突变株MCR；比较化合物对野生型MoPn和MCR的抑制效果，发现MCR对INP0007更敏感，而且CF0001的抗衣原体作用与抑制T3SS和铁饥饿无关，MCR全基因组测序发现的4个点突变也与T3SS无关，这些结果都证明CF0001是一类与INP0007作用机制完全不同的新型抗衣原体化合物；逐个分析MCR点突变与CF0001抗性之间的关系时发现，任何一个单独的点突变都不能形成CF0001抗性；设计合成了一批CF0001对羟亚苄基酰肼类衍生物，其中化合物4d的抗衣原体活性最强，它的IC50值为3.55 μM，强于CF0001的7.30 μM。为了进一步寻找具有抗衣原体活性的小分子化合物，本研究继续筛选了一系列不同来源的化合物；首先发现了海洋来源的3个双吲哚类化合物，其中化合物1的活性最强，IC50值为46.6 ~ 96.4 μM，它们通过持续的药物作用抑制衣原体的增殖过程，不影响EB的感染能力；其次筛选到一类吡咯3取代衍生物，它们都具有较好的抗衣原体活性，IC50值在1 μM附近，强于上述CF0001衍生物4d，它们也通过持续的药物作用抑制衣原体的增殖过程，对EB的感染能力没有直接影响；最后筛选到一类吡咯4取代衍生物，虽然它们的细胞毒性都很低，但抗衣原体活性普遍不如3取代衍生物，最强化合物10的IC50值为1.70 ~ 3.87 μM，它们既能抑制衣原体的增殖过程，也能直接降低EB的感染能力。本研究为抗衣原体药物的研发提供了一些新的方向和思路。