角膜营养不良是一组遗传遗传性疾病,其TGFBI基因突变会影响Bowman膜和角膜基质。突变型TGFBIp高度易于聚集并沉积在角膜中。根据突变的类型,蛋白质沉积物可能会有所不同(淀粉样蛋白,无定形粉状骨料或两者的混合形式),使角膜不透明,从而降低视敏度。发现突变蛋白的聚集是特异性的,具有不同于角膜的独特聚集机制。据报道,与WT蛋白相比,突变蛋白的蛋白水解加工不同。突变蛋白的蛋白水解加工产生高度淀粉样多肽片段。目前的治疗选择,可供患者使用,与高复发率相关的组织置换手术。对于角膜营养不良患者的简单治疗选择的临床需要已经变得非常必要,以防止蛋白质聚集或溶解预先形成的聚集体。这里,我们报告了使用弱亲和色谱(WAC)从MaybridgeRO3片段库中筛选2500种化合物。通过15N-HSQCNMR测定验证来自WAC的初级命中,并鉴定结合的特定区域。重组突变蛋白(R555W和H572R的第4个FAS-1结构域)与通过NMR测定鉴定的先导化合物一起通过胰蛋白酶进行有限的蛋白水解。铅化合物(MO07617,RJF00203和,BTB05094)可有效延迟/防止R555W突变体中淀粉样肽的产生,化合物(RJF00203和BTB05094)可有效延迟/防止H572R突变体中淀粉样肽的产生。因此,本文报道的先导化合物在进一步验证和/或修饰后,可能被建议作为潜在的治疗选择,通过抑制TGFBI-角膜营养不良中淀粉样肽的形成来预防/延迟聚集。
Corneal dystrophies are a group of genetically inherited disorders with mutations in the TGFBI gene affecting the Bowman\'s membrane and the corneal stroma. The mutant TGFBIp is highly aggregation-prone and is deposited in the cornea. Depending on the type of mutation the protein deposits may vary (amyloid, amorphous powdery aggregate or a mixed form of both), making the cornea opaque and thereby decreases visual acuity. The aggregation of the mutant protein is found to be specific with a unique aggregation mechanism distinct to the cornea. The proteolytic processing of the mutant protein is reported to be different compared to the WT protein. The proteolytic processing of mutant protein gives rise to highly amyloidogenic peptide fragments. The current treatment option, available for patients, is tissue replacement surgery that is associated with high recurrence rates. The clinical need for a simple treatment option for corneal dystrophy patients has become highly essential either to prevent the protein aggregation or to dissolve the preformed aggregates. Here, we report the screening of 2500 compounds from the Maybridge RO3 fragment library using weak affinity chromatography (WAC). The primary hits from WAC were validated by 15N-HSQC NMR assays and specific regions of binding were identified. The recombinant mutant proteins (4th FAS-1 domain of R555W and H572R) were subjected to limited proteolysis by trypsin together with the lead compounds identified by NMR assays. The lead compounds (MO07617, RJF00203 and, BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the R555W mutant and compounds (RJF00203 and BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the H572R mutant. Thus the lead compounds reported here upon further validation and/or modification might be proposed as a potential treatment option to prevent/delay aggregation by inhibiting the formation of amyloidogenic peptides in TGFBI-corneal dystrophy.