Structure-guided development of fungal specific calcineurin inhibitors

摘要 免疫受损患者的系统性真菌感染具有极高的死亡率。 目前的抗真菌药物不足以保护患者免受日益增加的抗真菌耐药性和 对于新的抗真菌药物，现在比以往任何时候都更加清晰。然而，这些真核病原体中的靶标之间的相似性 而它们的人类宿主使新的抗真菌药物的开发具有挑战性。天然产物 FK506通过结合抑制真菌和人类的丝氨酸-苏氨酸特异性蛋白磷酸酶钙调神经磷酸酶 与免疫亲和素FKBP12结合，随后与钙调神经磷酸酶结合。病原真菌中的隐球菌属 新生杆菌、白色念珠菌和烟曲霉，钙调神经磷酸酶是一种关键的毒力因子 分别在人体体温下生长、血清中生长和酵母菌-菌丝二型转变。在……里面 人类需要钙调神经磷酸酶来激活T细胞和产生IL-2。事实上，FK506在临床上被用作 强效免疫抑制药。尽管真菌和哺乳动物的钙调神经磷酸酶和FKBP12是高度保守的，我们 最近发现了FKBP12在80s环上的关键氨基酸差异，这些差异位于FKBP12- FK506-钙调神经磷酸酶界面。最近开发的FK506类似物APX879被修改为 接近FKBP12 80S环的FK506(C22酮氧)。此模拟显示显著减少 免疫抑制活性在体外和隐球菌病的动物模型中仍保持抗真菌活性。我们的 中心假设是，通过结构指导的合理设计，FK506类似的钙调神经磷酸酶抑制剂可以 通过在FKBP12的80s环中引入差异相互作用，提高了真菌的特异性。在……里面 目标1，将基于预测的相互作用设计和合成定义的FK506类似物文库 具有已知的来自真菌和哺乳动物的钙调神经磷酸酶三元络合物的晶体结构。这个结构-- 活性关系将通过测试抗真菌活性的光谱和 该文库的免疫抑制活性。在AIM中，2种先导化合物将在小鼠体内进行疗效测试 隐球菌病模型和体内免疫抑制。通过定义小分子之间的相互作用 将开发钙调神经磷酸酶、FKBP12和FK506化合物，将钙调神经磷酸酶抑制作用转移到 体内进入较高抗真菌活性和较低免疫抑制活性的治疗窗口。

Abstract Systemic fungal infections in immunocompromised patients have exceedingly high mortality rates. Current antifungal drugs are not sufficient to protect patients from increasing antifungal resistance and a need for new antifungals is now clearer than ever. However, similarities between targets in these eukaryotic pathogens and their human hosts have made the development of new antifungal drugs challenging. The natural product FK506 inhibits the serine-threonine specific protein phosphatase calcineurin in both fungi and humans by binding to the immunophilin FKBP12 and subsequently binding to calcineurin. In the pathogenic fungi Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus, calcineurin is a key virulence factor required for growth at human body temperature, growth in serum, and the yeast-hyphal dimorphic transition, respectively. In humans, calcineurin is required for T-cell activation and IL-2 production. In fact, FK506 is used clinically as a potent immunosuppressant. Although fungal and mammalian calcineurin and FKBP12 are highly conserved, we have recently identified key amino acid differences in the 80s loop of FKBP12 that are located at the FKBP12- FK506-calcineurin interface. A recently developed FK506 analog, APX879, is modified at a single moiety of FK506 (C22 keto oxygen) that approaches the FKBP12 80s loop. This analog exhibits significantly reduced immunosuppressive activity yet retains antifungal activity in vitro and in an animal model of cryptococcosis. Our central hypothesis is that with structure-guided rational design, FK506 analog calcineurin inhibitors can be generated with increased fungal specificity by introducing differential interactions in the 80s loop of FKBP12. In Aim 1, a defined library of FK506 analogs will be designed and synthesized based on the predicted interactions with known crystal structures for the calcineurin ternary complexes from fungi and mammals. The structure- activity relationship will then be determined by testing the spectrum of antifungal activity and the immunosuppressive activity of this library. In Aim 2 lead compounds will be tested for in vivo efficacy in murine models of cryptococcosis and in vivo immunosuppression. By defining the small molecule interactions between calcineurin, FKBP12, and FK506, compounds will be developed that shift the efficacy of calcineurin inhibition in vivo into the therapeutic window of higher antifungal activity and reduced immunosuppressive activity.