Structure-guided development of fungal specific calcineurin inhibitors

摘要 免疫功能低下患者的全身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抗真菌药物耐药性增加的影响， 寻找新的抗真菌药物的可能性比以往任何时候都要清楚。然而，这些真核病原体中靶点之间的相似性 以及它们的人类宿主使得新的抗真菌药物的开发具有挑战性。天然产物 FK 506通过结合丝氨酸-苏氨酸特异性蛋白磷酸酶钙调神经磷酸酶抑制真菌和人类中的丝氨酸-苏氨酸特异性蛋白磷酸酶钙调神经磷酸酶 亲免素FKBP 12，随后结合钙调磷酸酶。在病原真菌隐球菌中 新型念珠菌、白色念珠菌和烟曲霉，钙调神经磷酸酶是一个关键的毒力因子， 分别在人体温度下生长、在血清中生长和酵母-菌丝二型转变。在 在人类中，钙调磷酸酶是T细胞活化和IL-2产生所必需的。事实上，FK 506在临床上被用作 强效免疫抑制剂虽然真菌和哺乳动物的钙调神经磷酸酶和FKBP 12是高度保守的，我们 最近已经确定了FKBP 12的80 s环中的关键氨基酸差异，这些差异位于FKBP 12- FK 506-钙调磷酸酶界面。最近开发的FK 506类似物APX 879在以下单个部分进行修饰： 接近FKBP 12 80 s环的FK 506（C22酮氧）。该类似物表现出显著降低的 免疫抑制活性在体外和隐球菌病的动物模型中仍保留抗真菌活性。我们 中心假设是，通过结构指导的合理设计，FK 506类似物钙调磷酸酶抑制剂可以 通过在FKBP 12的80 s环中引入差异相互作用而产生具有增加的真菌特异性。在 目的1，基于预测的相互作用，设计并合成确定的FK 506类似物库 已知来自真菌和哺乳动物的钙调神经磷酸酶三元复合物的晶体结构。结构- 然后通过测试抗真菌活性谱和抗真菌活性谱来确定活性关系。 该文库的免疫抑制活性。在目的2中，将测试先导化合物在鼠中的体内功效。 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、FKBP 12和FK 506等化合物，以改变钙调神经磷酸酶抑制的功效。 体内进入更高的抗真菌活性和降低的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.