Pathogen-Specific Regulation of Protein Assembly

描述（由申请人提供）：世界人口的六分之一患有传染病，如果没有其他原因，而只是未来几乎肯定会出现突变株和新病原体，则需要涉及不太可能产生耐药性的新机制的新药剂。胆色素原合成酶（PBGS; EC 4.2.1.24）是卟啉生物合成途径中的必需酶。我们发现PBGS活性的调节依赖于蛋白质的组装状态，其中某些形式具有功能活性，而其他形式则没有。PBGS已被证明存在于多种高活性八聚体和低活性六聚体中，其相互转换是在两种不同的二聚体构象的水平上。我们预测，通过控制体内非添加剂组装状态来调节酶功能可能是自然界和蛋白质类中常见的，称为morpheein概念。我们已经确定了改变PBGS组装状态并抑制功能的小分子。例如，“morphlock-1”（MW 413）是通过计算分析无活性豌豆（豌豆）PBGS六聚体的结构选择的，因为预测它结合在由三个单独的蛋白质亚基形成的空腔中。该空腔存在于非活性六聚体中，而不存在于活性八聚体中。体外研究表明，morphlock-1稳定了六聚体，导致活性八聚体PBGS完全消失，并且在相同的浓度和条件下影响功能丧失。考虑到亚基之间蛋白质-蛋白质界面所涉及的结合位点的性质，我们认为不太可能对使用这种方法发现的化合物产生耐药性。我们还鉴定了三种化合物，其阻断NIAID类别B优先病原体小肠结肠炎耶尔森氏菌中生命必需的PBGS，但不阻断人PBGS，并且在Y.小肠结肠炎菌区域抑制测定。这些探针提供了令人兴奋的验证，即蛋白质组装状态的病原体特异性调节与病原体的杀灭相关，但由于它们的大尺寸和非专有来源，它们很可能不是候选药物。因此，SBIR第一阶段资助的目的是利用我们迄今为止开发的信息，结合X射线晶体学的新见解（如果有的话），并在出现时进行进一步筛选，以制备化合物库，提供行业标准度量和标准定义的先导化合物和临床前候选药物。我们将使用的方法包括迭代效价结构活性关系开发、选择性和eADME（早期吸收、分布、代谢、排泄）测试和评价。在第一阶段SBIR的第二年年底，我们将确定2-3种化合物用于第二阶段的临床前和临床开发。此外，在这项工作的过程中，我们还将审查可能的扩展超过Y。在一些实施方案中，所述抗肿瘤药物包括抗小肠结肠炎菌、恶性疟原虫、铜绿假单胞菌、霍乱弧菌和刚地弓形虫。 公共卫生相关性：世界人口的六分之一患有传染病，需要涉及新机制的小分子疗法，特别是那些不太可能产生耐药性的小分子疗法。我们在此描述了一种用于发现抗感染剂的新方法，其中可以通过调节蛋白质的组装状态来实现病原体中必需酶靶标的物种特异性功能丧失。我们首先寻求将我们的策略应用于发现治疗小肠结肠炎耶尔森氏菌（NIAID B类优先病原体）的药物。

DESCRIPTION (provided by applicant): One sixth of the world's population has an infectious disease, and new agents involving novel mechanisms less likely to engender resistance are needed if for no other reason than the almost certain emergence of mutant strains and new pathogens in the future. Porphobilinogen synthase (PBGS; EC 4.2.1.24) is an essential enzyme in the porphyrin biosynthesis pathway. We have found that regulation of the activity of PBGS is dependent upon the assembly state of the protein, in which certain forms are functionally active and others not. PBGS has been shown to exist in a manifold of high-activity octamers and low-activity hexamers whose interconversion is at the level of two different dimer conformations. We predict that regulation of enzyme function by control of nonadditive assembly states in vivo may be common through nature and protein class, termed the morpheein concept. We have identified small-molecules that alter the assembly state of PBGS and inhibit function. For example, "morphlock-1" (MW 413) was selected by computational analysis of the structure of the inactive Pisum sativum (pea) PBGS hexamer because it was predicted to bind in a cavity formed by three of the individual protein subunits. This cavity is present in the inactive hexamer and not in the active octamer. In vitro studies have demonstrated morphlock-1 stabilized the hexamer, causing complete disappearance of the active octameric PBGS, and at the same concentration and conditions effected loss of function. Given the nature of the binding site involved in the protein-protein interface between subunits, we believe it is unlikely that resistance will develop to compounds discovered using this approach. We have also identified three compounds that block the PBGS, essential for life, in the NIAID Category B priority pathogen Yersinia enterocolitica, but not human PBGS, and are active in a Y. enterocolitica zone inhibition assay. These probes provide exciting validation that pathogen-specific regulation of protein assembly state correlates with killing of the pathogen, but are most likely not drug candidates due to their large size and non-proprietary source. Therefore, it is the purpose of this SBIR Phase I grant to use the information we have developed so far, taken together with new insight from X-ray crystallography, if any, and further screening as it emerges, to prepare compound libraries that provide leads and preclinical drug candidates as defined by industry-standard metrics and criteria. The methods that we will use include iterative potency structure activity relationship development, selectivity, and eADME (early absorption, distribution, metabolism, excretion) testing and evaluation. At the end of the second year of this Phase I SBIR, we will have identified 2-3 compounds for preclinical and clinical development in Phase II. In addition, during the course of this work we will also examine possible extension beyond Y. enterocolitica to Plasmodium falciparum, Pseudomonas aeruginosa, Vibrio cholerae, and Toxoplasma gondii. PUBLIC HEALTH RELEVANCE: One sixth of the world's population has an infectious disease and small-molecule therapeutics involving novel mechanisms are needed, especially those less likely to engender resistance. We describe here a new approach for the discovery of anti- infective agents in which species-specific loss of function of an essential enzyme target in a pathogen can be achieved, by regulation of the assembly state of the protein. We first seek to apply our strategy to the discovery of agents to treat Yersinia enterocolitica, an NIAID Category B priority pathogen.