Pathogen-Specific Regulation of Protein Assembly

描述（由申请人提供）：世界上六分之一的人口患有传染病，如果没有其他原因，除了未来几乎肯定会出现突变株和新的病原体外，需要涉及不太可能产生耐药性的新机制的新药物。卟胆色素原合成酶（Porphobilinogen synthase， PBGS; EC 4.2.1.24）是卟啉生物合成途径中必需的酶。我们发现，PBGS活性的调节取决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，其相互转化在两种不同的二聚体构象水平上。我们预测，通过控制体内非加性组装状态来调节酶的功能可能是通过自然和蛋白质类共同的，称为morphein概念。我们已经确定了改变PBGS组装状态并抑制其功能的小分子。例如，通过对无活性Pisum sativum（豌豆）PBGS六聚体的结构进行计算分析，选择了“morphlock-1”（MW 413），因为它可以结合在由三个单独的蛋白质亚基形成的空腔中。这种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导致功能丧失。考虑到亚基之间蛋白质-蛋白质界面的结合位点的性质，我们认为用这种方法发现的化合物不太可能产生耐药性。我们还发现了三种化合物，它们在NIAID B类优先病原体小肠结肠炎耶尔森菌中阻断PBGS，而不是人类PBGS，并且在小肠结肠炎耶尔森菌区抑制试验中具有活性。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死相关，但由于其体积大且来源非专有，很可能不是候选药物。因此，SBIR I期授权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，准备化合物文库，根据行业标准指标和标准提供先导和临床前候选药物。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）测试和评估。在第一阶段SBIR的第二年结束时，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，我们还将研究从肠结肠炎耶夫氏菌延伸到恶性疟原虫、铜绿假单胞菌、霍乱弧菌和刚地弓形虫的可能性。

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.