Novel, P. jirovecii Specific Antipneumocystis Agents

描述（由申请人提供）：肺囊虫肺炎（PCP）是艾滋病患者中最常见和危及生命的机会性感染，是造成广泛发病率和死亡率的原因。导致人类感染的病原体是耶氏肺囊虫，由于缺乏体外培养系统和动物模型，它仍然是一个难以捉摸的目标。卡氏肺囊虫是一种感染小鼠而不感染人类的生物，在培养和动物模型中被用作人类病原体的替代品，以评估潜在的治疗方案。甲氧苄啶/磺胺甲恶唑是二氢叶酸还原酶（DHFR）抑制剂甲氧苄啶和二氢叶酸合酶（DHPS）抑制剂的联合治疗，是治疗PCP最有效的一线药物。由于对磺胺成分的不良反应，以及最近发现的DHPS酶的耐药突变，以及随之而来的不良反应和二线药物的失败率，这促使人们广泛寻找替代治疗方案，以治疗这种艾滋病定义的、危及生命的感染。我们最近从人类病原体P. jrovecii中分离并鉴定了DHFR，并表明它在治疗剂的抑制活性方面与P. carinii DHFR有很大不同。此外，我们首次鉴定出两种化合物是pjDHFR的个位数纳摩尔抑制剂，其中一种化合物对人类病原体DHFR的选择性比人类DHFR高2190倍。我们还从先前的研究中得到证据，表明这类化合物能有效地渗透完整的生物体。本申请的具体目的是：1)合成I-XI系列化合物；2)评价化合物作为pjDHFR和hDHFR抑制剂的作用；3)评价Aim 2筛选的类似物对人胚胎肺成纤维细胞的细胞毒性；4)基于Aims 2和3在大鼠卡氏疟原虫模型中评估选定的类似物（2至4个）；5)利用pjDHFR和hDHFR对两种母体化合物以及本研究化合物进行x射线晶体结构测定，从而对类似物的效力和选择性的结构原因进行分子理解。这项研究将提供一个独特的机会，以确定有效的和选择性的DHFR抑制剂的结构要求，从P. jrovecii，使用我们已经确定的特殊铅类似物，导致人类PCP的病原体，这也将有助于未来的类似物设计。此外，它将首次允许使用来自人类病原体的DHFR而不是来自替代品来选择用于动物模型研究的化合物。我们期望这项研究也可能提供潜在的化合物，用于临床治疗人类PCP以及耐药菌株，这些化合物是基于pjDHFR抑制数据的选择而获得的，这对临床环境更有意义。这些药物可以单独使用或联合使用来治疗PCP，从而提供针对新靶点的新药物。

DESCRIPTION (provided by applicant): Pneumocystis pneumonia (PCP) is the most common and life threatening opportunistic infection in AIDS patients and is responsible for extensive morbidity and mortality. The pathogen that causes the infection in humans is Pneumocystis jirovecii and has remained an elusive target due to a lack of in vitro culture systems and animal models. Pneumocystis carinii, the organism that infects mice and not humans, has been used as a surrogate for the human pathogen both in culture and in animal models to evaluate potential treatment options. Trimethoprim/sulfamethoxazole the combination of a dihydrofolate reductase (DHFR) inhibitor trimethoprim and a dihydropteroate synthase (DHPS) inhibitor, sulfamethoxazole is the most effective first- line treatment for PCP. The failure of this option due to adverse reactions to the sulfa component as well as recently identified resistant mutants of the DHPS enzyme and the attending adverse reactions and failure rate of second-line agents have prompted an extensive search for alternate treatment options for this AIDS defining, life threatening infection. We have very recently isolated and characterized the DHFR from the human pathogen, P. jirovecii and showed it to be quite different from P. carinii DHFR with respect to inhibitory activities of therapeutic agents. In addition, we have, for the very first time, identified two compounds that are single digit nanomolar inhibitors of pjDHFR with one compound having a remarkable 2190-fold selectivity for the human pathogen DHFR compared to the human DHFR. We also have evidence from a previous study that compounds of this class efficiently penetrate intact organism. The specific aims of this application are: 1) to synthesize compounds in Series I-XI; 2) to evaluate the compounds as inhibitors of pjDHFR and hDHFR; 3) to evaluate selected analogs from Aim 2 for cytotoxicity in human embryonic lung fibroblasts; 4) to evaluate selected analogs (two to four) on the basis of Aims 2 and 3 in a rat model of P. carinii; and 5) to carry out X-ray crystal structure determination of the two parent compounds along with compounds from this study with pjDHFR and hDHFR to afford a molecular understanding of the structural reasons for the potency and selectivity of the analogs. This study will afford a unique opportunity to determine the structural requirements of potent and selective inhibitors of DHFR from P. jirovecii, the pathogen that causes PCP in humans using exceptional lead analogs that we have identified, that will also help in future analog design. In addition, it will allow, for the first time, a selection of compounds for animal model studies using the DHFR from the human pathogen rather than from a surrogate. We expect the study to also perhaps afford potential compounds, for clinical use against PCP in humans as well as against resistant strains, obtained on the basis of selection that originates with pjDHFR inhibition data that is much more meaningful to the clinical setting. These agents could be used alone or in combination to treat PCP thus providing novel agents against a new target.