Novel, P. jirovecii Specific Antipneumocystis Agents

描述（由申请人提供）：肺孢子虫肺炎（PCP）是艾滋病患者中最常见且危及生命的机会性感染，是导致广泛发病率和死亡率的原因。引起人类感染的病原体是肺孢子虫，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。卡氏肺孢子虫是一种感染小鼠而非人类的生物体，已在培养物和动物模型中用作人类病原体的替代物，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蝶酸合酶（DHPS）抑制剂的组合，磺胺甲恶唑是PCP最有效的一线治疗药物。由于对磺胺组分的不良反应以及最近鉴定的DHPS酶的耐药突变体和二线药物的伴随不良反应和失败率，该选择的失败促使广泛寻找这种AIDS定义的、威胁生命的感染的替代治疗选择。我们最近已经从人类病原体Jirovecii中分离并表征了DHFR，并表明其在治疗剂的抑制活性方面与卡氏肺孢子虫DHFR完全不同。此外，我们首次鉴定了两种化合物，它们是pjDHFR的单位纳摩尔抑制剂，其中一种化合物对人类病原体DHFR的选择性是人类DHFR的2190倍。我们还从以前的研究中得到证据，证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）合成系列I-XI中的化合物; 2）评价化合物作为pDHFR和hDHFR的抑制剂; 3）评价来自目标2的所选类似物在人胚肺成纤维细胞中的细胞毒性; 4）评价所选类似物（二至四）的基础上的目的2和3在大鼠模型的卡氏肺吸虫;和5）进行X-用pjDHFR和hDHFR对两个母体化合物沿着本研究化合物进行X射线晶体结构测定，得到分子理解类似物的效力和选择性的结构原因。这项研究将提供一个独特的机会，以确定来自P. jirovecii的DHFR的有效和选择性抑制剂的结构要求，P. jirovecii是导致人类PCP的病原体，使用我们已经确定的特殊铅类似物，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而不是来自替代物的DHFR选择用于动物模型研究的化合物。我们预计，该研究还可能提供潜在的化合物，用于临床对抗人类中的PCP以及抵抗耐药菌株，这些化合物是基于对临床环境更有意义的pjDHFR抑制数据的选择而获得的。这些药物可以单独或联合使用来治疗PCP，从而提供针对新靶点的新型药物。

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.