Pneumocystis jirovecii Targeted Antiopportunistic Agents

描述(申请人提供)：尽管存在高效抗逆转录病毒疗法(HAART或ART)，但机会性感染(OIS)仍然是艾滋病毒感染者相当大的发病率和死亡率的主要原因，正如疾控中心在2009年4月10日的MMWR上所述。这一点对于主要的OI--肺炎(PCP)尤其重要，这种肺炎是由日本肺孢子虫引起的。甲氧苄氨嘧啶/磺胺甲恶唑(TMP/SMX)是治疗PCP的一线药物，由二氢叶酸还原酶(DHFR)抑制剂(TMP)和二氢翼酸合成酶(DHPS)抑制剂(SMX)组成。由于对磺胺类药物和TMP的不良反应和耐药性以及二线药物的失败率和不良反应，该方案失败，迫切需要替代药物。我们小组从人类病原体Jirovecii(PjDHFR)中分离并鉴定了难以捉摸的DHFR，并表明它与其替代物卡氏肺孢子虫(PcDHFR)(发生在啮齿动物中)在治疗药物的抑制活性方面不同。此外，我们还鉴定了两个系列的化合物(1，3，5和6)，它们对pjDHFR的选择性是hDHFR的19-99倍，对pjDHFR具有皮摩尔和纳摩尔的KI和IC50值。据我们所知，我们是唯一能够获得pjDHFR的群体，以及对pjDHFR具有高选择性(19-99倍)和效力(皮摩尔和纳摩尔)的化合物(与hDHFR相比)。最近，我们还克隆并表达了临床上相关的、抗TMP的pjDHFR双突变体，发现我们的化合物在面对TMP的500倍耐药时，对双突变体pjDHFR仍保持纳米分子抑制，并将这些用于我们的评估。这是治疗日本血吸虫感染的药物评估中的一个范例改变事件，到目前为止，该药物在DHFR和体内模型中使用的都是代用的卡氏肺孢子虫。其具体目标是：1)合成系列I-XII中的建议化合物；2)评价化合物作为hDHFR和野生型及抗药性突变体pjDHFR的抑制剂的作用；3)评价AIM 2中精选的类似物在人胚胎肺成纤维细胞中的毒性；4)评价AIMS 2和3中精选的类似物(5)在血清结合、代谢稳定性和药代动力学方面的作用，并在日本血吸虫感染的小鼠模型中进行开发和评价。科迪博士将对系列1、3和5以及系列I-XII中具有pjDHFR和hDHFR的部分化合物进行X射线晶体结构测定，以便从分子水平上了解类似物的选择性和效力。这项研究将确定有效和选择性抑制pjDHFR的结构要求，并将有助于未来的药物设计和药效团的产生。此外，它还将首次提供基于来自人类病原体(P.jirovecii)而不是替代者(P.carinii)的DHFR的日本血吸虫动物模型中化合物的评估。这项研究应确定用于临床评估的潜在化合物，以对抗PCP和耐药PCP单独或联合使用。

DESCRIPTION (provided by applicant): Despite the availability of highly active antiretroviral therapy (HAART or ART), opportunistic infections (OIs) remain the leading cause of considerable morbidity and mortality in HIV infected persons as stated by the CDC in MMWR, April 10, 2009. This is particularly important with the leading OI, pneumonia (PCP), caused by Pneumocystis jirovecii. Trimethoprim/sulfamethoxazole (TMP/SMX), the combination of a dihydrofolate reductase (DHFR) inhibitor (TMP) and a dihydropteroate synthase (DHPS) inhibitor (SMX) for PCP is the first- line agent. The failure of this option due to adverse reactios and resistance to the sulfa drug as well as TMP along with the failure rate and adverse reactions of second-line agents necessitates the urgent need for alternate agents. Our group has isolated and characterized the elusive DHFR from the human pathogen Pneumocystis jirovecii (pjDHFR) and shown it to be distinct and different from its surrogate Pneumocystis carinii (pcDHFR) (occurs in rodents), with respect to inhibitory activities of therapeutic agents. n addition, we have identified two series of compounds (1, 3, 5 and 6) with selectivity for pjDHFR over hDHFR of 19-99-fold with picomolar and nanomolar Ki and IC50 values for pjDHFR. To our knowledge, we are the only group with access to pjDHFR and compounds that have both high selectivity (19-99-fold) and potency (picomolar and nanomolar) against pjDHFR (compared to hDHFR). Recently we have also cloned and expressed clinically relevant, TMP-resistant double mutants of pjDHFR and found that our compounds retain nanomolar inhibition against the double mutant pjDHFR in the face of TMP's 500-fold resistance, and will also use these in our evaluations. This is a paradigm changing event in the evaluation of drugs for P. jirovecii infection that up until now has utilized the surrogate P. carinii for DHFR as well as in vivo models. The Specific Aims are to: 1) synthesize proposed compounds in Series I-XII; 2) evaluate the compounds as inhibitors of hDHFR and wild type and resistant mutant pjDHFR; 3) evaluate selected analogs from Aim 2 in human embryonic lung fibroblasts for toxicity; 4) evaluate selected analogs (5) from Aims 2 and 3 for serum binding, metabolic stability and pharmacokinetics and develop and evaluate in a mouse model of P. jirovecii infection. X-ray crystal structure determination of 1, 3 and 5 and selected compounds from Series I-XII with pjDHFR and hDHFR will be done by Dr. Cody to afford a molecular understanding of the selectivity and potency of the analogs. This study will determine the structural requirements for potent and selective inhibition of pjDHFR and will assist in future drug design and pharmacophore generation. In addition it will provide, for the first time, the evaluation of compounds in a P. jirovecii animal model based on DHFR from the human pathogen (P. jirovecii) rather than a surrogate (P. carinii). The study should identify potential compounds for clinical evaluation against PCP and resistant PCP to be used alone or in combination.