Pneumocystis jirovecii Targeted Antiopportunistic Agents

描述（由申请人提供）：尽管有高效抗逆转录病毒疗法（HAART 或 ART），但正如 CDC 在 MMWR（2009 年 4 月 10 日）中所述，机会性感染 (OI) 仍然是 HIV 感染者发病率和死亡率相当高的主要原因。这对于由耶氏肺孢子虫引起的主要 OI 肺炎 (PCP) 尤为重要。甲氧苄氨嘧啶/磺胺甲恶唑 (TMP/SMX)、二氢叶酸还原酶 (DHFR) 抑制剂 (TMP) 和二氢叶酸合酶 (DHPS) 抑制剂 (SMX) 的组合是 PCP 的一线药物。由于磺胺类药物和 TMP 的不良反应和耐药性导致这一选择的失败，以及二线药物的失败率和不良反应，迫切需要替代药物。我们的团队从人类病原体耶氏肺孢子虫 (pjDHFR) 中分离并鉴定了难以捉摸的 DHFR，并表明其在治疗药物的抑制活性方面与其替代品卡氏肺孢子虫 (pcDHFR)（发生在啮齿类动物中）截然不同。此外，我们还鉴定了两个系列的化合物（1、3、5 和 6），其对 pjDHFR 的选择性比 hDHFR 高 19-99 倍，pjDHFR 的 Ki 和 IC50 值为皮摩尔和纳摩尔。据我们所知，我们是唯一能够获得 pjDHFR 和对 pjDHFR（与 hDHFR 相比）具有高选择性（19-99 倍）和效力（皮摩尔和纳摩尔）的化合物的团队。最近，我们还克隆并表达了临床相关的、TMP 抗性的 pjDHFR 双突变体，发现我们的化合物在面对 TMP 500 倍的抗性时，仍保留了对双突变体 pjDHFR 的纳摩尔抑制作用，并且也将在我们的评估中使用它们。这是针对耶氏疟原虫感染的药物评估中的一个范式改变事件，迄今为止，该药物评估一直使用卡氏疟原虫作为 DHFR 以及体内模型的替代物。具体目标是： 1) 合成系列 I-XII 中提出的化合物； 2)评价化合物作为hDHFR以及野生型和抗性突变体pjDHFR的抑制剂； 3) 评估来自目标2的选定类似物在人胚胎肺成纤维细胞中的毒性； 4) 评估目标 2 和 3 中选定的类似物 (5) 的血清结合、代谢稳定性和药代动力学，并在耶氏疟原虫感染的小鼠模型中进行开发和评估。 Cody 博士将使用 pjDHFR 和 hDHFR 对 1、3 和 5 以及系列 I-XII 中选定的化合物进行 X 射线晶体结构测定，以从分子角度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生成。此外，它将首次基于来自人类病原体（P.jirovecii）而不是替代物（P.carinii）的DHFR，对P.jirovecii动物模型中的化合物进行评估。该研究应确定可单独或联合使用的潜在化合物，用于针对 PCP 和耐药 PCP 的临床评估。 公共卫生相关性：疾病控制中心 (CDC) 在其最近的报告中指出，艾滋病患者的感染仍然是这些患者生活质量差和死亡的主要原因。我们已经发现了能够选择性杀死这些引起感染的最重要生物体的药物，其选择性比正常细胞高 19-99 倍。这项研究旨在优化药物，并在小鼠模型中针对导致人类感染的微生物进行评估，以提供针对这些感染的选择性药物，包括耐药形式，为人体试验做好准备。

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. PUBLIC HEALTH RELEVANCE: The Center for Disease Control (CDC) in its recent report has indicated the infections in persons with AIDS remains a major cause of poor quality of life and death for these patients. We have discovered drugs that are able to selectively kill the most important of these infection causing organisms with very high selectivity of 19- 99-fold over normal cells. This study seeks to optimize the drugs and evaluating them against the organisms that cause the human infection in mouse models of these infections to afford selective agents against these infections, including drug resistant forms, to be ready for testing in human trials.