Small molecule targeting of MLK3 for heart failure

 描述（由申请人提供）：心力衰竭（HF）是大多数心血管疾病的最终表现，是一种预后不良的毁灭性疾病。病理性心脏重塑（肥大、纤维化）是临床结局的已知预测因素。我们和其他人已经证明，心脏损伤后心脏成纤维细胞（CF）（“支持细胞”）的病理激活释放各种促肥大/炎症介质，其靶向心肌细胞（CM）（“功能细胞”）和成纤维细胞，以加剧重塑。阐明这种病理性交流的机制可能具有治疗前景。混合谱系激酶（MLKs）是JNK和p38 MAPK上游的应激激活的双特异性MAPKKKs家族。MLK 3特别地直接涉及HIV相关的神经变性（HAND），其中它介导小胶质细胞（“支持细胞”）活化神经元（“功能细胞”）损伤。HAND和HF的相似致病机制，即有害的“支持细胞-功能细胞”通讯，表明MLK 3在HF中具有相似的病理作用。然而，MLK在心脏中的功能作用在很大程度上仍然未知。我们已经产生了全局MLK 3-/-和CF-靶向的条件性MLK 3fl/fl小鼠，其中我们没有发现基础心脏表型;然而，它们在心肌损伤后受到保护。我们已经合成了MLK 3特异性抑制化合物，包括URMC-099（CNS-渗透剂）和URMC-128（CNS-不渗透剂）。URMC-099在体外减少病理性CF活化;其在HF的药理学和手术模型中显著减弱心脏肥大并减少间质纤维化，在MLK 3全局或条件无效小鼠中没有附加益处。我们的假设是MLK 3在病理性CF激活和HF进展中起重要作用，并且其抑制具有治疗HF的希望。为了解决我们的假设，我们提出了以下目的：目的1：在野生型、MLK 3-/-和诱导型CM或CF MLK 3-/-小鼠中的临床相关HF模型中确定MLK 3抑制或消融的治疗功效和特异性。该目的将研究MLK 3药理学抑制（使用我们的结构上不同的CNS渗透剂或非渗透剂化合物）和/或消融（整体、诱导型CM、诱导型驻留或激活的CF限制性无效小鼠）在缺血再灌注（I/R）HF中的治疗功效和特异性。目的2：阐明MLK 3抑制的细胞机制，并验证MLK 3作为小鼠和人HF的治疗靶点。该目的将使用经受致病性损伤的原代啮齿动物CF和CM（目的1）来检验MLK 3抑制或消融将减弱病理性CF激活和随后的CF/MF-CM病理性串扰的假设。重要的是，类似的测定将在人HF心脏细胞中进行。体外和体内MLK 3功能获得研究将补充这些研究。我们的研究结果可能阐明一种新的方法来治疗HF和一个新的范例的病理“支持细胞功能细胞”的串扰在病理生理学的多种疾病状态。

 DESCRIPTION (provided by applicant): Heart failure (HF), the final manifestation of most cardiovascular pathologies, is a devastating disease with poor prognosis. Pathologic cardiac remodeling (hypertrophy, fibrosis) is a known predictor of clinical outcome. We and others have demonstrated that pathologic activation of the cardiac fibroblast (CF) ("support cell") after cardiac injury releases various pro-hypertrophic/inflammatory mediators that target both cardiomyocytes (CM) ("functional cell") and fibroblasts to exacerbate remodeling. Elucidating mechanisms of this pathologic communication may hold therapeutic promise. Mixed Lineage Kinases (MLKs) are a family of stress-activated dual-specificity MAPKKKs upstream of JNK and p38 MAPKs. MLK3 in particular has been directly implicated in HIV-associated neurodegeneration (HAND), where it mediates microglial ("support cell") activation neuronal ("functional cell") damage. The similar pathogenic mechanisms of HAND and HF, namely deleterious "support cell-functional cell" communication, suggest a similar pathologic role for MLK3 in HF. However, the functional role(s) of MLKs in the heart remain largely unknown. We have generated global MLK3-/- and CF-targeted conditional MLK3fl/fl mice, in which we have found no basal cardiac phenotype; however, they are protected following myocardial injury. We have synthesized MLK3-specific inhibitory compounds, including URMC-099 (CNS-penetrant) and URMC-128 (CNS-impenetrant). URMC-099 reduced pathologic CF activation in vitro; it significantly attenuated cardiac hypertrophy and reduced interstitial fibrosis in pharmacologic and surgical models of HF, with no additive benefit in MLK3 global or conditional null mice. Our hypothesis is that MLK3 plays an important role in pathologic CF activation and HF progression and that its inhibition holds therapeutic promise for HF. To address our hypothesis, we propose the following Aims: Aim 1: Determine the therapeutic efficacy and specificity of MLK3 inhibition or ablation in clinically-relevant HF models in wild type, MLK3-/- and inducible CM or CF MLK3-/- mice. This aim will investigate the therapeutic efficacy and specificity of MLK3 pharmacologic inhibition (using our structurally distinct CNS-penetrant or -impenetrant compounds) and/or ablation (global, inducible CM, inducible resident or activated CF-restricted null mice) in ischemia-reperfusion (I/R) HF. Aim 2: Elucidate the cellular mechanisms underlying MLK3 inhibition and provide validation of MLK3 as a therapeutic target in both mouse and human HF. This aim will test the hypothesis that MLK3 inhibition or ablation will attenuate pathologic CF activation and subsequent CF/MF-CM pathologic crosstalk, using primary rodent CF and CM (Aim 1) subjected to pathogenic insults. Importantly, similar assays will ensue in human HF cardiac cells. MLK3 gain of function studies in vitro and in vivo will complement these studies. Our results may elucidate a new approach for treating HF and a novel paradigm of pathologic "support cell-functional cell" cross talk in the pathophysiology of multiple disease states.