Cyclic Peptide Protease Inhibitors for the Treatment of Prostate Cancer

尽管新的治疗方法对延长患有癌症的男性的生活质量和数量产生了影响 前列腺癌(PCA)，迫切需要新的治疗方法来对抗这种疾病的侵袭性形式。 最近，我们在开发靶向于膜的治疗性多肽方面有了一些重要的发现。 PCa侵袭性亚型中结合金属蛋白酶氨基肽酶N(APN)的表达准确可靠地 通过询问APN在前列腺癌组织中的表达，我们通过噬菌体展示鉴定了一种新的抗体，该抗体具有特异性 在福尔马林固定的石蜡包埋组织中识别APN。这种抗体使我们能够筛查前列腺癌组织 具有前所未有的保真度的微阵列(TM)。患者活检组织和组织的免疫组织化学分析 TMAS发现APN在非雄激素受体(AR)驱动的PCa中优先过度表达 AR驱动的前列腺癌。我们还在种族差异TMA和患者活检中记录了 APN在非裔美国人的前列腺癌中表达，而在高加索人患者中不表达。都是非洲人- 美国男性和患有非AR驱动的前列腺癌的男性代表了表现出侵袭性疾病的患者群体 总体存活率很低。利用基于APN底物特异性的合理设计方法，我们 鉴定了一个环肽(Leu-His-Ser-Pro-Trp=cLHSPW)，它是APN酶的微摩尔抑制物 活动。这种多肽被发现在高剂量下对表达APN的PCA异种移植具有适度的治疗作用。 通过连接物将两个cLHSPW多肽连接在一起，在体外和体内产生了具有增强效力的二聚体 活着。该二聚体是APN的纳米分子抑制物，并被癌细胞迅速内化。什么时候 在与单体相同的高剂量下，二聚体在体内导致完全的肿瘤磨损。这些 研究结果提示，高阶多价cLHSPW多肽可能是有效的APN靶向治疗药物。在……里面 在这项提议中，我们将使用大环框架合成二聚体、三聚体和四聚体cLHSPW肽。 除了易于合成外，大环骨架还可以螯合放射性金属以进行正电子发射。 断层扫描(PET)。为了开发一类新的前列腺癌治疗药物，多肽的体外治疗效果 将被表征，并将在体内进行PET成像以监测生物分布(目标1)，能力 用于消除异种皮下移植瘤负荷的多肽将被检查(AIM 2)和X光 对与多肽形成络合物的APN进行结晶学和冷冻电子显微镜观察，以获得 机制洞察力，并最终导致优化抑制剂的开发(目标3)。我们的初步数据 强烈表明，我们已经开发出有效的治疗方法，有可能导致戏剧性的转变 在如何积极地对待PCA方面。

Although new therapies have had an impact prolonging the quality and quantity of life of men suffering from prostate cancer (PCa), novel treatments are urgently needed to combat aggressive forms of the disease. Recently, we made several key discoveries in the development of therapeutic peptides targeting the membrane bound metalloprotease aminopeptidase N (APN) in aggressive subtypes of PCa. To accurately and reliably interrogate the expression of APN in PCa tissues, we identified a novel antibody by phage display that specifically recognized APN in formalin-fixed paraffin embedded tissues. This antibody allowed us to screen PCa tissue microarrays (TMAs) with a fidelity never before seen. Immunohistochemistry analysis of patient biopsies and TMAs found that APN was preferentially overexpressed in non-androgen receptor (AR) driven PCa compared to AR-driven prostate adenocarcinoma. We also documented in a race disparity TMA and patient biopsies that APN was expressed in the PCa of African-American men while absent in Caucasian patients. Both African- American men and men with non-AR driven PCa represent patient populations who exhibit aggressive disease with poor overall survival. Using a rational design approach based off of the substrate specificity of APN, we identified a cyclic peptide (Leu-His-Ser-Pro-Trp = cLHSPW) that was a micromolar inhibitor of APN’s enzymatic activity. This peptide was found to be moderately therapeutic in APN-expressing PCa xenografts at high doses. Tethering of two cLHSPW peptides together by a linker created a dimer with enhanced potency in vitro and in vivo. The dimer was a nanomolar inhibitor of APN and was rapidly internalized by the cancer cell. When administered at the same high dose as the monomer, the dimer led to complete tumor attrition in vivo. These findings suggest that higher order multivalent cLHSPW peptides may be effective APN-targeted therapeutics. In this proposal, we will synthesize dimeric, trimeric, and tetrameric cLHSPW peptides using macrocyclic frames. In addition to ease of synthesis, the macrocyclic frames can also chelate radiometals for positron-emission tomography (PET). To develop a new class of PCa therapeutics, the in vitro therapeutic efficacy of the peptides will be characterized and PET imaging will be performed in vivo to monitor biodistribution (Aim 1), the ability of the peptides to eliminate tumor burden in subcutaneous xenografts will be examined (Aim 2) and X-ray crystallography and cryo-electron microscopy will be performed on APN complexed with the peptides to obtain mechanistic insights and ultimately lead to the development of optimized inhibitors (Aim 3). Our preliminary data strongly suggest that we have developed potent therapeutics that have the potential to result in a dramatic shift in how aggressive PCa is treated.