Identification of LSD1 inhibitors targeting epigenetic regulation in tumor cells

描述(由申请人提供)：最近发现的赖氨酸特异性脱甲基酶1(LSD1)阐明了表观遗传控制基因表达的重要细胞机制。特别是，二甲基赖氨酸4，组蛋白H3(H3K4me2)是一个位于基因启动子上的转录激活染色质标记，LSD1对该标记的异常去甲基化可能广泛抑制在人类癌症中重要的肿瘤抑制基因的表达。我们和其他人已经进行了研究，证实LSD1是一个令人兴奋的新治疗靶点。我们最近报道了一系列对重组人LSD1有有效抑制作用的(双)胍类和(双)双胍类化合物。这些抑制剂显著增加H3K4me2水平，启动染色质重塑，并诱导肿瘤抑制基因的重新表达，使它们适合于类似物的开发。我们首先在体外证明了LSD1抑制剂的抗肿瘤作用，最近在体内证明了它们显著的抗肿瘤作用。这些研究提供了抑制LSD1可以导致显著的抗肿瘤作用的证据或原理。这一提议的中心假设是，可以识别和开发抑制LSD1的化合物用于人类癌症的治疗。沿着这些思路，这项建议的具体目标是：具体目标1.设计和合成多个系列的类似物作为LSD1的潜在抑制剂。我们将使用包括类似物合成和高通量评估的系统药物化学方法来生产合理设计的小分子LSD1抑制剂文库。这些类似物将在结构上与我们的先导化合物或LSD1底物相关。我们还将使用虚拟筛选来从商业数据库中识别线索，并为合成和筛选提供更有效的类似物。具体目标2.新合成的类似物作为LSD1抑制剂的评价及其在培养的肿瘤细胞中的表观遗传效应的研究。每个类似物都将被评估为纯化的LSD1的抑制剂，并将确定抑制动力学。所有类似物的细胞效应将在HCT116肿瘤细胞系中进行监测。每种化合物都将单独进行评估，并与DNA甲基转移酶抑制剂和/或I/II类组蛋白脱乙酰酶抑制剂结合使用。我们将监测特定的染色质标记和基因产物，以确定每种化合物是否会导致肿瘤抑制基因重新表达，并将测量细胞生长和活力。具体目的3.LSD1抑制剂及其联合治疗的体内评价。有希望的化合物和联合治疗将在人HCT116肿瘤移植瘤的剂量和疗效研究中取得进展。使用这种方法，很有可能识别出有效的LSD1抑制剂，这些药物有可能成为一类重要的新型抗肿瘤药物。 与公共卫生相关：最近发现的赖氨酸特异性脱甲基酶1(LSD1)已被证明在基因表达的表观遗传控制中发挥重要作用，LSD1水平的升高会导致在人类癌症中重要的肿瘤抑制因子的表达减少。我们的团队已经发现了一系列作为LSD1抑制剂并重新激活这些肿瘤抑制基因的双胍类化合物，使这些化合物成为一类潜在的新型抗肿瘤药物。在这项建议中，我们描述了多个新的相关类似物的设计和化学合成，这些类似物将可逆地抑制或不可逆失活LSD1，并提议进行实验，以确定它们对基因表达、细胞功能的影响，以及它们在培养和小鼠异种移植模型中抑制肿瘤细胞生长的能力。

DESCRIPTION (provided by applicant): The recent discovery of the enzyme lysine-specific demethylase 1 (LSD1) has illuminated an important cellular mechanism for epigenetic control of gene expression. In particular, dimethyl lysine 4, histone H3 (H3K4me2) is a transcription activating chromatin mark at gene promoters, and aberrant demethylation of this mark by LSD1 may broadly repress the expression of tumor suppressor genes that are important in human cancer. We and others have conducted studies verifying that LSD1 is an exciting new therapeutic target. We recently reported a series of (bis)guanidines and (bis)biguanides that are potent inhibitors of recombinant human LSD1. These inhibitors significantly increase H3K4me2 levels, initiate chromatin remodeling and induce the re-expression of tumor suppressor genes, making them suitable leads for analogue development. We were the first to demonstrate antitumor effects of LSD1 inhibitors in vitro and have recently demonstrated their significant antitumor effects in vivo. These studies provide proof or principle that inhibition of LSD1 can lead to significant antitumor effects. The central hypothesis of this proposal is that compounds that inhibit LSD1 can be identified and developed for use in the treatment of human cancer. Along these lines, the specific aims of this proposal are: Specific aim 1. Design and synthesis of multiple series of analogues as potential inhibitors of LSD1. We will use a systematic medicinal chemistry approach that includes analogue synthesis and high-throughput evaluation to produce rationally designed libraries of small-molecule LSD1 inhibitors. These analogues will be structurally related to our lead compounds, or to the LSD1 substrate. We will also use virtual screening to identify leads from commercial databases, and to suggest more potent analogues for synthesis and screening. Specific aim 2. Evaluation of newly synthesized analogues as LSD1 inhibitors and study of their epigenetic effects in cultured tumor cells. Each analogue will be evaluated as an inhibitor of purified LSD1, and the kinetics of inhibition will be determined. The cellular effects of all analogues will be monitored in the HCT116 tumor cell line. Each compound will be evaluated alone, and in combination with a DNA methyltransferase inhibitor and/or a class I/II histone deacetylase inhibitor. We will monitor specific chromatin marks and gene products to determine whether each compound causes tumor suppressor gene re-expression, and cell growth and viability will be measured. Specific aim 3. Evaluation of LSD1 inhibitors and combination treatments in vivo. Promising compounds and combination treatments will be advanced to dosing and efficacy studies in human HCT116 tumor xenografts. Using this approach, there is a high probability of identifying potent LSD1 inhibitors that have the potential to become an important new class of antitumor agent. PUBLIC HEALTH RELEVANCE: The recently discovered enzyme lysine-specific demethylase 1 (LSD1) has been shown to play an important role in epigenetic control of gene expression, and elevated levels of LSD1 lead to a reduced expression of tumor suppressor factors that are important in human cancer. Our group has discovered a series of guanidines and biguanides that act as potent LSD1 inhibitors and reactivate these tumor suppressor genes, making these compounds a potential new class of antitumor agents. In this proposal, we describe the design and chemical synthesis of multiple new series of related analogues that will reversibly inhibit or irreversibly inactivate LSD1, and propose experiments to determine their effects on gene expression, cellular function and their ability to inhibit tumor cell growth in culture and in a mouse xenograft model.