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去甲基酶1 （LSD1）阐明了基因表达表观遗传控制的重要细胞机制。特别是二甲基赖氨酸4，组蛋白H3 （H3K4me2）是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会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（双）胍类和（双）双胍类化合物，它们是重组人LSD1的有效抑制剂。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，使其成为类似物开发的合适先导物。我们首先在体外证明了LSD1抑制剂的抗肿瘤作用，最近又证明了它们在体内的显著抗肿瘤作用。这些研究提供了抑制LSD1可导致显著抗肿瘤作用的证据或原理。该提案的中心假设是，抑制LSD1的化合物可以被识别并开发用于治疗人类癌症。按照这些思路，本建议的具体目标是：LSD1抑制剂多系列类似物的设计与合成。我们将使用系统的药物化学方法，包括类似物合成和高通量评价，以产生合理设计的小分子LSD1抑制剂文库。这些类似物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从商业数据库中识别线索，并建议更有效的类似物进行合成和筛选。具体目标2。新合成的LSD1抑制剂类似物的评价及其在培养肿瘤细胞中的表观遗传效应研究。每个类似物将被评估为纯化LSD1的抑制剂，并确定抑制动力学。所有类似物的细胞效应将在HCT116肿瘤细胞系中进行监测。每种化合物将单独评估，并与DNA甲基转移酶抑制剂和/或I/II类组蛋白去乙酰化酶抑制剂联合评估。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导致肿瘤抑制基因重新表达，并测量细胞生长和活力。具体目标3。体内LSD1抑制剂及联合治疗的评价。有前景的化合物和联合治疗将推进到人类HCT116肿瘤异种移植的剂量和疗效研究。利用这种方法，很有可能发现有效的LSD1抑制剂，这些抑制剂有可能成为一类重要的新型抗肿瘤药物。

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.