Development of a direct DUX4 inhibitor for Facioscapulohumeral Muscular Dystrophy (FSHD)

摘要/摘要 面肩肱骨营养不良(FSHD)是第三种最常见的肌营养不良症，其影响范围超过 三万美国人。FSHD是一种进行性疾病，患者最初会失去面部、肩部的肌肉细胞 退化前的上臂包括几乎所有的骨骼肌，其中20%成为轮椅 被绑住了。95%的FSHD患者表现出高度多态的D4Z4重复序列(FSHD1)的收缩，其中包含 转录因子(Tf)双同源盒4(DUX4)的开放阅读框架。DUX4错误表达是 与成肌细胞毒性有关，被认为是FSHD的驱动因素。据认为，DUX4可以诱导 成肌细胞通过上调包括MBD3L2、TRIM43、ZSCAN4和LEUTX在内的非靶基因而死亡 通常在肌肉中表达。尽管目前还没有FDA批准的治疗FSHD的方法，但洛斯帕莫特是一种小型的 分子p38抑制FSHD患者细胞中DUX4的转录，并证实其临床意义 在几个结果指标中受益。然而，losmapimod并不是DUX4和FSHD所特有的，因此 开发具有不同抑制模式的多靶向治疗将提高患者的成功率 治疗FSHD的长期副作用最小。我们假设锁定DUX4将阻止多个 并减少肌肉细胞的死亡。虽然像DUX4这样的转铁蛋白是有吸引力的治疗靶点，但它们 具有挑战性的小分子靶标，因为它们缺乏清晰的结合口袋，具有较大的表面积 对于蛋白质-蛋白质相互作用很重要，并且包含大的内在无序的结构域。在阿勒泰 治疗公司，我们开发了一个平台，能够识别内在的小捆绑口袋 先前不可用药的TF中的无序结构域，使靶向DUX4的新的可用药方法成为可能 以及开发有效和高度特异的DUX4抑制剂(DUX4i)。我们完成了硅胶筛查和 确定了通过靶向无序的连接器域来减少DUX4 DNA结合的抑制剂。重要的是，这些 DUX4i具有最小的细胞毒性，减少了DUX4的靶基因，并挽救了DUX4驱动的细胞活性，重要的是 治疗FSHD。我们提出三个目标，以确定和描述最有希望的线索，并继续我们的 努力开发一种基于抑制DUX4的FSHD可行治疗方法。圆满完成了我们的 该提案旨在提名一名主要的DUX4候选药物，目标如下：1)确定DUX4靶标 用我们的DUX4is抑制基因，并在更广泛的正常细胞群中测量细胞毒性2)确定 我们的DUX4对其他含有同源异型盒的基因如PAX3/PAX7的特异性和测量潜力 对正常成肌细胞分化的非特异性抑制3)DUX4在小鼠体内的药效测定 将人FSHD成肌细胞接种于胫前肌(FSHD异种移植模型)。然后，我们将继续进行 SBIR第二阶段拨款将包括药物化学努力和额外的动物研究，并最终 用于FSHD的一流DUX4抑制剂的商业化。第一种直接DUX4抑制剂的研制 将使Altay治疗公司启动FSHD的临床试验，最终带来亟需的治疗方法。

Abstract/Summary Facioscapulohumeral dystrophy (FSHD) is the third most common form of muscular dystrophy affecting over 30,000 Americans. FSHD is a progressive disease where patients initially lose muscle cells in the face, shoulders and upper arms before degeneration expands to include nearly all skeletal muscles and 20% become wheelchair bound. 95% of FSHD patients display a contraction of the highly polymorphic D4Z4 repeat (FSHD1) containing an open reading frame for the transcription factor (TF) Double Homeobox 4 (DUX4). DUX4 misexpression is associated with myoblast toxicity and is thought to be the driver of FSHD. It is believed that DUX4 induces myoblast death by upregulating target genes including MBD3L2, TRIM43, ZSCAN4, and LEUTX that are not normally expressed in muscle. Although there are no FDA approved therapies for FSHD, losmapimod, a small molecule p38 inhibitor, was shown to inhibit DUX4 transcription in FSHD patient cells and demonstrated clinical benefit in several outcome measures. However, losmapimod is not specific to DUX4 and FSHD, therefore developing multiple targeted therapies with different modes of inhibition would increase the success rate in treating FSHD with minimum long term side effects. We hypothesize that targeting DUX4 will block multiple pathways and reduce muscle cell death. Although TFs like DUX4 are attractive therapeutic targets, they are challenging to target with small molecules because they lack clear binding pockets, have large surface areas important for protein-protein interactions and contain large intrinsically disordered domains. At Altay Therapeutics, we developed a platform that enables identification of small binding pockets within intrinsically disordered domains in previously undruggable TFs, allowing a novel druggable approach for targeting DUX4 and development of potent and highly specific DUX4 inhibitors (DUX4i). We completed in-silico screening and identified inhibitors that reduced DUX4 DNA binding by targeting the disordered linker domain. Importantly, these DUX4i had minimal cytotoxicity, reduced DUX4 target genes and rescued DUX4 driven cell viability, important to treat FSHD. We propose three aims to identify and characterize the most promising lead and continue our efforts to develop a viable treatment for FSHD based on inhibiting DUX4. The successful completion of our proposal is intended to nominate a lead DUX4 drug candidate with the following aims, 1) Determine DUX4 target gene inhibition with our DUX4is and measure cytotoxicity in a broader panel of normal cells 2) Determine specificity of our DUX4is against other homeobox-containing genes such as PAX3/PAX7 and measure potential non-specific inhibition of normal myoblast differentiation 3) Measure in vivo efficacy of DUX4is in mice implanted with human FSHD myoblasts in the tibialis anterior muscles (xenograft model of FSHD). We will then pursue an SBIR phase 2 grant that will include medicinal chemistry efforts and additional animal studies and ultimately commercialization of a first-in-class DUX4 inhibitor for FSHD. The development of the first direct DUX4 inhibitor will position Altay Therapeutics to initiate clinical trials in FSHD to ultimately bring a much-needed therapy.