Advancing Small Molecule Read Through Compounds to Prevent and/or Treat Heritable Pulmonary Artery Hypertension

Coment Treateutics是一家为新生儿、儿科和孤儿药物开发新疗法的公司 “利基”市场，以解决严重的未得到满足的医疗需求。我们团队建议早期开发一种新的 治疗遗传性肺动脉高压(HPAH)，一种罕见的遗传性疾病，涉及 进行性肺动脉压升高、右心衰竭和死亡。没有有效的方法 治疗。HPAH通常由无义编码的信使核糖核酸中的提前终止密码子(PTCs)引起。 II型骨形成蛋白受体(BMPR2)基因突变。某些抗生素具有 有能力诱导PTCs的“通读”，从而产生全长蛋白质，但毒性不可接受。至 为了避免这种毒性，我们首先在一个包含7万个化合物的小分子文库上进行了高通量筛选， 使用指示命中的通读测试。我们衍生并申请了HITS专利，实现了体外、体外和 在BMPR2以外的致病基因中使用PTCs的体内概念验证，并发表了这些结果。 接下来，我们从衍生化合物中选择了一种名为GJ103的分子进行进一步开发， 基于通读效率和良好的物理和溶解特性，并开始在hPAH中进行测试， 使用从hPAH患者获得的含有与疾病相关的PTCs的血液生长内皮细胞 在BMPR2中。GJ103处理后BMPR2蛋白表达明显增强。进一步用小鼠细胞进行测试 用人类细胞证实了这一结果。接下来，我们与莫雷尔实验室(剑桥大学， 英国)在他们创造的小鼠身上测试GJ103，这些小鼠由于两种临床相关的胡说八道中的一种而患上hPAH Bmpr2(Bmpr2+/R899X和Bmpr2+/R584X小鼠)突变。在进行了初步的剂量发现研究后， GJ103在体内诱导了高达一半的野生型BMPR2蛋白水平。初步急性和慢性毒性测试 没有显示出毒性的证据。GJ103诱导的BMPR2蛋白在小鼠体内的功能研究 体外和体内的脂多糖通透性分析和共聚焦显微镜研究表明，该蛋白似乎 适当地向细胞表面输送内皮细胞和平滑肌细胞。骨形态发生蛋白的下游上调 BMP途径基因靶标(Id1、Id2和VE-1) 钙粘蛋白)提示BMP信号的功能激活。在这里，我们建议测试关键问题，以便使用 GJ103治疗hPAH：GJ103诱导的BMPR2表达能否阻止或延缓hPAH的发生 如果及早开始治疗，Bmpr2+/R899X或Bmpr2+/R584X小鼠？在目标1中，我们将与 Soban实验室(UCLA)测试GJ103是否可以诱导BMPR2的表达足以减缓或停止 HPAH在小鼠体内的发育。因变量包括检测右心血流动力学和重塑 GJ103对Bmpr2+/R899X和Bmpr2+/R584X小鼠进行超声和直接插管。目标2建议 BMPR2蛋白与热汉实验室(LA BioMed)的伴随表达分析 如果GJ103成功。我们的研究可能代表了患有hPAH的个体的第一个希望。

Advent Therapeutics is a company that develops new therapies for neonatal, pediatric, and Orphan Drug “niche” markets to address serious unmet medical needs. Our team proposes early stage development of a new treatment for hereditable pulmonary arterial hypertension (hPAH), a rare, hereditary disorder that involves progressive elevation of pulmonary arterial pressures, right heart failure, and death. There are no effective treatments. hPAH is often caused by premature termination codons (PTCs) in the mRNA encoded by nonsense mutations in the type II bone morphogenetic protein receptor (BMPR2) gene. Certain antibiotics possess the capacity to induce “readthrough” of PTCs and thus produce a full-length protein, but are unacceptably toxic. To obviate this toxicity, we first performed a high-throughput screen on a 70,000-compound small molecule library, using a readthrough assay that indicated hits. We derivatized and patented hits, achieved in vitro, ex vivo, and in vivo proof-of-concept using PTCs in disease-causing genes other than BMPR2, and published these results. We next selected a molecule named GJ103 from among the derivatized compounds for further development, based on readthrough efficiency and favorable physical and solubility characteristics, and began testing in hPAH, using blood outgrowth endothelial cells obtained from patients with hPAH that contained disease-relevant PTCs in BMPR2. GJ103 treatment induced significant expression of BMPR2 protein. Further testing with mouse cells corroborated the results with human cells. Next, we collaborated with the Morell lab (University of Cambridge, UK) to test GJ103 in mice they created that develop hPAH by virtue of one of two clinically-relevant nonsense mutations in Bmpr2 (Bmpr2+/R899X and Bmpr2+/R584X mice). After performing preliminary dose-finding studies, GJ103 induced up to half of wild-type BMPR2 protein levels in vivo. Preliminary acute and chronic toxicity testing failed to show evidence of toxicity. BMPR2 protein induced by GJ103 demonstrated its functional effects in an LPS permeability assay in vitro and in vivo, and confocal microscopy studies indicated the protein appeared to traffic properly to the cell surface of endothelial and smooth muscle cells. Downstream upregulation of BMP signaling intermediaries (SMADs and phosphoSMADs) and BMP pathway gene targets (Id1, Id2, and VE- cadherin) indicated functional activation of BMP signaling. Here we propose to test the critical question for using GJ103 to treat hPAH: can GJ103-induced expression of BMPR2 prevent or slow development of hPAH in Bmpr2+/R899X or Bmpr2+/R584X mice if treatment is initiated early enough? In Aim 1, we will collaborate with the Soban lab (UCLA) to test whether GJ103 can induce expression of BMPR2 sufficient to slow or halt development of hPAH in mice. Dependent variables include testing right heart hemodynamics and remodeling using echo and direct catheterization in Bmpr2+/R899X and Bmpr2+/R584X mice treated with GJ103. Aim 2 proposes concomitant expression analyses with the Rehan lab (LA BioMed) of BMPR2 protein, which should be elevated if GJ103 is successful. Our studies might represent the first hope for individuals suffering from hPAH.