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

Advent Therapeutics是一家为新生儿、儿科和孤儿药开发新疗法的公司 “利基”市场，以解决严重未满足的医疗需求。我们的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， 肺动脉压进行性升高、右心衰竭和死亡。没有有效 治疗。hPAH通常是由无义编码的mRNA中的提前终止密码子（PTC）引起的。 II型骨形态发生蛋白受体（BMPR 2）基因突变。某些抗生素具有 诱导PTC的“通读”并因此产生全长蛋白的能力，但具有不可接受的毒性。到 考虑到这种毒性，我们首先对70，000个化合物小分子文库进行了高通量筛选， 使用指示命中的通读测定。我们衍生和专利命中，实现在体外，离体， 在BMPR 2以外的致病基因中使用PTC进行体内概念验证，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GJ 103的分子用于进一步开发， 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， 使用从含有疾病相关PTC的hPAH患者获得的血液生长内皮细胞 在BMPR 2中。GJ 103处理诱导BMPR 2蛋白的显著表达。进一步测试小鼠细胞 证实了人类细胞的结果。接下来，我们与莫雷尔实验室（剑桥大学， UK）在小鼠中测试GJ 103，这些小鼠通过两种临床相关的无意义之一来产生hPAH Bmpr 2突变（Bmpr 2 +/R899 X和Bmpr 2 +/R584 X小鼠）。在进行初步剂量探索研究后， GJ 103在体内诱导高达野生型BMPR 2蛋白水平的一半。初步急性和慢性毒性试验 未能显示出毒性的证据。GJ 103诱导的BMPR 2蛋白证实了其在细胞内的功能作用。 体外和体内LPS渗透性测定以及共聚焦显微镜研究表明，该蛋白似乎 适当地运输到内皮细胞和平滑肌细胞的细胞表面。BMP的下游上调 信号传导中间体（SMAD和磷酸化SMAD）和BMP途径基因靶点（Id 1、Id 2和VE-1）。 钙粘蛋白）指示BMP信号传导的功能活化。在这里，我们提出测试的关键问题，使用 GJ 103治疗hPAH：GJ 103诱导的BMPR 2表达能否预防或减缓hPAH的发展？ Bmpr 2 +/R899 X或Bmpr 2 +/R584 X小鼠是否足够早开始治疗？在目标1中，我们将与 Soban实验室（UCLA）测试GJ 103是否可以诱导BMPR 2的表达，足以减缓或停止 小鼠中hPAH的发展。因变量包括检测右心血流动力学和重构 在用GJ 103处理的Bmpr 2 +/R899 X和Bmpr 2 +/R584 X小鼠中使用超声和直接导管插入术。目标2建议 与Rehan实验室（LA BioMed）进行的BMPR 2蛋白的伴随表达分析， 如果GJ 103成功的话。我们的研究可能代表了hPAH患者的第一个希望。

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.