High Throughput Screening (HTS) to Discover Graft-Versus-Host Disease Inhibitors

描述(申请人提供)：异基因造血干细胞移植(HSCT)是一种治疗许多恶性疾病的潜在疗法，但其临床应用受到移植物抗宿主病(GVHD)的阻碍。不幸的是，目前还没有针对GVHD患者血液中升高的蛋白质的治疗方法，也没有努力开发针对GVHD的药物。这是一个重要的问题，因为目前的治疗仅限于针对效应细胞的非特异性靶向。我们的长期目标是开发针对GVHD患者中调节失调的蛋白质的药物，作为潜在的GVHD新疗法。我们在这一应用中的目标是通过对小分子文库的高通量筛选(HTS)来发现能够抑制这些升高的蛋白质的化学探针，并在生物模型系统中进行评估。这些化合物将有助于发现潜在的GVHD特异性药物，从而更好地利用许多血液病患者的治疗。我们的中心假设是GVHD的主要血浆生物标记物，肿瘤致瘤性抑制2(ST2)可以被小分子化合物靶向以减轻GVHD。这一假说是基于我们发表的数据形成的，该数据将ST2的可溶性形式(Sst2)描述为预测GVHD治疗无反应的最重要的生物标志物。Sst2作为IL-33的诱饵受体，是已知的唯一的ST2配体，驱动Th2细胞向Th1表型转变。这种表型变化导致GVHD，抑制Sst2和IL-33的结合可以缓解GVHD。因此，我们开始了一项试点HTS活动，使用AlphaScreen检测试验来发现抑制ST2和IL33相互作用的化学探针。我们优化的检测条件是稳健的，给出了z‘因子e 0.7，并允许发现17个匹配项。这项研究的基本原理是，可能会有比飞行员屏幕上的探测器更好的探测器，因此需要大屏幕。此外，一个包括从更广泛的化合物库中进行潜在命中的电子浓缩和在GVHD生物模型中测试化学探针的平台将是相关的。三个具体目标是：1)HTS的实施，以发现抑制ST2/IL33途径的化学探针，该途径在移植物抗宿主病中过度表达。2)利用基于HTS确认的HITS的药效团模型生成的焦点化合物文库来丰富HITS，并在ST2中鉴定可能的结合位点。3)用体外人体试验和体内GVHD小鼠模型验证HITS。这种综合的方法是创新的，因为它应用了176,000种不同小分子的HTS技术，在电子致富HITS和靶点结合位点评估方面产生了针对GVHD难治性生物标志物的化学探针的识别和开发用于治疗。这项拟议的研究具有重要意义，因为GVHD特异性抑制剂的发现将为GVHD生物标记物作为可用药靶点的治疗评估提供基础。这些药物的应用将促使适当的效应性T细胞发生表型变化，从而提高疗效，降低毒性。

DESCRIPTION (provided by applicant): Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for many malignant diseases but its clinical utility has been impeded by graft-versus-host disease (GVHD). Unfortunately, there are currently no therapies that target proteins that are elevated in the blood of patients presenting with GVHD, and no effort has been made to develop drugs specific for GVHD. This represents an important problem, because currently therapies are limited to the nonspecific targeting of effector cells. Our long-term goal is to develop drugs targeting proteins that are dysregulated in patients with GVHD as potential novel GVHD therapies. Our objective in this application is to discover chemical probes that can inhibit these elevated proteins through high throughput screening (HTS) of small molecules libraries and be evaluated in the biological model systems. These compounds will allow for discovery of potential GVHD-specific drugs and therefore better harnessing of treatment in many patients with hematological cancers. Our central hypothesis is that the lead plasma biomarker of GVHD, suppression of tumorigenicity 2 (ST2) can be targeted with small molecule compounds to alleviate GVHD. This hypothesis was formed based on our published data characterizing the soluble form of ST2 (sST2) as the most significant biomarker to predict the non-response to GVHD therapy. sST2 acts as a decoy receptor for IL-33, the only known ligand for ST2, driving Th2 cells toward a Th1 phenotype. This phenotypic change leads to GVHD, and an inhibitor of the binding association between sST2 and IL-33 could alleviate GVHD. We therefore embark on a pilot HTS campaign using the AlphaScreen detection assay to discover chemical probes that inhibit the ST2 and IL33 interaction. Our optimized assay condition was robust giving z'-factors e 0.7 and permitted discovery of 17 hits. The rationale for this study is that the likelihood for better probes than those in the pilot screen warrants a large screen. In addition, a platform including in silico enrichment of potential hits from broader compound libraries and testing of the chemical probes in biological models of GVHD will be relevant. The three specific aims are: 1) HTS implementation to discover chemical probes that inhibit the ST2/IL33 pathway that is overexpressed in graft-versus-host disease. 2) Enrich hits using focus compound libraries generated from pharmacophore models based on confirmed hits from HTS and identification of putative binding sites in ST2. 3) Hits validation with in vitro huma assays and in vivo murine models of GVHD. This integrative approach is innovative because it applies HTS technology of 176,000 diverse small molecules with in silico enrichment of hits and target binding site evaluation yielding the identification and development of chemical probes against biomarkers of GVHD refractoriness to therapy. The proposed research is significant because discovery of GVHD-specific inhibitors will provide the foundation for therapeutic evaluation of GVHD biomarkers as druggable targets. The application of these drugs will drive the phenotypic changes in appropriate effector T cells with increased efficacy and lowered toxicity.