Division of Rheumatology, Immunology, and Allergy and Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
PLoS Genet. 2013 May;9(5):e1003487. doi: 10.1371/journal.pgen.1003487. Epub 2013 May 16.
Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.
虽然在小鼠和人类中的遗传和非遗传研究提示 CD40 通路参与了类风湿关节炎（RA），但目前尚无抑制 CD40 信号转导的药物用于 RA 或任何其他疾病的临床治疗。在这里，我们试图了解通过先前的全基因组关联研究（GWAS）发现的 CD40 风险变异在 RA 中的生物学后果，并基于人类遗传发现对 CD40 信号转导进行高通量药物筛选。首先，我们在 7222 例血清阳性 RA 患者和 15870 例对照中精细定位 CD40 风险位点，在 500 例 RA 病例和 650 例对照中对 CD40 编码外显子进行深度测序，以确定一个可解释整个关联信号的单核苷酸多态性（rs4810485，P=1.4×10(-9)）。其次，我们证明与非风险等位基因纯合的个体相比，RA 风险等位基因纯合的个体原发性人 CD19+B 淋巴细胞表面的 CD40 约多 33%（P=10(-9)），这一发现通过对 1469 例健康对照个体外周血单个核细胞的表达数量性状基因座（eQTL）分析得到证实。第三，我们使用逆转录病毒 shRNA 感染来干扰人 B 淋巴细胞系（BL2）表面的 CD40 数量，并观察到 CD40 蛋白的量与 NF-κB 转录因子的 RelA（p65）亚单位磷酸化之间存在直接相关性。最后，我们在 BL2 细胞中建立了一种基于三聚体 CD40 配体（tCD40L）激活的 NF-κB 荧光素酶报告基因检测的高通量筛选，并对 1982 种化学化合物和 FDA 批准的药物进行了高通量筛选。经过一系列对照筛选和对原代人 CD19+B 细胞的测试，我们鉴定出 2 种以前未涉及炎症或 CD40 介导的 NF-κB 信号转导的新型化学抑制剂。我们的研究证明了一个概念验证，即人类遗传学可用于指导基于表型的高通量小分子筛选的开发，以鉴定复杂特征（如 RA）中的潜在新型疗法。
