Session Type: Plenary Sessions
Background/Purpose: Deriving therapeutic targets from human genetics linked with biological alterations of risk alleles may provide a more successful approach to drug development than traditional efforts that focus on biological insight alone. Here, we successfully translate a SNP association from a genome-wide association study (GWAS) in rheumatoid arthritis (RA) into a high-throughput screen (HTS) based on cellular phenotype in a human B cell line to identify inhibitors of CD40-mediated NF-κB signaling.
Methods: We fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls genotyped on the Immunochip, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls. We use flow cytometry to measure CD40 protein levels on the surface of primary CD19+ from 90 healthy control individuals. We use gene expression arrays to measure CD40 RNA levels in peripheral blood mononuclear cells from 1,469 healthy control individuals. We use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2). 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. Counter-screens of the top “hit” compounds were performed in the BL2 line activated with both tCD40L and LPS, and in an additional B cell line, Ramos, activated with tCD40L and TNF. Two known and two novel compounds were tested for inhibition of tCD40-NFkB signaling in primary human CD19+ B cells by measuring CD86 expression with flow cytometry.
Results: A single common SNP at the CD40 locus explains the entire signal of association (rs4810485, P=1.4×10-9), without any evidence for independent rare variants contributing to RA risk. Subjects homozygous for the common 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 PBMC’s (P<10-15). We observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Using our luciferase reporter assay, we identify 81 “hit” compounds (out of 1,982) that consistent inhibit luciferase activity following tCD40L activation. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 “known” and 2 “novel” chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. One known inhibitor is tranilast, a drug currently in a phase II clinical trial of RA; the other is a corticosteroid derivative. The two novel compounds represent promising tool compounds to develop new therapies to treat RA.
Conclusion: 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.
R. M. Plenge,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/genome-wide-association-study-to-high-throughput-cell-based-phenotypic-screen-identifies-novel-chemical-inhibitors-of-cd40-signaling/