Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors
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Author/Creator ORCID
Date
2019-07-05
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Citation of Original Publication
Miller TW, Amason JD, Garcin ED, Lamy L, Dranchak PK, Macarthur R, et al. (2019) Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors. PLoS ONE 14(7): e0218897. https://doi.org/10.1371/journal.pone.0218897
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Abstract
CD47 is an immune checkpoint molecule that downregulates key aspects of both the innate
and adaptive anti-tumor immune response via its counter receptor SIRPα, and it is
expressed at high levels in a wide variety of tumor types. This has led to the development of
biologics that inhibit SIRPα engagement including humanized CD47 antibodies and a soluble SIRPα decoy receptor that are currently undergoing clinical trials. Unfortunately, toxicological issues, including anemia related to on-target mechanisms, are barriers to their
clinical advancement. Another potential issue with large biologics that bind CD47 is perturbation of CD47 signaling through its high-affinity interaction with the matricellular protein
thrombospondin-1 (TSP1). One approach to avoid these shortcomings is to identify and
develop small molecule molecular probes and pretherapeutic agents that would (1) selectively target SIRPα or TSP1 interactions with CD47, (2) provide a route to optimize pharmacokinetics, reduce on-target toxicity and maximize tissue penetration, and (3) allow more
flexible routes of administration. As the first step toward this goal, we report the development
of an automated quantitative high-throughput screening (qHTS) assay platform capable of
screening large diverse drug-like chemical libraries to discover novel small molecules that
inhibit CD47-SIRPα interaction. Using time-resolved Fo¨rster resonance energy transfer
(TR-FRET) and bead-based luminescent oxygen channeling assay formats (AlphaScreen),
we developed biochemical assays, optimized their performance, and individually tested
them in small-molecule library screening. Based on performance and low false positive rate,
the LANCE TR-FRET assay was employed in a ~90,000 compound library qHTS, while the
AlphaScreen oxygen channeling assay served as a cross-validation orthogonal assay for
follow-up characterization. With this multi-assay strategy, we successfully eliminated compounds that interfered with the assays and identified five compounds that inhibit the CD47-
SIRPα interaction; these compounds will be further characterized and later disclosed.
Importantly, our results validate the large library qHTS for antagonists of CD47-SIRPα interaction and suggest broad applicability of this approach to screen chemical libraries for other
protein-protein interaction modulators.