Quantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitors

Simple item page
dc.contributor.authorMiller, Thomas W.
dc.contributor.authorAmason, Joshua D.
dc.contributor.authorGarcin, Elsa D.
dc.contributor.authorLamy, Laurence
dc.contributor.authorDranchak, Patricia K.
dc.contributor.authorMacarthur, Ryan
dc.contributor.authorBraisted, John
dc.contributor.authorRubin, Jeffrey S.
dc.contributor.authorBurgess, Teresa L.
dc.contributor.authorFarrell, Catherine L.
dc.contributor.authorRoberts, David D.
dc.contributor.authorInglese, James
dc.date.accessioned2019-10-02T15:01:33Z
dc.date.available2019-10-02T15:01:33Z
dc.date.issued2019-07-05
dc.description.abstractCD47 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.en_US
dc.description.sponsorshipThis work was supported in part by the Avon Foundation for Women (02-2015-045, 02- 2014-051, 02-2015-095, 02-2017-034; TWM, TLB, CLF), the National Cancer Institute (1U01CA218259-01A1; TWM, TLB, CLF, JSR) the Intramural Research Programs of NCATS (Project 1ZIATR000053-03, JI) and NCI (Project ZIASC009172, DDR), the Maryland Industrial Partnerships Program (MIPs, #5914; EDG, TWM) and Cooperative Research and Development Agreement 02921 with the National Institutes of Health. This work was also conducted at the Advanced Light Source (ALS), a national user facility operated by Lawrence Berkeley National Laboratory on behalf of the Department of Energy, Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies (IDAT) program, supported by DOE Office of Biological and Environmental Research. Additional support comes from the National Institute of Health project ALS-ENABLE (P30 GM124169) and a High-End Instrumentation Grant S10OD018483. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.en_US
dc.format.extent31 pagesen_US
dc.genreJournal Articlesen_US
dc.identifierdoi:10.13016/m23oej-sf7y
dc.identifier.citationMiller 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.0218897en_US
dc.identifier.urihttps://doi.org/10.1371/journal.pone.0218897
dc.identifier.urihttp://hdl.handle.net/11603/14958
dc.language.isoen_USen_US
dc.publisherPLOSen_US
dc.relation.isAvailableAtThe University of Maryland, Baltimore County (UMBC)
dc.relation.ispartofUMBC Chemistry & Biochemistry Department Collection
dc.relation.ispartofUMBC Faculty Collection
dc.rightsThis item is likely protected under Title 17 of the U.S. Copyright Law. Unless on a Creative Commons license, for uses protected by Copyright Law, contact the copyright holder or the author.
dc.rightsCC0 1.0 Universal (CC0 1.0) Public Domain Dedication*
dc.rights.urihttps://creativecommons.org/publicdomain/zero/1.0/*
dc.subjectLibrary screeningen_US
dc.subjectSmall moleculesen_US
dc.subjectBiotinen_US
dc.subjectCentrifugesen_US
dc.subjectOxygenen_US
dc.subjectProtein concentration assaysen_US
dc.subjectHigh throughput screeningen_US
dc.subjectProtein-protein interactionsen_US
dc.titleQuantitative high-throughput screening assays for the discovery and development of SIRPα-CD47 interaction inhibitorsen_US
dc.typeTexten_US

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Quantitative high-throughput screening.pdf
Size:
4.77 MB
Format:
Adobe Portable Document Format
Description:
Download
License bundle
Now showing 1 - 1 of 1
No Thumbnail Available
Name:
license.txt
Size:
2.56 KB
Format:
Item-specific license agreed upon to submission
Description:
Download

Collections

UMBC Chemistry & Biochemistry Department
UMBC Faculty Collection