title: Integration of Affinity Selection-Mass Spectrometry and Functional Cell-Based Assays to Rapidly Triage Druggable Target Space within the NF-κB Pathway.
abstract: The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection-mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway.
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA victoria.kutilek@merck.com.
authors.forename: Victoria D
authors.initials: VD
authors.lastname: Kutilek
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Christine L
authors.initials: CL
authors.lastname: Andrews
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Richards
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Zangwei
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Tianxiao
authors.initials: T
authors.lastname: Sun
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Hashke
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Nadya
authors.initials: N
authors.lastname: Smotrov
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Rafael
authors.initials: R
authors.lastname: Fernandez
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Elliott B
authors.initials: EB
authors.lastname: Nickbarg
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Chad
authors.initials: C
authors.lastname: Chamberlin
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Berengere
authors.initials: B
authors.lastname: Sauvagnat
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Curran
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Boinay
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Saradjian
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Samantha J
authors.initials: SJ
authors.lastname: Allen
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Noel
authors.initials: N
authors.lastname: Byrne
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA Current address: AbbVie, North Chicago, IL USA.
authors.forename: Nathaniel L
authors.initials: NL
authors.lastname: Elsen
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Rachael E
authors.initials: RE
authors.lastname: Ford
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Dawn L
authors.initials: DL
authors.lastname: Hall
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Kornienko
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA Current address: Medimmune, Gaithersburg, MD, USA.
authors.forename: Keith W
authors.initials: KW
authors.lastname: Rickert
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Sujata
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Shipman
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Lumb
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA Current address: Arvinas, New Haven, CT, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Coleman
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Dandliker
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Ilona
authors.initials: I
authors.lastname: Kariv
authors.affiliation: Department of Pharmacology, Screening and Protein Sciences, Merck & Co, Kenilworth, NJ, USA.
authors.forename: Bruce
authors.initials: B
authors.lastname: Beutel
mesh_terms: D055808:Drug Discovery; D057166:High-Throughput Screening Assays / Q000379:methods*; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011485:Protein Binding; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship; D048015:TNF Receptor-Associated Factor 5 / Q000037:antagonists & inhibitors / Q000737:chemistry; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors / Q000737:chemistry
chemical_list: D008024:Ligands; D016328:NF-kappa B; C498143:RELA protein, human; D048015:TNF Receptor-Associated Factor 5; C000617531:TRAF5 protein, human; D051996:Transcription Factor RelA
keywords: ALIS; NF-κB pathway; NF-κB1/p105; TRAF5; affinity selection; high-throughput screening; mass spectrometry; phenotypic screening