title: Definitive Metabolite Identification Coupled with Automated Ligand Identification System (ALIS) Technology: A Novel Approach to Uncover Structure-Activity Relationships and Guide Drug Design in a Factor IXa Inhibitor Program.
abstract: A potent and selective Factor IXa (FIXa) inhibitor was subjected to a series of liver microsomal incubations, which generated a number of metabolites. Using automated ligand identification system-affinity selection (ALIS-AS) methodology, metabolites in the incubation mixture were prioritized by their binding affinities to the FIXa protein. Microgram quantities of the metabolites of interest were then isolated through microisolation analytical capabilities, and structurally characterized using MicroCryoProbe heteronuclear 2D NMR techniques. The isolated metabolites recovered from the NMR experiments were then submitted directly to an in vitro FIXa enzymatic assay. The order of the metabolites' binding affinity to the Factor IXa protein from the ALIS assay was completely consistent with the enzymatic assay results. This work showcases an innovative and efficient approach to uncover structure-activity relationships (SARs) and guide drug design via microisolation-structural characterization and ALIS capabilities.
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Xianshu
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Gary E
authors.initials: GE
authors.lastname: Martin
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Huifang
authors.initials: H
authors.lastname: Yao
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Jackie
authors.initials: J
authors.lastname: Shang
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Randal M
authors.initials: RM
authors.lastname: Bugianesi
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Kenneth P
authors.initials: KP
authors.lastname: Ellsworth
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Sonatore
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Peter
authors.initials: P
authors.lastname: Nizner
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Edward C
authors.initials: EC
authors.lastname: Sherer
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Hill
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Ian W
authors.initials: IW
authors.lastname: Knemeyer
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Wayne M
authors.initials: WM
authors.lastname: Geissler
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Dandliker
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Roy
authors.initials: R
authors.lastname: Helmy
authors.affiliation: Department of Medicinal Chemistry, ‡Department of Process and Analytical Chemistry, and ¶Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc. , PO Box 2000, Rahway, New Jersey 07065, United States.|Department of Pharmacology and ⊥Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck Research Laboratories, Merck & Co., Inc. , 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States.|Department of Pharmacokinetics, Pharmacodynamics and Drug Metabolism, and §Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
authors.forename: Harold B
authors.initials: HB
authors.lastname: Wood
mesh_terms: D000818:Animals; D001331:Automation; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D015949:Factor IXa / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D005343:Fibrinolytic Agents; D008024:Ligands; D015949:Factor IXa