title: Targeting substrate-site in Jak2 kinase prevents emergence of genetic resistance.
abstract: Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. Here, we report a novel mechanism of JAK2 kinase inhibition by fedratinib (TG101348) that prevents emergence of genetic resistance. Using in vitro drug screening, we identified 211 amino-acid substitutions conferring resistance to ruxolitinib (INCB018424) and cross-resistance to the JAK2 inhibitors AZD1480, CYT-387 and lestaurtinib. In contrast, these resistant variants were fully sensitive to fedratinib. Structural modeling, coupled with mutagenesis and biochemical studies, revealed dual binding sites for fedratinib. In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM). Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants. Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site.
authors.affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.
authors.forename: Meenu
authors.initials: M
authors.lastname: Kesarwani
authors.affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.
authors.forename: Erika
authors.initials: E
authors.lastname: Huber
authors.affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Kincaid
authors.affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.
authors.forename: Chris R
authors.initials: CR
authors.lastname: Evelyn
authors.affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.
authors.forename: Jacek
authors.initials: J
authors.lastname: Biesiada
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology University of Cincinnati College of Medicine, University of Cincinnati, Ohio 45229 USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Rance
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology University of Cincinnati College of Medicine, University of Cincinnati, Ohio 45229 USA.
authors.forename: Mahendra B
authors.initials: MB
authors.lastname: Thapa
authors.affiliation: Division of Hematology-Oncology UCSF School of Medicine, San Francisco, California, 94143 USA.
authors.forename: Neil P
authors.initials: NP
authors.lastname: Shah
authors.affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.
authors.forename: Jarek
authors.initials: J
authors.lastname: Meller
authors.affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Cincinnati Children's Hospital Medical Center, Cancer Blood Disease Institute, Divisions of Experimental Hematology and Cancer Pathology, Cincinnati, Ohio, 45229 USA.|Department of Molecular Genetics, Biochemistry and Microbiology University of Cincinnati College of Medicine, University of Cincinnati, Ohio 45229 USA.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Azam
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D001665:Binding Sites; D020134:Catalytic Domain; D003062:Codon; D004351:Drug Resistance / Q000235:genetics*; D018432:Drug Resistance, Multiple / Q000235:genetics; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009570:Nitriles; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology; D011743:Pyrimidines; D011759:Pyrrolidines / Q000737:chemistry / Q000494:pharmacology; D013379:Substrate Specificity; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology; D000096926:Benzenesulfonamides
chemical_list: D000255:Adenosine Triphosphate; D003062:Codon; D053614:Janus Kinase 2; D009570:Nitriles; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D011759:Pyrrolidines; D013449:Sulfonamides; C528327:fedratinib; D000096926:Benzenesulfonamides; C540383:ruxolitinib