ABSTRACT
Inhibition of the hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) represents a promising strategy for discovering next-generation treatments for renal anemia. We identified a pyrimidine core with HIF-PHD inhibitory activity based on scaffold hopping of FG-2216 using crystal structures of HIF-PHD2 in complex with compound. By optimizing the substituents at the 2- and 6- positions of the pyrimidine core, we discovered DS44470011, which improves the effectiveness of erythropoietin (EPO) release in cells. Oral administration of DS44470011 to cynomolgus monkeys increased plasma EPO levels.
Subject(s)
Anemia , Hypoxia-Inducible Factor-Proline Dioxygenases , Macaca fascicularis , Prolyl-Hydroxylase Inhibitors , Animals , Anemia/drug therapy , Hypoxia-Inducible Factor-Proline Dioxygenases/antagonists & inhibitors , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Administration, Oral , Humans , Prolyl-Hydroxylase Inhibitors/pharmacology , Prolyl-Hydroxylase Inhibitors/chemistry , Prolyl-Hydroxylase Inhibitors/chemical synthesis , Pyrimidines/chemistry , Pyrimidines/pharmacology , Pyrimidines/chemical synthesis , Structure-Activity Relationship , Molecular Structure , Erythropoietin , Drug Discovery , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesisABSTRACT
The design and discovery of a new series of (5-alkynyl-3-hydroxypicolinoyl)glycine inhibitors of prolyl hydroxylase (PHD) are described. These compounds showed potent in vitro inhibitory activity toward PHD2 in a fluorescence polarization-based assay. Remarkably, oral administration of 17, with an IC50 of 64.2 nM toward PHD2, was found to stabilize HIF-α, elevate erythropoietin (EPO), and alleviate anemia in a cisplatin-induced anemia mouse model with an oral dose of 25 mg/kg. Rat and dog studies showed that 17 has good pharmacokinetic properties, with oral bioavailabilities of 55.7 and 54.0%, respectively, and shows excellent safety profiles even at a high dose of 200 mg/kg in these animals. Based on these results, 17 is currently being evaluated in a phase I clinical trial for anemia.
Subject(s)
Anemia/drug therapy , Glycine/analogs & derivatives , Glycine/therapeutic use , Picolinic Acids/therapeutic use , Prolyl-Hydroxylase Inhibitors/therapeutic use , Anemia/chemically induced , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cisplatin , Dogs , Drug Design , Erythropoietin/metabolism , Female , Glycine/pharmacokinetics , Glycine/toxicity , Male , Mice, Inbred C57BL , Molecular Structure , Picolinic Acids/chemical synthesis , Picolinic Acids/pharmacokinetics , Picolinic Acids/toxicity , Prolyl-Hydroxylase Inhibitors/chemical synthesis , Prolyl-Hydroxylase Inhibitors/pharmacokinetics , Prolyl-Hydroxylase Inhibitors/toxicity , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
Prolyl hydroxylation domain (PHD) enzymes catalyze the hydroxylation of the transcription factor hypoxia-inducible factor (HIF) and serve as cellular oxygen sensors. HIF and the PHD enzymes regulate numerous potentially tissue-protective target genes which can adapt cells to metabolic and ischemic stress. We describe a fluorescent PHD inhibitor (1-chloro-4-hydroxybenzo[g]isoquinoline-3-carbonyl)glycine which is suited to fluorescence-based detection assays and for monitoring PHD inhibitors in biological systems. In cell-based assays, application of the fluorescent PHD inhibitor allowed co-localization with a cellular PHD enzyme and led to live cell imaging of processes involved in cellular oxygen sensing.
Subject(s)
Benzylisoquinolines/pharmacology , Fluorescent Dyes/pharmacology , Molecular Imaging/methods , Optical Imaging/methods , Prolyl Hydroxylases/metabolism , Prolyl-Hydroxylase Inhibitors/pharmacology , Benzylisoquinolines/chemical synthesis , Benzylisoquinolines/chemistry , Biocatalysis/drug effects , Dose-Response Relationship, Drug , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Molecular Structure , Prolyl-Hydroxylase Inhibitors/chemical synthesis , Prolyl-Hydroxylase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Collagen is the most abundant protein in animals. A variety of indications are associated with the overproduction of collagen, including fibrotic diseases and cancer metastasis. The stability of collagen relies on the posttranslational modification of proline residues to form (2S,4R)-4-hydroxyproline. This modification is catalyzed by collagen prolyl 4-hydroxylases (CP4Hs), which are Fe(II)- and α-ketoglutarate (AKG)-dependent dioxygenases located in the lumen of the endoplasmic reticulum. Human CP4Hs are validated targets for treatment of both fibrotic diseases and metastatic breast cancer. Herein, we report on 2,2'-bipyridinedicarboxylates as inhibitors of a human CP4H. Although most 2,2'-bipyridinedicarboxylates are capable of inhibition via iron sequestration, the 4,5'- and 5,5'-dicarboxylates were found to be potent competitive inhibitors of CP4H, and the 5,5'-dicarboxylate was selective in its inhibitory activity. Our findings clarify a strategy for developing CP4H inhibitors of clinical utility.
Subject(s)
Carboxylic Acids/chemistry , Hypoxia-Inducible Factor-Proline Dioxygenases/antagonists & inhibitors , Iron Chelating Agents/chemistry , Procollagen-Proline Dioxygenase/antagonists & inhibitors , Prolyl-Hydroxylase Inhibitors/chemistry , Pyridines/chemistry , Animals , Binding, Competitive , Carboxylic Acids/chemical synthesis , Collagen/antagonists & inhibitors , Collagen/biosynthesis , Dose-Response Relationship, Drug , Enzyme Assays , Humans , Hypoxia-Inducible Factor-Proline Dioxygenases/chemistry , Iron/metabolism , Iron Chelating Agents/chemical synthesis , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Kinetics , Procollagen-Proline Dioxygenase/chemistry , Prolyl-Hydroxylase Inhibitors/chemical synthesis , Pyridines/chemical synthesis , Recombinant Proteins/chemistryABSTRACT
We have discovered a novel complex crystal structure of the PHD2 enzyme with its inhibitor, the 2,8-diazaspiro[4.5]decan-1-one analogue 4b. The widely reported salt bridge between Arg383 of the enzyme and its inhibitors in all complex structures published thus far was not observed in our case. In our complex structure compound 4b forms several novel interactions with the enzyme, which include a hydrogen bond with Arg322, a π-cation interaction with Arg322, a π-π stacking with Trp389, and a π-π stacking with His313. Guided by the structural information, SAR studies were performed on the 2,8-diazaspiro[4.5]decan-1-one series leading to the discovery of compound 9p with high potency and good oral pharmacokinetic profile in mice.
