ABSTRACT
Inhibitors of mitochondrial branched chain aminotransferase (BCATm), identified using fragment screening, are described. This was carried out using a combination of STD-NMR, thermal melt (Tm), and biochemical assays to identify compounds that bound to BCATm, which were subsequently progressed to X-ray crystallography, where a number of exemplars showed significant diversity in their binding modes. The hits identified were supplemented by searching and screening of additional analogues, which enabled the gathering of further X-ray data where the original hits had not produced liganded structures. The fragment hits were optimized using structure-based design, with some transfer of information between series, which enabled the identification of ligand efficient lead molecules with micromolar levels of inhibition, cellular activity, and good solubility.
Subject(s)
Mitochondria/enzymology , Transaminases/antagonists & inhibitors , Adipocytes/drug effects , Adipocytes/enzymology , Crystallography, X-Ray , High-Throughput Screening Assays , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Protein Binding , Structure-Activity RelationshipABSTRACT
The hybridization of hits, identified by complementary fragment and high throughput screens, enabled the discovery of the first series of potent inhibitors of mitochondrial branched-chain aminotransferase (BCATm) based on a 2-benzylamino-pyrazolo[1,5-a]pyrimidinone-3-carbonitrile template. Structure-guided growth enabled rapid optimization of potency with maintenance of ligand efficiency, while the focus on physicochemical properties delivered compounds with excellent pharmacokinetic exposure that enabled a proof of concept experiment in mice. Oral administration of 2-((4-chloro-2,6-difluorobenzyl)amino)-7-oxo-5-propyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 61 significantly raised the circulating levels of the branched-chain amino acids leucine, isoleucine, and valine in this acute study.
Subject(s)
Mitochondrial Proteins/antagonists & inhibitors , Pyrazoles/chemistry , Pyrimidinones/chemistry , Transaminases/antagonists & inhibitors , Adipocytes/drug effects , Adipocytes/enzymology , Animals , Crystallography, X-Ray , Humans , Isoleucine/blood , Leucine/blood , Mice, Inbred BALB C , Mice, Inbred C57BL , Models, Molecular , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Pyrimidinones/chemical synthesis , Pyrimidinones/pharmacology , Structure-Activity Relationship , Transaminases/chemistry , Valine/bloodABSTRACT
[reaction: see text] In this, the first of two Letters, we describe how a P3/P4 urea linking unit was used to greatly enhance the biochemical and replicon potency of inhibitors based upon the pyrrolidine-5,5-trans-lactam template. Compound 7b demonstrated a 100 nM IC(50) in the replicon cell-based surrogate HCV assay.
Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/enzymology , Lactams/chemistry , Protease Inhibitors/chemical synthesis , Pyrrolidines/chemistry , Urea/chemistry , Viral Nonstructural Proteins/antagonists & inhibitors , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Crystallography, X-Ray , Hepacivirus/drug effects , Hepacivirus/physiology , Inhibitory Concentration 50 , Molecular Conformation , Molecular Structure , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effectsABSTRACT
[reaction: see text] In this, the second of two letters, we describe the elaboration of the pyrrolidine-5,5-trans-lactam template to delineate the requirements for optimal substitution of the pyrrolidine and lactam nitrogen atoms. Central to the strategy is the use of rapid iterative synthesis in conjunction with X-ray crystal structure determination of ligand-protein complexes.
