Discovery and Optimization of the First ATP Competitive Type-III c-MET Inhibitor.

Recent clinical reports have highlighted the need for wild-type (WT) and mutant dual inhibitors of c-MET kinase for the treatment of cancer. We report herein a novel chemical series of ATP competitive type-III inhibitors of WT and D1228V mutant c-MET. Using a combination of structure-based drug design and computational analyses, ligand 2 was optimized to a highly selective chemical series with nanomolar activities in biochemical and cellular settings. Representatives of the series demonstrate excellent pharmacokinetic profiles in rat in vivo studies with promising free-brain exposures, paving the way for the design of brain permeable drugs for the treatment of c-MET driven cancers.

Discovery of a selective c-MET inhibitor with a novel binding mode.

The c-MET receptor tyrosine kinase has received considerable attention as a cancer drug target yet there remains a need for inhibitors which are selective for c-MET and able to target emerging drug-resistant mutants. We report here the discovery, by screening a DNA-encoded chemical library, of a highly selective c-MET inhibitor which was shown by X-ray crystallography to bind to the kinase in an unprecedented manner. These results represent a novel mode of inhibiting c-MET with a small molecule and may provide a route to targeting drug-resistant forms of the kinase whilst avoiding potential toxicity issues associated with broad kinome inhibition.

Subcutaneous delivery of FGF21 mRNA therapy reverses obesity, insulin resistance, and hepatic steatosis in diet-induced obese mice.

Fibroblast growth factor 21 (FGF21) is a promising therapeutic agent for treatment of type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH). We show that therapeutic levels of FGF21 were achieved following subcutaneous (s.c.) administration of mRNA encoding human FGF21 proteins. The efficacy of mRNA was assessed following 2-weeks repeated s.c. dosing in diet-induced obese (DIO), mice which resulted in marked decreases in body weight, plasma insulin levels, and hepatic steatosis. Pharmacokinetic/pharmacodynamic (PK/PD) modelling of several studies in both lean and DIO mice showed that mRNA encoding human proteins provided improved therapeutic coverage over recombinant dosed proteins in vivo. This study is the first example of s.c. mRNA therapy showing pre-clinical efficacy in a disease-relevant model, thus, showing the potential for this modality in the treatment of chronic diseases, including T2D and NASH.

Pharmacological treatment with FGF21 strongly improves plasma cholesterol metabolism to reduce atherosclerosis.

AIMS: Fibroblast growth factor (FGF) 21, a key regulator of energy metabolism, is currently evaluated in humans for treatment of type 2 diabetes and non-alcoholic steatohepatitis. However, the effects of FGF21 on cardiovascular benefit, particularly on lipoprotein metabolism in relation to atherogenesis, remain elusive. METHODS AND RESULTS: Here, the role of FGF21 in lipoprotein metabolism in relation to atherosclerosis development was investigated by pharmacological administration of a half-life extended recombinant FGF21 protein to hypercholesterolaemic APOE*3-Leiden.CETP mice, a well-established model mimicking atherosclerosis initiation and development in humans. FGF21 reduced plasma total cholesterol, explained by a reduction in non-HDL-cholesterol. Mechanistically, FGF21 promoted brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning, thereby enhancing the selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT and into browned WAT, consequently accelerating the clearance of the cholesterol-enriched remnants by the liver. In addition, FGF21 reduced body fat, ameliorated glucose tolerance and markedly reduced hepatic steatosis, related to up-regulated hepatic expression of genes involved in fatty acid oxidation and increased hepatic VLDL-triglyceride secretion. Ultimately, FGF21 largely decreased atherosclerotic lesion area, which was mainly explained by the reduction in non-HDL-cholesterol as shown by linear regression analysis, decreased lesion severity, and increased atherosclerotic plaque stability index. CONCLUSION: FGF21 improves hypercholesterolaemia by accelerating triglyceride-rich lipoprotein turnover as a result of activating BAT and browning of WAT, thereby reducing atherosclerotic lesion severity and increasing atherosclerotic lesion stability index. We have thus provided additional support for the clinical use of FGF21 in the treatment of atherosclerotic cardiovascular disease.

Fragment-Based Discovery of Novel Allosteric MEK1 Binders.

The MEK1 kinase plays a critical role in key cellular processes, and as such, its dysfunction is strongly linked to several human diseases, particularly cancer. MEK1 has consequently received considerable attention as a drug target, and a significant number of small-molecule inhibitors of this kinase have been reported. The majority of these inhibitors target an allosteric pocket proximal to the ATP binding site which has proven to be highly druggable, with four allosteric MEK1 inhibitors approved to date. Despite the significant attention that the MEK1 allosteric site has received, chemotypes which have been shown structurally to bind to this site are limited. With the aim of discovering novel allosteric MEK1 inhibitors using a fragment-based approach, we report here a screening method which resulted in the discovery of multiple allosteric MEK1 binders, one series of which was optimized to sub-µM affinity for MEK1 with promising physicochemical and ADMET properties.

Structural Basis for Targeting the Folded P-Loop Conformation of c-MET.

We report here a fragment screen directed toward the c-MET kinase from which we discovered a series of inhibitors able to bind to a rare conformation of the protein in which the P-loop adopts a collapsed, or folded, arrangement. Preliminary SAR exploration led to an inhibitor (7) with nanomolar biochemical activity against c-MET and promising cell activity and kinase selectivity. These findings increase our structural understanding of the folded P-loop conformation of c-MET and provide a sound structural and chemical basis for further investigation of this underexplored yet potentially therapeutically exploitable conformational state.

Structural and Molecular Insight into Resistance Mechanisms of First Generation cMET Inhibitors.

Many small molecule inhibitors of the cMET receptor tyrosine kinase have been evaluated in clinical trials for the treatment of cancer and resistance-conferring mutations of cMET are beginning to be reported for a number of such compounds. There is now a need to understand specific cMET mutations at the molecular level, particularly concerning small molecule recognition. Toward this end, we report here the first crystal structures of the recent clinically observed resistance-conferring D1228V cMET mutant in complex with small molecule inhibitors, along with a crystal structure of wild-type cMET bound by the clinical compound savolitinib and supporting cellular, biochemical, and biophysical data. Our findings indicate that the D1228V alteration induces conformational changes in the kinase, which could have implications for small molecule inhibitor design. The data we report here increases our molecular understanding of the D1228V cMET mutation and provides insight for future inhibitor design.

The Structure of Murine N1-Acetylspermine Oxidase Reveals Molecular Details of Vertebrate Polyamine Catabolism.

N1-Acetylspermine oxidase (APAO) catalyzes the conversion of N1-acetylspermine or N1-acetylspermidine to spermidine or putrescine, respectively, with concomitant formation of N-acetyl-3-aminopropanal and hydrogen peroxide. Here we present the structure of murine APAO in its oxidized holo form and in complex with substrate. The structures provide a basis for understanding molecular details of substrate interaction in vertebrate APAO, highlighting a key role for an asparagine residue in coordinating the N1-acetyl group of the substrate. We applied computational methods to the crystal structures to rationalize previous observations with regard to the substrate charge state. The analysis suggests that APAO features an active site ideally suited for binding of charged polyamines. We also reveal the structure of APAO in complex with the irreversible inhibitor MDL72527. In addition to the covalent adduct, a second MDL72527 molecule is bound in the active site. Binding of MDL72527 is accompanied by altered conformations in the APAO backbone. On the basis of structures of APAO, we discuss the potential for development of specific inhibitors.