Cell death plays a causal role in heart failure, and its inhibition poses a promising approach that has not been thoroughly explored. In past approaches to target discovery, clinical failures have reflected deficiencies in mechanistic understanding, and failure to translate laboratory findings to a human setting. We previously identified MAP4K4/HGK as an activator of the TAK1 - JNK pathway in cardiac cell death, whose inhibition by dominant-negative mutations or shRNA knockdown confers protection in cultured rat neonatal cardiomyocytes.Methods
Compound activity against MAP4K4 recombinant purified kinase domain was carried out by HTRF Transcreener ADP assay (Cisbio), knockdown of MAP4K4 was achieved by virus-mediated gene transfer of shRNAmirs into human induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Selectivity was assessed against a panel of 140 human kinases (Dundee, MRC protein phosphorylation and ubiquitination unit). Cell death was assessed by hypodiploid DNA content, Draq7 uptake and Caspase 3 activity.Results
Firstly confirming the relevance of MAP4K4 in a human setting, shRNAmir-mediated knockdown was protective against H202-induced cardiomyocyte cell death in human iPSC-derived cardiomyocytes. To address this therapeutic potential, we initiated a small molecule drug discovery programme by first screening a library of known kinase inhibitors for activity against recombinant MAP4K4 kinase domain. Hits with pIC50 > 7 (series 1) that shared similar features of charge distribution were used to generate a consensus pharmacophore model for inhibition of MAP4K4. Virtual interrogation of a database of more than one million compounds identified novel compounds fitting this model. The top 40 ranked compounds were tested for activity against MAP4K4 and the most potent from each of two chemical series (series 2) taken forward. Series 2 had much improved selectivity (only 3–5 of 141 kinases inhibited > 50% at 1 M) over both series 1 and two small molecule inhibitors described by Pfizer. In addition, series 2 were not toxic to cardiomyocytes up to 10 M. Importantly both compounds from series 2 showed protection against H202- or C2-ceramide mediated cell death in rat neonatal cardiomyocytes.Conclusions
Thus we have obtained promising lead compounds for optimisation and mechanistic studies in both murine and human models, and shown that inhibition of MAP4K4 in vitro , whether by knockdown of expression or pharmacological inhibition is protective against cell death in cardiomyocytes. MAP4K4 is therefore a potential target for pharmacological inhibition of cell death in cardiac disease.