Identification and characterisation of small molecule inhibitors of the atypical protein kinase C family (aPKC)


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Christelle Soudy1, Frederic Elustondo1, Christian Dillon1, Alex Boakes1, Carine Rosse2, Mark Linch2, Sven Kjaer2, Brenda Kostelecky2, Sarah Kaye1, Caroline Barton1, Aurore Lejeune1, Neil McDonald2, Peter Parker2, Caroline Foxton1, Tim Hammonds1, Lloyd Kelland1, Jon Roffey1

1Cancer Research Technology Discovery Laboratories, University College London, London, UK, 2London Research Institute, London, UK

Abstract

Protein Kinase C (PKC) is a family of at least 12 serine/threonine kinases that are involved in the transduction of signals regulating cellular proliferation, differentiation, polarity, and survival. The PKC family has been divided into three structurally and functionally distinct sub-types: classic isoforms (PKCα, PKCβ, PKCγ); novel isoforms (PKCδ, PKCε, PKCη, PKCθ); and atypical isoforms (PKCζ, PKCι). Many classic and novel PKC isoforms are targets of tumour-promoting phorbol esters; thus aberrant PKC signalling may play a key role in tumourigenesis. Recent evidence has demonstrated PKCι to be an oncogene in non-small cell lung cancer (NSCLC), and there is increasing evidence linking aberrant PKCζ signalling to prostate cancer. This and other data strongly implicates inhibition of the aPKCs as a novel target for an anti-tumour agent. Using an active kinase domain of human PKCι, we screened a diverse set of 55,000 compounds by fluorescence polarisation. Through a process of Hit to Lead we further developed a number of distinct chemical series which were shown to inhibit PKCι via an allosteric or ATP-competitive mechanism of action. Some of our most potent compounds from each series, e.g. CRT0066055 (IC50PKCι = 83nM) and CRT0099431 (IC50PKCι=16nM), were taken forward for selectivity screening against a broad panel of kinases. The ATP-competitive inhibitor series were relatively selective for the atypical PKC isoforms, whereas the allosteric inhibitors show exquisite selectivity for PKCι. In addition, both types of inhibitor show good in vitro ADME properties, and exhibit promising results in cell-based assays which warrant progression to lead optimization and studies in vivo.

Protein Kinase C (PKC) is a family of at least 12 serine/threonine kinases that are involved in the transduction of signals regulating cellular proliferation, differentiation, polarity, and survival. The PKC family has been divided into three structurally and functionally distinct sub-types: classic isoforms (PKCα, PKCβ, PKCγ); novel isoforms (PKCδ, PKCε, PKCη, PKCθ); and atypical isoforms (PKCζ, PKCι). Many classic and novel PKC isoforms are targets of tumour-promoting phorbol esters; thus aberrant PKC signalling may play a key role in tumourigenesis. Recent evidence has demonstrated PKCι to be an oncogene in non-small cell lung cancer (NSCLC), and there is increasing evidence linking aberrant PKCζ signalling to prostate cancer. This and other data strongly implicates inhibition of the aPKCs as a novel target for an anti-tumour agent. Using an active kinase domain of human PKCι, we screened a diverse set of 55,000 compounds by fluorescence polarisation. Through a process of Hit to Lead we further developed a number of distinct chemical series which were shown to inhibit PKCι via an allosteric or ATP-competitive mechanism of action. Some of our most potent compounds from each series, e.g. CRT0066055 (IC50PKCι = 83nM) and CRT0099431 (IC50PKCι=16nM), were taken forward for selectivity screening against a broad panel of kinases. The ATP-competitive inhibitor series were relatively selective for the atypical PKC isoforms, whereas the allosteric inhibitors show exquisite selectivity for PKCι. In addition, both types of inhibitor show good in vitro ADME properties, and exhibit promising results in cell-based assays which warrant progression to lead optimization and studies in vivo.

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