Experimental Therapeutics Programme

Biology Section

Head of Section:  Carmen Blanco
Research highlights

During 2016, our Section was involved in several projects :

Cyclin-dependent kinase 8 (CDK8)

This is a funded project (grant no. SAF2013-44267-R). We have identified ETP-27, a highly selective picomolar CDK8/CDK19 inhibitor with picomolar cellular inhibition of P-STAT1-S727. The main off-target identified is Haspin; ETP-27 demonstrates a cellular selectivity of > 30 fold for CDK8 vs. Haspin. This compound shows good solubility, permeability, metabolic stability in human and rat microsomes that is moderate in the mouse, and no alerts in terms of CYP-P450 inhibition and hERG binding, as well as important toxicity related parameters. Moreover, the compound is orally bioavailable as observed in pharmacokinetic (PK) studies in mice; in mechanistic PK-PD studies, modulation iomarker PSTAT1-S727 was observed between 1 and 4 h after oral treatment. ETP-27 has shown promising results in an efficacy study in MOLM13 xenografts, where it demonstrated a 50% tumour growth inhibition after 10 mg/kg twice-a-day treatment over 16 days. Our next steps will focus on the in vivo testing of new compounds resulting from the fine optimisation of ET-27 in order to achieve an even higher in vivo exposure.

Microtubule-associated serine/threonine protein kinase-like (MASTL)

This project is undertaken in collaboration with the CNIO Cell Division and Cancer Group. We have previously reported the production of active human full length MASTL protein to run biochemical assays. We used it to validate the hits coming from the phenotypic screening and to perform a biochemical screening of our ETP-640 library. In a single point screening assay, with the cut-off value set at 40% inhibition, we achieved a hit rate of 0.02. Hits were confirmed in a dose response assay and a number of analogues were tested. We have identified a 300nM hit, which is a good starting point for the exploration of Structure-ActivityRelationships (SAR) and for pharmacophore development. This information will be used later on for hit generation and subsequent hit-to-lead (HtL) exploration of novel inhibitors.

Kinase X

This project is a collaborative undertaking with VIB (Belgium). We have identified a lead compound with nanomolar inhibition activity for the target, good solubility, permeability and high selectivity against a panel of 456 kinases. Moreover, the compound is orally bioavailable, well tolerated in mice and, in a distribution study, was detected in several tissues for up to 8 hours. This lead compound is being used at VIB for in vivo validation studies.

Kinase Y

In this second project, also in collaboration with VIB, we have characterised at the biochemical level, 30 compounds synthesised in hit generation activities.

Telomeric repeat binding factor 1 (TRF1)

This project is carried out in collaboration with the CNIO Telomeres and Telomerase Group. A phenotypic assay to measure the association of TRF1 to telomeres has been used to test 72 compounds that include analogues of a second hit, ETP-946, identified in the initial screening; chemical probes derived from it have been generated. Moreover, we have set up a cellular thermal shift assay with over-expressed human TRF1 to validate if this series of compounds interacts directly with TRF1, and with potential to serve as a platform for identifying compounds that directly interact with TRF1.

Cancer stem cells (CSC) and gluconeogenesis

These projects are carried out in collaboration with the CNIO Tumour Suppression Group. For the CSC project, we collaborated in the deconvolution studies of the identified hits, evaluating their activity in kinase and receptor panels, as well as providing technical support for the assays using chemical probes. For the gluconeogeneis project, we have performed pharmacokinetic profiles of the hits obtained in the screening; the most promising compound has been tested in vivo by our collaborator.

Brain metastasis screening

The CNIO Brain Metastasis Group has developed an ex vivo assay to search for drugs that kill human brain metastasis in mice. ETP-Biology has provided support in running the experiments and also to validate the in vitro screening results obtained with the approved or in clinical trial ETP-antitumour library. Two classes of drugs are under further characterisation.

Focal adhesion kinase (FAK)

This project is undertaken in collaboration with the CNIO Cell Signalling and Adhesion Group. We have set up a biochemical assay with purified protein produced by our collaborator, using both the catalytic domain alone and the full length protein. We have tested 80 compounds coming from a virtual screening analysis; the hit rate obtained was of 0.21 with a cut off value set at 90% inhibition. Five of the compounds with an IC50 below 500nM have been selected for crystallisation studies.

Support to other CNIO Groups

We have given support to Manuel Serrano’s Group by analysing, with liquid chromatography-tandem mass spectrometry (LC-MS/MS), in tumour and host-mouse plasma samples, the levels of a standard-of-care-drug administered in nanoparticles in order to improve the delivery of chemotherapeutics to their site of action.