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Experimental Therapeutics Programme

Director: Joaquín Pastor
Secretary: Natalia Catalá

The current ETP-CNIO pipeline encompasses targeted and phenotypic projects. The following highlights summarise some of our achievement during 2016.

Our most advanced targeted project is dedicated to CDK8 inhibitors. We have selected our first lead, ETP-27, which has yielded positive results in PK-PD studies and has shown early signs of efficacy in a MOLM13 xenograft model after oral administration. Currently, we have embarked on the fine optimisation of the in vivo exposure within this series.

Additional targeted projects, focused on Mastl and Haspin inhibitors, are undertaken in collaboration with Marcos Malumbres (CNIO Cell Division and Cancer Group). The Mastl project started with a cell-based screening of several ETP-libraries, where a few 'high-micromolar' hits emerged as potential Mastl inhibitors. However, a direct target engagement experiment to unequivocally identify Mastl as their molecular target was not possible due to the unavailability of isolated active Mastl protein. ETP’s Biology Team has been able to set up highly efficient conditions for the purification, isolation and production of 'full length active Mastl kinase'. This important achievement has enabled, for the first time, the biochemical profiling of the identified hits and an additional targeted biochemical screening of ETP-libraries. As a result of these activities, we have identified several families of compounds as biochemical Mastl inhibitors, including ETP-750 with an IC50 of around 300 nM. These results shall pave the way for the discovery and development of advanced Mastl inhibitors in the near future. It is worth mentioning that the production of the protein was carried out by the CNIO Crystallography and Protein Engineering Unit.

Using the same cell-based assay, Malumbres' Group identified compounds that efficiently produce 'mitotic cell death', a new avenue for cancer therapy. The knowledge of the biochemical profile of these hits obtained by ETP has contributed to the identification of Haspin kinase as the target responsible for the observed phenotype. Currently, ETP is working on the discovery of Haspin inhibitors where we have already identified highly potent compounds in the low-nanomolar range.

We are also collaborating with P. Carmeliet (VIB-KU Leuven, Belgium) for the discovery of novel inhibitors of a particular enzyme in the field of vascular normalisation. We have carried out a screening campaign and we are now working on hit generation activities to identify catalytic inhibitors of this enzyme and their associated intellectual property.

Phenotypic screenings have proven to be advantageous for the discovery of innovative molecular targets and modulators, as well as to establish their link with disease. Nevertheless, the molecular target responsible for a desired phenotype needs to be identified and this deconvolution phase implies an extra step of complexity in the process. As mentioned above, we are working on several phenotypic projects:

ETP collaborates with Manuel Serrano (CNIO Tumour Suppression Group) in a project dedicated to the discovery of novel targets and modulators against Cancer Stem Cells (CSCs). We have been focusing on target deconvolution activities around previously identified hits that have the ability to selectively kill CSCs and to inhibit the tumour-initiating capacity of pancreatic CSCs. Our Medicinal Chemistry Team has designed and synthesised chemical probes around those molecules by using 'minimalist linkers'. These chemical fragments bear a photoactivatable group to achieve crosslinking of the modified hits with targeted proteins, and a special chemical group to perform 'click chemistry' that is useful to attach the 'crosslinked complex' to a reporter tag for imaging and/or pull-down experiments. The treatment of cells and cell lysates with these molecules has enabled the identification of a target candidate, which is currently under additional validation studies.

ETP is collaborating with Maria A. Blasco (CNIO Telomeres and Telomerase Group) in the discovery of TRF1 inhibitors, a project that also requires a 'target deconvolution' phase. During this year, we focused our attention on a series of TRF1 inhibitors with an unknown and perhaps innovative mechanism of TRF1 modulation. We have profiled the main hit, ETP-946, against large panels of enzymes and receptors representing more than 600 targets. Among them, we have retrieved 4 potential candidates, which are currently undergoing validation studies. The chemical modification of ETP-946 with 'minimalist linkers' has yielded potential affinity probes that are currently under evaluation. Last but not least, ETP has set up a 'thermal stability assay' to study the stabilisation of hTRF1 overexpressed in HEK293 cells in the presence of ETP-946 and analogues. These experiments will inform us about direct interactions of those compounds with TRF1.

Finally, I’d like to mention that other 'phenotypic exploratory projects' are also currently at the screening phase; these are undertaken in collaboration with the CNIO Researchers Manuel Valiente, Óscar Fernández-Capetillo and Massimo Squatrito.