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Cellular pharmacology

by florent_r0s1 - published on , updated on

Team leader : Jean-Louis Baneres

Within the IBMM, the role of the “Cellular Pharmacology” group consists in setting up original pharmacological tools to secure better knowledge of the molecular mechanisms regulating the physiological equilibria. The long-term objective being the exploitation of these tools in human therapy.
In close and constant collaboration with the chemical, analysis and modelling teams, the activity of the group covers several levels:

  • the implementation of reliable biological and pharmacological models, which are straightforward to use (functional cells isolated from animals’ tissues, transfected cellular lineages, membranar preparations,…)
  • the more fundamental study of the action mechanism of original compounds on clearly set targets (peptide hormone receptors, enzymes, …) and the usage of these “pharmacological tools” for better understanding of the physiological mechanisms controlled by these targets;
  • fine-tuning new pharmacological models in vitro, in order to extend pharmacological screening to new fields of physiology and pharmacology.

    In the past, the group has worked quite a lot on certain peptide hormones of the digestive system such as gastrin, cholecystokinin, bradykinin, bombesin, ghrelin, -MSH, etc…, and consequently has partaken, in close collaboration with the chemists, of the development of non peptide or pseudopeptide derivatives of these hormones, with an agonist or antagonist activity.
    Quite often, these hormones are pleiotropic agents exhibiting a wide spectrum of own biological activities (acid secretion, secretion of pancreatic enzymes, secretion of growth hormone, …) accompanied by neuromodulating activity at the CNS of a trophic effect on certain cellular types.
    Certain clinical data suggest that some of these peptide hormones are involved in the regulation of the cellular growth and hence in cancerogenesis phenomena; several teams of clinicians, toxicologists and galenists related to oncopharmacology are developing thematics in this direction within the IBMM.

Scientific interests


  • in vitro pharmacological studies:
    * drug design, evaluation of the activity of original ligands on various GPCRs (cck1, cck2 receptors, MC1R receptor, ghrelin receptor), Kd, ED50, IC50 determination.
    **molecular mechanisms controlling the function of GPCRs receptors: ligand/receptor interaction, ligand directed selectivity in GPCRs signalling, receptor interaction with intracellular partners, conformational diversity and functional selectivity, receptor dimerisation and conformational selectivity, functional analysis of wild type and mutant GPCRs,
  • photobiology, oncopharmacology , pharmacotoxicology

Technical skills

  • classical in vitro pharmacology: binding studies with radiolabelled ligands (saturation binding, competitive binding), radiolabeling of peptides with iodine 125, estimation of dose response curves, second messengers measurements, AMPc, IP3, Ca2+…, gene reporter strategy using SRE, CRE and fos luciferase assays,
  • molecular biology techniques: production of recombinant receptors, site directed mutagenesis of GPCRs,
  • physical and biophysical techniques: in vitro refolding of recombinant receptors (refolding in amphipols, refolding in lipid/detergent micelles, reconstitution in nanodiscs), radioactivity measurements, fluorescence measurements, circular dichroism, fluorescence energy transfer techniques (FRET, BRET), ACPtag and SNAPtag technologies.


CELL CYCLE BIOSENSORS & INHIBITORS

Morris M. DR2 CNRS
Pellerano M. IE CNRS

INTRODUCTION – Cyclin-dependent Kinases & Cancer
One of the major hallmarks of cancer concerns deregulation of the mechanisms underlying cell proliferation, associated with genetic amplification, overexpression or hyperactivation of cell cycle regulators. In particular Cyclin-dependent kinases (CDK/cyclins) participate in a wide variety of essential biological processes including transcription, neuronal differentiation and metabolism, aside from the role they were first identified for in cell cycle progression. These kinases are hyperactivated in most human cancers, thereby contributing to sustain uncontrolled proliferation, and constitute attractive pharmacological targets for anticancer therapeutics. However there are currently no means of reporting on the activity of these targets directly in tumours. Moreover the only inhibitors of these kinases which are currently in clinical trials target the ATP-binding pocket.

Figure 1

Specifically our research focuses on detection and targeting of CDK4/CyclinD in lung cancer, melanoma and lymphoma, and on CDK5/p25 in neuronal cancers.

FLUORESCENT BIOSENSORS for probing and imaging cyclin-dependent kinases
In order to propose sensitive and adapted strategies for probing CDK/Cyclins, we have developed an original technology based on a toolbox of peptide and protein-based fluorescent biosensors which offers a straightforward means of sensing alterations in kinase levels, activity or conformation in vitro, in living cells and in vivo, when introduced into cells in an appropriate fashion, thanks to synthetic probes whose spectral properties are sensitive to environmental changes.

Figure 2

These biosensors constitute sensitive tools for reporting on alterations associated with hyperactivation or inhibitrion of CDK/Cyclin kinases in real time in vitro, in living cells and in vivo. They offer a powerful technology for diagnostic purposes, to monitor response to therapeutics and to develop pharmacological screening strategies.

*Link to the Biosensor Workgroup / GDR2588
*Linl to Biotechnology Journal Special Issue – Feb 2014
*Link to PMBTS Volume « Fluorescent Biosensors » 2013

TARGETING Cyclin-dependent kinases
In order to propose novel inhibitors of cyclin-dependent kinases for anticancer therapeutics we are developing two approaches :
1-Rational design of peptide and peptidomimetic inhibitors targeting protein/protein interfaces which are essential for CDK/Cyclin kinase function
2-High throughput screening of chemical compound libraries for small molecules that target protein/protein interfaces or for allosteric inhibitors that perturb kinase function through conformational interference, thanks to specific fluorescent biosensors

Publications associated with projects
[1] Morris M.C. Fluorescent Biosensors of Intracellular Targets: from genetically-encoded reporters to modular polypeptide probes. Cell Biochem.Biophys. 56,19-37(2010)
[2] Kurzawa L, Pellerano M, Coppolani JB, Morris MC.Fluorescent peptide bioprobe for quantification of cyclin-dependent kinases in living cells, PloS ONE, 6(10):e26555.
[3] Van, T.N.N. & Morris, M.C. Fluorescent sensors of protein kinases: from basics to biomedical applications.Progr. Mol. Biol. Trans. Science, 113, 217-274 (2013)
[4] Morris MC. Fluorescent Biosensors - Probing Protein Kinase Function in Cancer and Drug Discovery. Biochim Biophys Acta 1834(7),1387-95 (2013)
[5] Prével, C., Pellerano, M., Van, T.N.N., Morris, M.C. Fluorescent Biosensors for High Throughput Screening of Protein Kinase Inhibitors. Biotechnology J. 9, 253-65 (2014)
[6] Van T.N.N., Pellerano, M., Lykaso S., Morris, M.C. Fluorescent protein biosensor for probing CDK/Cyclin activity in vitro and in living cells. ChembioChem (2014) 15 (15): 2298–2305.
[7] Prével C., Kurzawa L., Van T.N.N. & Morris MC (2014) Fluorescent biosensors for drug discovery- New tools for Old Targets – Screening for Inhibitors of Cyclin-dependent kinases. European Journal of Medicinal Chemistry. 10.1016/j.ejmech.2014.10.003
[8] Peyressatre M., Prével C., Pellerano M., Morris M.C. (2015) Targeting Cyclin-Dependent Kinases in Human Cancers : from small molecules to peptide inhibitors. Cancers 23;7(1):179-237.
[9] Morgan Pellerano, Delphine Naud-Martin, Marie-Paule Teulade-Fichou, Florence Mahuteau-Betzer & Morris M.C. (2016) TP2-Rho is a Sensitive Solvatochromic Red-shifted Probe for Monitoring the Interactions between CDK4 and Cyclin D ChemBioChem, special Issue Protein/Protein Interactions, in press
[10] Prével C., Pellerano M., Gonzalez Vera JA, Henri P., Meunier L., Vollaire J., Josserand V. & Morris M.C. (2016) Monitoring CDK4 activity in cell extracts, melanoma xenografts and skin biopsies with a peptide biosensor, Biosensors & Bioelectronics, 2016 Apr 22;85:371-380. doi: 10.1016/j.bios.2016.04.050. [Epub ahead of print]

FUNDING SOURCES
GSO Emergence Technologies pour la Santé (Juillet 2015-2016) “Detection et suivi du glioblastoma par quantification de l’activité de CDK5”.
INCA - Programme translationnel PRT-K 2014 (Juin 2015-2018) « FOLDLUNGK4 – Ciblage de la kinase CDK4/Cycline D par des foldamères et application thérapeutique au cancer du poumon »
INCA Projet Libre Recherches Biomédicales (Dec 2010-Dec 2014) : “ Développement de biosenseurs pour la mesure en imagerie optique non-invasive de l’activité kinase cycline-dépendante dans des modèles de tumeurs chez l’animal”.
ANR (Dec 2013-Dec 2017) « NANOMULTISENS : Nanosenseurs multifonctionnels pour la detection de protéines kinases intracellulaires constituant des biomarqueurs cancéreux»