Final Report Summary - JAGV2013MMG (Development of Fluorescent Polypeptide Biosensors for Detection of Protein Kinase Cancer Biomarkers and Drug Discovery Programs)
Cancer currently suffers from an overall lack of efficient targeted therapies and, perhaps more importantly, from a lack of early and personalized diagnostic tools. The development of personalized medicine and targeted therapies calls for sensitive and selective tools to detect and quantify specific cancer biomarkers in pathological versus healthy cells, for early-stage diagnostics, monitoring disease progression and response to therapies. Today, there is an urgent need for innovative sensing technologies, which would allow for direct, sensitive, non-invasive and cost-effective detection of cancer biomarkers.
Hyperactivation of cyclin-dependent kinases (CDK/Cyclins) contributes to cancer cell proliferation in several human cancers. As such, and given their central role in coordination of cell division, as well as their prognostic value as indicators of poor disease outcome, these enzymes constitute established cancer biomarkers and attractive pharmacological targets for the development of cancer therapeutics. Unfortunately, diagnostic approaches for detecting alterations in the activities of these kinases in a standardized fashion are poorly developed, and they involve indirect and invasive approaches. To circumvent this issue, Dr May Morris, has developed an original and innovative approach to probe the activity of cyclin-dependent kinases, based on fluorescent peptide biosensors (CDKACT technology). This technology has been successfully validated using recombinant CDK/Cyclins and cell extracts expressing these kinases, as well as to monitor CDK/Cyclin activity in living cells by fluorescence imaging. However this first generation of biosensors remains limited by technological bottlenecks, in particular with respect to sensitivity and signal-to-noise ratio. Optimization of these criteria is a sine qua none for the development of biomedical applications, as well as, for a more widespread application of biosensor technology to drug discovery programs.
On the other hand, today there is clearly an unmet need for new therapeutic strategies to inhibit CDK/Cyclin activity based on drugs that do not target the ATP-binding pocket, and instead bind to less conserved sites. In this respect, allosteric inhibitors that interfere with conformational transitions required for kinase activation would are more likely to be selective. With the aim of screening for allosteric CDK/Cyclin inhibitors Dr May Morris has developed the so-called CDKCONF technology, based on conformational biosensors, which enable selective identification of molecules that affect the conformational dynamics of CDKs. This technology was successfully applied to screen a library of small chemical compounds, leading to the identification of novel allosteric inhibitors of CDK2 and inhibit cancer cell proliferation. However, the applicability of this biosensor technology is limited, as it only exhibited rather subtle changes in fluorescence, which affects sensitivity, reliability and signal to noise ratio. Consequently, it would be of an unquestionable value for a more extensive use in drug discovery, to develop variants of these biosensors which could give a robust signal for easy assay readout to be used for identification of inhibitors of other kinases.
The first main objective of this research programme was to design and engineer sensitive and selective biosensors to probe and quantify the hyperactivity of cyclin-dependent kinases that constitute cancer biomarkers of particular relevance in lung cancer, melanoma and glioblastoma, respectively. The second main objective of this programme consisted in developing new, sensitive and selective conformational biosensors to screen for non-ATP-competitive inhibitors of CDK/Cyclins which may serve for cancer therapeutics.
Specifically, several peptide biosensors were developed to report on CDK4 activity and conjugated to different fluorescent probes to evaluate their response and sensitivity. A TAMRA-CDKACT4 biosensor was further implemented to probe CDK4 kinase in melanoma cell extracts, in melanoma mouse xenografts and in skin biopsies, thereby providing promising perspectives for cancer diagnostics (Prével et al. BSBE 2016). Furthermore, in an attempt to improve the robustness and sensitivity of biosensor response, a lanthanide biosensor was developed, that responds to CDK4 kinase activity upon sensitization of a DOTA[Tb3+] complex incorporated into a CDK4-specific substrate peptide, by a unique tryptophan residue in an adjacent phosphoaminoacid binding moiety.This lanthanide biosensor responds to CDK4 activity in melanoma cell extracts through a significant and dose-dependent increase in luminescence, thereby providing much greater sensitivity than the previously developed CDKACT4 biosensors (Gonzalez-Vera et al. ChemComm 2017).
A quinolimide-based solvatochromic fluorophore was also developed and conjugated to a CDK5 derived- peptide, which was demonstrated to constitute a sensitive probe for monitoring the interaction between CDK5 kinase and its regulatory subunit p25 both in vitro and in glioblastoma cells successfully probed the interaction with endogenous p25 (Gonzalez-Vera et al. ChemComm 2016).
A CDK5-specific conformational biosensor was engineered based on CDKCONF technology previously developed in the lab with the aim of identifying allosteric modulators of CDK5 conformational dynamics. This biosensor was optimized, then applied to screen three small molecule libraries. Hits were further characterized in kinase activity assays in order to identify their ability to inhibit CDK5/p25, then in glioblastoma proliferation assays, and finally characterized gain insight into their mechanism of action with respect to substrate (TAU) and p25 binding to CDK5.
This multidisciplinary project has allowed Dr Juan A. González Vera, to receive training in Chemical Biology, Organic Chemistry, Biochemistry, Biophysics, Cell Biology, and Fluorescent Imaging Technologies at the IBMM-UMR5247 in Montpellier, under supervision of Dr May Morris. The work performed has lead to several publications in peer-reviewed journals (Prével C. et al., Biosens. Bioelectron., 2016; González-Vera J. A. et al., Chem. Commun., 2016; González-Vera J. A. et al., Chem. Commun., 2017), one review (González-Vera J. A. et al., Proteomes, 2015) and another publication in preparation (González-Vera J. A. & Peyressatre M. et al.). These studies were also communicated in several national and international conferences in the form of posters or oral presentations.
Finally, the results of this research should have a a strong scientific, technological and economic impact, by contributing to develop smart and personalized diagnostics by improving strategies for early detection of enzymatic cancer biomarkers, and providing new tools for monitoring cancer progression, whilst also providing new lead compounds with selective inhibition profiles for cancer therapeutics.
Hyperactivation of cyclin-dependent kinases (CDK/Cyclins) contributes to cancer cell proliferation in several human cancers. As such, and given their central role in coordination of cell division, as well as their prognostic value as indicators of poor disease outcome, these enzymes constitute established cancer biomarkers and attractive pharmacological targets for the development of cancer therapeutics. Unfortunately, diagnostic approaches for detecting alterations in the activities of these kinases in a standardized fashion are poorly developed, and they involve indirect and invasive approaches. To circumvent this issue, Dr May Morris, has developed an original and innovative approach to probe the activity of cyclin-dependent kinases, based on fluorescent peptide biosensors (CDKACT technology). This technology has been successfully validated using recombinant CDK/Cyclins and cell extracts expressing these kinases, as well as to monitor CDK/Cyclin activity in living cells by fluorescence imaging. However this first generation of biosensors remains limited by technological bottlenecks, in particular with respect to sensitivity and signal-to-noise ratio. Optimization of these criteria is a sine qua none for the development of biomedical applications, as well as, for a more widespread application of biosensor technology to drug discovery programs.
On the other hand, today there is clearly an unmet need for new therapeutic strategies to inhibit CDK/Cyclin activity based on drugs that do not target the ATP-binding pocket, and instead bind to less conserved sites. In this respect, allosteric inhibitors that interfere with conformational transitions required for kinase activation would are more likely to be selective. With the aim of screening for allosteric CDK/Cyclin inhibitors Dr May Morris has developed the so-called CDKCONF technology, based on conformational biosensors, which enable selective identification of molecules that affect the conformational dynamics of CDKs. This technology was successfully applied to screen a library of small chemical compounds, leading to the identification of novel allosteric inhibitors of CDK2 and inhibit cancer cell proliferation. However, the applicability of this biosensor technology is limited, as it only exhibited rather subtle changes in fluorescence, which affects sensitivity, reliability and signal to noise ratio. Consequently, it would be of an unquestionable value for a more extensive use in drug discovery, to develop variants of these biosensors which could give a robust signal for easy assay readout to be used for identification of inhibitors of other kinases.
The first main objective of this research programme was to design and engineer sensitive and selective biosensors to probe and quantify the hyperactivity of cyclin-dependent kinases that constitute cancer biomarkers of particular relevance in lung cancer, melanoma and glioblastoma, respectively. The second main objective of this programme consisted in developing new, sensitive and selective conformational biosensors to screen for non-ATP-competitive inhibitors of CDK/Cyclins which may serve for cancer therapeutics.
Specifically, several peptide biosensors were developed to report on CDK4 activity and conjugated to different fluorescent probes to evaluate their response and sensitivity. A TAMRA-CDKACT4 biosensor was further implemented to probe CDK4 kinase in melanoma cell extracts, in melanoma mouse xenografts and in skin biopsies, thereby providing promising perspectives for cancer diagnostics (Prével et al. BSBE 2016). Furthermore, in an attempt to improve the robustness and sensitivity of biosensor response, a lanthanide biosensor was developed, that responds to CDK4 kinase activity upon sensitization of a DOTA[Tb3+] complex incorporated into a CDK4-specific substrate peptide, by a unique tryptophan residue in an adjacent phosphoaminoacid binding moiety.This lanthanide biosensor responds to CDK4 activity in melanoma cell extracts through a significant and dose-dependent increase in luminescence, thereby providing much greater sensitivity than the previously developed CDKACT4 biosensors (Gonzalez-Vera et al. ChemComm 2017).
A quinolimide-based solvatochromic fluorophore was also developed and conjugated to a CDK5 derived- peptide, which was demonstrated to constitute a sensitive probe for monitoring the interaction between CDK5 kinase and its regulatory subunit p25 both in vitro and in glioblastoma cells successfully probed the interaction with endogenous p25 (Gonzalez-Vera et al. ChemComm 2016).
A CDK5-specific conformational biosensor was engineered based on CDKCONF technology previously developed in the lab with the aim of identifying allosteric modulators of CDK5 conformational dynamics. This biosensor was optimized, then applied to screen three small molecule libraries. Hits were further characterized in kinase activity assays in order to identify their ability to inhibit CDK5/p25, then in glioblastoma proliferation assays, and finally characterized gain insight into their mechanism of action with respect to substrate (TAU) and p25 binding to CDK5.
This multidisciplinary project has allowed Dr Juan A. González Vera, to receive training in Chemical Biology, Organic Chemistry, Biochemistry, Biophysics, Cell Biology, and Fluorescent Imaging Technologies at the IBMM-UMR5247 in Montpellier, under supervision of Dr May Morris. The work performed has lead to several publications in peer-reviewed journals (Prével C. et al., Biosens. Bioelectron., 2016; González-Vera J. A. et al., Chem. Commun., 2016; González-Vera J. A. et al., Chem. Commun., 2017), one review (González-Vera J. A. et al., Proteomes, 2015) and another publication in preparation (González-Vera J. A. & Peyressatre M. et al.). These studies were also communicated in several national and international conferences in the form of posters or oral presentations.
Finally, the results of this research should have a a strong scientific, technological and economic impact, by contributing to develop smart and personalized diagnostics by improving strategies for early detection of enzymatic cancer biomarkers, and providing new tools for monitoring cancer progression, whilst also providing new lead compounds with selective inhibition profiles for cancer therapeutics.