Mass Spectrometry-guided structural analysis of protein kinase inhibitor complexes
Lead Research Organisation:
University of Liverpool
Department Name: Institute of Integrative Biology
Abstract
Background: Protein phosphorylation is catalyzed by protein kinases, which have become hugely important drug targets in 'personalized' human diseases such as cancer. A plethora of physical techniques are now available to measure ligand (drug)-binding propensity amongst kinases. Protein kinases and pseudokinases (catalytically deficient variants with a similar overall fold) represent important current drug targets in the clinic, with >30 kinase inhibitors approved for cancer and inflammatory conditions and hundreds more in development. However, kinase inhibitors often suffer from a lack of specificity, and unwanted side-effects arise due to off-target binding to unintended protein conformations presented to them in the cell. Unfortunately, we know little about structural factors that influence dynamic kinase interactions with drugs. In this studentship, we will exploit technological advances to examine and quantify how specific kinase conformations bind different classes of drugs.
The challenge: An inherent issue with studying kinases by X-ray crystallography is our inability to measure structural dynamics or kinase:ligand interactions that are intransigent to crystallisation. Our solution is to marry state-of-the-art structural Mass Spectrometry (MS) with X-ray crystallography, training a new generation of cross-disciplinary scientist. Structural MS is a rapidly developing field which takes advantage of the fact that macromolecular complexes, phosphorylation, ligand binding and conformational information are all preserved in the gas phase. MS-based affinity (quantitative Kd information) pertaining to conformation can inform complementary structural approaches (such as X-Ray crystallography), and reveal, or confirm, novel drug binding modes and allosteric networks in kinases. By marrying structural insights with bioinformatics (including publically available drug-binding data), we have created a strong case for funding for an MRC DiMeN DTP studentship.
This project is a collaboration between MRC, CR-UK and BBSRC-funded groups at the Universities of Liverpool and Leeds, and will employ quantitative physical approaches to study protein kinases in complex with small molecule ligands, focusing on approved or late-phase clinical drugs. By comparing ligand-bound and unbound populations of 'open' and 'closed' (lying between 'inactive' and 'active') conformational states that exist amongst highly dynamic cohorts of kinase signaling complexes, including poorly studied pseudokinases, our project represents a unique multidisciplinary training opportunity that addresses MRC strategic research and skill priorities. It represents a three-way collaboration between an experienced supervisory team of biochemical and biophysical scientists with very successful portfolios of student training.
Our three scientific goals are:
1) Evaluation of stability and dynamics of functional protein kinase and pseudokinase complexes using native Ion Mobility-Mass Spectrometry
2) Development of a conformation-based bioinformatic pipeline to report drug binding mode(s) using combined gas phase (MS) and X-ray crystallography approaches
3) Translation of findings into a quantitative analysis of drug-binding parameters for kinase complexes and clinical kinase inhibitors
Key outcomes for the student include:
1) Immersion in state-of-the art research studying protein kinases and their interaction with drugs
2) Member of a training network involving the Centre for Proteome Research (Liverpool) and the Astbury Centre for Structural Molecular Biology (Leeds)
3) A portfolio of training in MRC priority skill sets, including computational/bioinformatic analysis of quantitative structural data for kinase:inhibitor complexes
The challenge: An inherent issue with studying kinases by X-ray crystallography is our inability to measure structural dynamics or kinase:ligand interactions that are intransigent to crystallisation. Our solution is to marry state-of-the-art structural Mass Spectrometry (MS) with X-ray crystallography, training a new generation of cross-disciplinary scientist. Structural MS is a rapidly developing field which takes advantage of the fact that macromolecular complexes, phosphorylation, ligand binding and conformational information are all preserved in the gas phase. MS-based affinity (quantitative Kd information) pertaining to conformation can inform complementary structural approaches (such as X-Ray crystallography), and reveal, or confirm, novel drug binding modes and allosteric networks in kinases. By marrying structural insights with bioinformatics (including publically available drug-binding data), we have created a strong case for funding for an MRC DiMeN DTP studentship.
This project is a collaboration between MRC, CR-UK and BBSRC-funded groups at the Universities of Liverpool and Leeds, and will employ quantitative physical approaches to study protein kinases in complex with small molecule ligands, focusing on approved or late-phase clinical drugs. By comparing ligand-bound and unbound populations of 'open' and 'closed' (lying between 'inactive' and 'active') conformational states that exist amongst highly dynamic cohorts of kinase signaling complexes, including poorly studied pseudokinases, our project represents a unique multidisciplinary training opportunity that addresses MRC strategic research and skill priorities. It represents a three-way collaboration between an experienced supervisory team of biochemical and biophysical scientists with very successful portfolios of student training.
Our three scientific goals are:
1) Evaluation of stability and dynamics of functional protein kinase and pseudokinase complexes using native Ion Mobility-Mass Spectrometry
2) Development of a conformation-based bioinformatic pipeline to report drug binding mode(s) using combined gas phase (MS) and X-ray crystallography approaches
3) Translation of findings into a quantitative analysis of drug-binding parameters for kinase complexes and clinical kinase inhibitors
Key outcomes for the student include:
1) Immersion in state-of-the art research studying protein kinases and their interaction with drugs
2) Member of a training network involving the Centre for Proteome Research (Liverpool) and the Astbury Centre for Structural Molecular Biology (Leeds)
3) A portfolio of training in MRC priority skill sets, including computational/bioinformatic analysis of quantitative structural data for kinase:inhibitor complexes
Organisations
People |
ORCID iD |
Claire Eyers (Primary Supervisor) | |
Lauren Tomlinson (Student) |
Publications
Tomlinson LJ
(2020)
Ion Mobility-Mass Spectrometry to Evaluate the Effects of Protein Modification or Small Molecule Binding on Protein Dynamics.
in Methods in molecular biology (Clifton, N.J.)
Tomlinson LJ
(2020)
Determination of Phosphohistidine Stoichiometry in Histidine Kinases by Intact Mass Spectrometry.
in Methods in molecular biology (Clifton, N.J.)
Studentship Projects
Project Reference | Relationship | Related To | Start | End | Student Name |
---|---|---|---|---|---|
MR/N013840/1 | 01/10/2016 | 30/09/2025 | |||
1947325 | Studentship | MR/N013840/1 | 01/10/2017 | 30/09/2021 | Lauren Tomlinson |
Description | Spooky Science school event |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | School pupils attended the university to engage in activities associated with different departments within the institute of integrative biology. |
Year(s) Of Engagement Activity | 2018,2019 |