Using structural and chemical biology to understand the roles and mechanisms of CDKs: generating hypotheses for drug discovery
Lead Research Organisation:
Newcastle University
Department Name: Translational and Clinical Res Institute
Abstract
The behaviour of a cell depends on the genes it expresses and on its commitment to either a dormant or a proliferating state. The cyclin-dependent kinases (CDKs) bind to members of the cyclin protein family to form complexes that regulate both the expression of genes and cell proliferation. Transcription describes the process by which a gene sequence is converted into mRNA. Transcriptional CDKs regulate this process, mostly by controlling the activity of an enzyme that synthesizes the mRNA. Transcriptional CDKs also regulate RNA processing events. CDKs that control the cell cycle are activated in response to growth promoting signals and control the timing of the duplication of the genome and its subsequent segregation to generate two identical copies when the cell divides. Just as CDK-cyclins are important in normal cells, so they can also contribute to the development of disease when they do not function properly. The first part of our research programme is to advance understanding of the structures and functions of CDK-containing complexes. We have selected CDK-cyclins to study based on their roles in the development of specific cancers. We aim to find proteins that these CDK-cyclins bind to, discover their 3D structures, and characterise how CDK activity is regulated within these structures. We can then study how these CDK complexes contribute to the development of disease when they do not function correctly. The techniques of X-ray crystallography and, in recent years, cryo-electron microscopy allow us to image protein complexes in atomic detail and we will use both methods. We use bacteria, insect or cultured mammalian cells to generate the proteins for study by crystallography or cryoEM. The proteins can also be used in functional assays to determine, for example, how tightly they bind to one another, and what effect mutations have on their properties.
Our second aim is to exploit insight into CDK-cyclin complexes to generate ideas for how they may better be targeted by inhibitors. Historically the development of CDK inhibitors has targeted the CDK ATP-binding site. These inhibitors outcompete ATP, a cofactor that CDKs normally use, and thereby block the CDK's catalytic activity. This approach cannot distinguish the different activities of their CDK target, which may depend on the complexes in which they are found. Consequently, ATP-competitive inhibitors can have unwanted effects that limit their use as drugs. The use of CDK inhibitors in cancer therapy has been pioneered by a first generation of mixed CDK4/6 inhibitors, but tumours are already developing resistance to these inhibitors. To improve the safety and increase the robustness of clinical response to CDK inhibitors, our programme will identify opportunities to inhibit CDKs that do not target the ATP-binding site; (i) by first identifying hotspots on the CDKs and cyclins through which they interact with other protein partners and then developing inhibitors that block those hotspots (so-called protein-protein interaction inhibitors or PPIs); and (ii) by exploiting our understanding of the structural changes that accompany CDK activation to design "allosteric inhibitors" that prevent CDK activation. We will use a set of small molecules called "FragLites" that are designed to find potential interaction hotspots on a protein through an X-ray crystallographic screen. We will also identify cyclic peptides that bind selectively and with high affinity to our CDK-cyclin targets. We will develop and characterise both our FragLite and cyclic peptides to identify more potent PPIs and allosteric inhibitors.
Overall, our programme will allow us to address a barrier between basic science and validated projects that drug discovery groups can adopt. The approaches will deliver both novel biological insight, and actionable approaches to novel ways of inhibiting CDKs for drug design.
Our second aim is to exploit insight into CDK-cyclin complexes to generate ideas for how they may better be targeted by inhibitors. Historically the development of CDK inhibitors has targeted the CDK ATP-binding site. These inhibitors outcompete ATP, a cofactor that CDKs normally use, and thereby block the CDK's catalytic activity. This approach cannot distinguish the different activities of their CDK target, which may depend on the complexes in which they are found. Consequently, ATP-competitive inhibitors can have unwanted effects that limit their use as drugs. The use of CDK inhibitors in cancer therapy has been pioneered by a first generation of mixed CDK4/6 inhibitors, but tumours are already developing resistance to these inhibitors. To improve the safety and increase the robustness of clinical response to CDK inhibitors, our programme will identify opportunities to inhibit CDKs that do not target the ATP-binding site; (i) by first identifying hotspots on the CDKs and cyclins through which they interact with other protein partners and then developing inhibitors that block those hotspots (so-called protein-protein interaction inhibitors or PPIs); and (ii) by exploiting our understanding of the structural changes that accompany CDK activation to design "allosteric inhibitors" that prevent CDK activation. We will use a set of small molecules called "FragLites" that are designed to find potential interaction hotspots on a protein through an X-ray crystallographic screen. We will also identify cyclic peptides that bind selectively and with high affinity to our CDK-cyclin targets. We will develop and characterise both our FragLite and cyclic peptides to identify more potent PPIs and allosteric inhibitors.
Overall, our programme will allow us to address a barrier between basic science and validated projects that drug discovery groups can adopt. The approaches will deliver both novel biological insight, and actionable approaches to novel ways of inhibiting CDKs for drug design.
Technical Summary
This programme will exploit our collaborations and strengths with CDK reagents, biochemical/biophysical assays, structural biology and small molecule and peptide chemistry to dissect the mechanisms by which CDKs regulate the cell cycle and transcription.
Our first aim is to advance understanding of the activity of CDK-containing complexes. To this end we will:
(i) Continue crystallographic and cryo-EM studies of CDK9-cyclin T-containing and p27KIP1-CDK2-cyclin A-containing complexes
(ii) Initiate crystallization trials of CDK11 and CDK11-cyclin L/D3 complexes.
(iii) Use structural and biophysical techniques to define the cyclin K-SETD1A interaction.
(iv) Carry out crystallographic fragment ("FragLite") screens against (i) CDK4/6-cyclin D1/3 complexes and (ii) CDK2-cyclin E. We will assess the functional significance of hotspots identified in this way by directed mutagenesis followed by cell free (SPR, ITC, HTRF) and cell-based assays for known protein partners. We will use affinity purification mass spectrometry to identify new interactors and initiate structural studies for tractable and biologically significant protein interactions identified.
Our second aim is to exploit structural insight into CDK-cyclin complexes to develop hypotheses for allosteric and PPI approaches to drug discovery. To this end we will:
(i) Exploit our FragLite map of cyclin T to generate molecular and chemical tools to dissect the impact of specific cyclin T interactions on P-TEFb-dependent transcriptional programs. We will compare the effects of this approach to that of conventional CDK9 inhibitors and use this workflow to guide studies to follow up our FragLite screen of cyclin K.
(ii) Carry out mRNA-display screens to identify potent CDK11 and cyclin L, CDK4, CDK6 and CDK-cyclin D3, and cyclin K-specific cyclic peptides. This work will enable functional studies to identify and characterise CDK and cyclin protein binding sites and roles.
Our first aim is to advance understanding of the activity of CDK-containing complexes. To this end we will:
(i) Continue crystallographic and cryo-EM studies of CDK9-cyclin T-containing and p27KIP1-CDK2-cyclin A-containing complexes
(ii) Initiate crystallization trials of CDK11 and CDK11-cyclin L/D3 complexes.
(iii) Use structural and biophysical techniques to define the cyclin K-SETD1A interaction.
(iv) Carry out crystallographic fragment ("FragLite") screens against (i) CDK4/6-cyclin D1/3 complexes and (ii) CDK2-cyclin E. We will assess the functional significance of hotspots identified in this way by directed mutagenesis followed by cell free (SPR, ITC, HTRF) and cell-based assays for known protein partners. We will use affinity purification mass spectrometry to identify new interactors and initiate structural studies for tractable and biologically significant protein interactions identified.
Our second aim is to exploit structural insight into CDK-cyclin complexes to develop hypotheses for allosteric and PPI approaches to drug discovery. To this end we will:
(i) Exploit our FragLite map of cyclin T to generate molecular and chemical tools to dissect the impact of specific cyclin T interactions on P-TEFb-dependent transcriptional programs. We will compare the effects of this approach to that of conventional CDK9 inhibitors and use this workflow to guide studies to follow up our FragLite screen of cyclin K.
(ii) Carry out mRNA-display screens to identify potent CDK11 and cyclin L, CDK4, CDK6 and CDK-cyclin D3, and cyclin K-specific cyclic peptides. This work will enable functional studies to identify and characterise CDK and cyclin protein binding sites and roles.
Organisations
- Newcastle University (Lead Research Organisation)
- UNIVERSITY OF EDINBURGH (Collaboration)
- University of Sussex (Collaboration)
- Leiden University Medical Center (Collaboration)
- DURHAM UNIVERSITY (Collaboration)
- Yale University (Collaboration)
- UNIVERSITY OF YORK (Collaboration)
- UNIVERSITY OF LEEDS (Collaboration)
- DIAMOND LIGHT SOURCE (Collaboration)
- UNIVERSITY OF CAMBRIDGE (Collaboration)
Publications
Hope I
(2023)
Emerging approaches to CDK inhibitor development, a structural perspective.
in RSC chemical biology
Martin M
(2023)
Modern Methods of Drug Design and Development
Martin MP
(2022)
Exiting the tunnel of uncertainty: crystal soak to validated hit.
in Acta crystallographica. Section D, Structural biology
Rowland RJ
(2023)
Cryo-EM structure of SKP1-SKP2-CKS1 in complex with CDK2-cyclin A-p27KIP1.
in Scientific reports
Description | EPSRC Centre for Doctoral Training in Molecular Sciences for Medicine |
Amount | £7,291,056 (GBP) |
Funding ID | EP/S022791/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 04/2019 |
End | 10/2027 |
Description | ICF: Novel small molecule inhibitors for the treatment of CCNE1-amplified cancers |
Amount | £1,408,791 (GBP) |
Funding ID | MR/W029499/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 07/2022 |
End | 07/2024 |
Description | CDK PTMs |
Organisation | Leiden University Medical Center |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | Generation of CDK1- and CDK2-cyclin complexes. |
Collaborator Contribution | Identification of novel post-translational modifications on CDKs. Characterisation by mass spectrometry |
Impact | Exchange of reagents. |
Start Year | 2015 |
Description | Characterisation of CDK-containing complexes by H/D exchange mass spectrometry |
Organisation | University of Leeds |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Provision of CDK-containing complexes for analysis by H/D exchange mass spectrometry. Accompanying biophysical characterisation of the complexes and cell-based mechanistic studies. |
Collaborator Contribution | Access to mass spectrometers and expertise in data collection and analysis. |
Impact | Mass spectrometry, biophysical methods, X-ray crystallography, cell based mechanistic studies Joint publication: 10.1016/j.jmb.2020.166795 Preliminary data contributed to a successful application to Instruct for project access and to a subsequent successful grant proposal |
Start Year | 2018 |
Description | Characterisation of cyclin D3-containing complexes |
Organisation | Durham University |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Preparation of CRABP2 (authentic and mutant sequences), and CDK4- and CDK6-cyclin D1/D3 containing complexes for biophysical characterisation of the interaction between cyclin D3 and CRABP2. Structure determination of CRABP2 mutant proteins. Accompanying cell-based studies to characterise the CRABP2-cyclin D3 interaction. |
Collaborator Contribution | Provision of constructs to express CRABP2. Synthesis and characterisation of retinoic acid and derivatives. Determination of CRABP2 mutant structures by X-ray crystallography. |
Impact | Synthetic chemistry, biophysics, X-ray crystallography, cell-based studies |
Start Year | 2016 |
Description | Characterisation of the interaction between CDK1 and RGC32 |
Organisation | University of Sussex |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Provision of CDK1-containing complexes, expertise in biophysical assays and associated reagents |
Collaborator Contribution | Knowledge of RGC32 biology. Provision of RGC32 for assay. |
Impact | No outputs. Research is multidisciplinary: cellular studies carried out at Sussex, biophysical and biochemical analyses at Newcastle. Preliminary results contributed to a subsequent successful grant application. |
Start Year | 2016 |
Description | Functional characterisation of nanobodies targeting cell cycle regulators |
Organisation | University of Cambridge |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Determination of a p16INK4a-nanobody crystal structure. Biophysical characterisation of nanobody binding to CDK4/6-p16INK4a (authentic and mutant) containing complexes. Cellular studies to assess nanobody impact on p16INK4a function. |
Collaborator Contribution | Generation of the anti-p16INK4a nanobodies and subsequent biophysical characterisation. Provision of vectors for cellular expression of nanobodies and p16INK4a. |
Impact | X-ray crystallography, biophysical methods (ITC, HTRF, SPR, DSC), cellular studies (including generation of gene knockout cell lines) Preliminary data contributed to a successful subsequent grant application. |
Start Year | 2017 |
Description | Identification of CDK1 substrates |
Organisation | University of Edinburgh |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | CDK1 kinase assays |
Collaborator Contribution | Identification of CDK1 substrates |
Impact | Multidisciplinary: Biochemical and biophysical assays of CDK1 activity and preparation of CDK1-containing protein complexes at Newcastle University; mass spectrometry/proteomics at University of Edinburgh, in 2020 moved to the University of Dundee Joint publication (2023) DOI: 10.1016/j.celrep.2023.112139 |
Start Year | 2017 |
Description | Structural characterisation of CDK-cyclin complexes |
Organisation | Yale University |
Country | United States |
Sector | Academic/University |
PI Contribution | Systems for recombinant expression of CDK and cyclin-containing complexes for biophysical, biochemical and structural studies |
Collaborator Contribution | System-specific expertise to guide protein target selection and construct design |
Impact | X-ray crystallography, biophysical methods, drug design, cell-based studies. Preliminary results have contributed to a subsequent successful grant application. |
Start Year | 2019 |
Description | Structure determination of CDK-containing complexes by cryo electron microscopy |
Organisation | University of York |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Preparation of protein complexes for cryo-electron microscopy |
Collaborator Contribution | Access to electron microscopes and provision of expertise in data collection and structure determination |
Impact | Joint publication: DOI: 10.1038/s41598-023-37609-9 Multidisciplinary, cryo-electron microscopy, protein biophysics and characterisation |
Start Year | 2021 |
Description | Structure determination of CDK-containing complexes by cryo-electron microscopy |
Organisation | University of Leeds |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Preparation of protein complex samples for cryo-electron microscopy. |
Collaborator Contribution | Access to electron microscopes, provision of expertise in data collection and structure determination |
Impact | Preliminary results were included in a successful grant application. 2022 Joint publication: https://doi.org/10.1038/s41467-022-28281-0 2023 Joint publication: https://doi.org/10.1038/s41598-023-37609-9 |
Start Year | 2019 |
Description | X-ray crystallographic fragment screening |
Organisation | Diamond Light Source |
Country | United Kingdom |
Sector | Private |
PI Contribution | Provision of protein crystals for fragment library screening; fragment library design and synthesis; downstream medicinal chemistry |
Collaborator Contribution | Provision of fragment libraries, X-ray data collection and downstream data processing and interrogation |
Impact | PMID: 37858530 Multi-disciplinary: protein crystallisation, structure determination, biophysical assays, medicinal chemistry |
Start Year | 2021 |
Description | Hosting work experience students |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Opportunity for sixth form students to gain hands-on experience in a research laboratory. Students rotate around the lab to learn different techniques. |
Year(s) Of Engagement Activity | 2022,2023 |