Dr Tomlinson

Profile

I studied for a PhD in developmental biology at the University of Edinburgh in the MRC Reproductive Biology Unit. After completing my PhD I moved to Leeds and worked as a PDRA in Prof Margaret Knowles's lab studying the function of growth factor receptors in bladder cancer.

In 2010 I set up two facilities in Leeds - a siRNA screening facility and a facility to produce non-antibody binding proteins. In 2015 I became a University Academic Fellow at Leeds to build a research group to fully exploit the use of Affimers for studying protein function. In 2018 I was promoted to Associate Professor and in 2023 became the Head of School for Molecular and Cellular Biology.

Responsibilities

• Head of School (Molecular & Cellular Biology)

Research interests

Other websites:

• astbury webpage
• laboratory website

The Adhiron^TM platform

Antibodies are the best-studied group of biological binding molecules to date. They are important in a wide variety of biological and medical applications, but numerous alternatives are being developed including protein, RNA and DNA aptamers. These alternatives can bind to target proteins and so have potential as molecular biology tools, therapeutic agents and as diagnostic tools for detection and imaging of proteins in patient samples. Our group was established to exploit a novel Adhiron library that we invented (PEDS 2014, eLife 2017). The Adhiron platform is based on a constant small 91 amino acid scaffold that constrains one or two randomised nine amino acid loop regions for molecular recognition. From 2016-2024 this was known as the type II Affimer^TM platform. The Adhiron scaffold protein is extremely stable with a Tm of 101^oC , and maintains the beta structure following loop insertion. We have developed a large naïve phage display library (>3x10¹⁰) of Adhiron reagents that is of very high quality (86 % full length clones). The loop regions in the library contain an even distribution of each of the 19 amino acids excluding cysteines. We use phage display as a flexible platform for isolation reagents (Science Signalling 2017). Our laboratory uses Adhiron reagents to understand protein function by blocking protein-protein interactions, identifying novel protein conformers to aid drug discovery and to help visualise proteins in cells.

We also work collaboratively with academics, clinicians and industrial partners on numerous projects including developing diagnostics, therapeutics and to aid drug discovery by identifying druggable domains.

Blocking protein-protein interactions

We have previously demonstrated that we can generate specific Affimer reagents to block human SUMO2 (hSUMO2), and for the first time we have developed reagents which differentiate between hSUMO1 and hSUMO2 isoforms (Science Signalling 2017). To confirm the ability to inhibit hSUMO2 binding we developed assays that test the Affimers ability to inhibit SUMO interactions. In vitro recombinant RNF4 ubiquitinates polymers of hSUMO2 (poly-hSUMO2_2-8). Affimers specific for GFP (irrelevant control) and for hSUMO1 were unable to inhibit RNF4s ability to ubiquitinate poly-hSUMO2_2-8, whereas Affimer reagents specific for hSUMO2 robustly inhibited this activity at less than 1 µM.

We have also used Adhiron reagents to study protein-protein interactions in the MAPK pathway. This pathway is dysregulated in many diseases including cancer, yet reagents that can inhibit specific protein interactions are lacking and therefore the basic understanding of what each of these domains is lacking. We have isolated Affimer reagents against numerous domains of proteins in this pathway, including Grb2, Sos and Ras. For Grb2 we have targeted the SH2 domain (eLife 2017) and the two SH3 domains (Biomolecules 2024). Interestingly we highlighted different roles for the two SH3 domains during signalling in cells (see figure 1)

Figure 1. A proposed mechanism for the interaction of Grb2 with SOS1. The SH2 domain allosterically blocks the CSH3 domain, so only the NSH3 domain can bind SOS1. (a) SOS1 proline-rich motif 3 (PRM 3) interacts with the Grb2 NSH3 domain. (b) Cytosolic Grb2-SOS1 complex translocates to the tyrosine-phosphorylated RTK where the SH2 domain binds. (c) Grb2 SH2 domain also interacts with phospholipids in the plasma membrane. (d) This releases the block on the CSH3 domain, (e) triggering cooperative interactions between the two SH3 domains. (f) This enables the CSH3 domain to interact with SOS1 proline-rich motif 4 (PRM 4), inducing activation of Ras. The image in the red box shows where Affimer N-D7 and C-C12 are predicted to bind and inhibit interactions with SOS1 PRM 3 and PRM 4, respectively. Note: SOS1 PRM 3’s sequence is EVPVPPPVPPRRRPESAPAESSPSKI, and SOS1 PRM 4’s sequence is LDSPPAIPPRQPTSK.

Enabling Drug Discovery

One of the surprising effects of using Adhiron reagents is their ability to inhibit protein function and identify novel protein conformers making them unique target validation tools. This is extremely important for identifying druggable pockets on protein surfaces with the added benefit of having a relative small binding interface that can be mimicked by small molecules. We now have numerous published examples of inhibiting protein function in combination with identifying novel protein conformers. The two examples below show co-crystal structure of Adhiron reagents bound to Ras GTPase (Figure 2, Nature Communications 2021) and PAK5 (Figure 3, Cell Reports 2023). For Ras we isolated numerous reagents that bound to two different pockets on Ras. Interestingly these are known druggable pockets on Ras and demonstrate the ability of the Adhirons to mimic the small molecule inhibitors. The Adhiron called K3 mimics the recently approved AMG510 anti-Ras small molecule but represents the first non-covalent inhibitor of this pocket (Figure 2B). The binding of the K3 Adhiron also opened the pocket creating improved druggable properties.

Figure 2. A. Affimer K3 (magenta) was co-crystallized with KRASGDP (cyan) and solved to a resolution of 2.1 Å. The switch I (deep blue), switch II (orange) and α3 helix (dark gray) are depicted, showing their relative positioning around variable region 1 of Affimer K3. B. Alterations in the conformation of the Switch II region (orange and α3 helix (black) (top row) and the corresponding alterations in the electrostatics (bottom row). Residues 41–45 of Affimer K3 (green) shown with KRASGDP (left-hand panels), overlaid with the co-crystallized KRAS:ARS1620 structure (middle panels, PBD: 5V9U) and KRAS:AMG510 structure (right-hand panels, PDB: 6OIM) (ARS1620 is shown in yellow and AMG510 is shown in magenta).

The Adhiron isolated against PAK5 bound to the P+1 pocket on the kinase and locked the protein in an intermediate activation state, blocking kinase activity (Figure 3). Despite extensive studies on kinases this represents a novel conformation, one that was previously uncharacterised. Again, like the other Adhirons the interface is relatively small, and therefore provides the potential for developing small molecules that block protein function based on the structure of the Adhiron.

Figure 3. PAK5-Af17 (green) binds at the hinge region between the N lobe (light gray) and C lobe (dark gray) of PAK5. The Affimer is shown to complex with PAK5 at the apex of the activation loop (purple), αC helix (blue), and glycine-rich loop (orange) via both variable regions (VRs) (PDB: 8C12). This site is distinct from the nucleotide binding site, as shown by the overlaid ADP nucleotide from PAK3 (yellow by element; PDB: 6FD3). The conserved salt bridge residues Glu-Lys are shown as sticks with the salt bridge as dotted black lines and distorted R-spine depicted via a yellow surface with the gatekeeper residue as a red surface, with kinase domain of PAK5, highlighting the accessible back-pocket.

This work demonstrates the ability to isolate reagents capable of being used as target validation tools, reagents to look proteins in novel conformations, and act as tools for drug discovery.

Qualifications

• PhD

Student education

See also:

• Faculty Graduate School
• FindaPhD Project details:
• Please contact me for information about PhD opportunites

Current postgraduate researchers

• Ajinkya Rao
• Sophie Saunders
• Dr Anna Tang

Projects

<li><a href="//phd.leeds.ac.uk/project/169-licamm-modulation-of-hypofibrinolysis-in-diabetes.">LICAMM Modulation of hypofibrinolysis in diabetes.</a></li>