Imperial College London

Prof Ed Tate

Faculty of Natural SciencesDepartment of Chemistry

Professor of Chemical Biology
 
 
 
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Contact

 

+44 (0)20 7594 3752e.tate Website CV

 
 
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Assistant

 

Ms Agnes Lee +44 (0)20 7594 9852

 
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Location

 

301BMolecular Sciences Research HubWhite City Campus

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Summary

 

Publications

Publication Type
Year
to

223 results found

Benns HJ, Storch M, Falco JA, Fisher FR, Tamaki F, Alves E, Wincott CJ, Milne R, Wiedemar N, Craven G, Baragaña B, Wyllie S, Baum J, Baldwin GS, Weerapana E, Tate EW, Child MAet al., 2022, CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery., Nat Microbiol

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

Journal article

Zhang L, Lovell S, De Vita E, Jagtap P, Lucy D, Goya Grocin A, Kjaer S, Borg A, Henning J, Miller A, Tate Eet al., 2022, A KLK6 activity-based probe reveals a role for KLK6 activity in pancreatic cancer cell invasion, Journal of the American Chemical Society

Journal article

Priyamvada L, Kallemeijn WW, Faronato M, Wilkins K, Goldsmith CS, Cotter CA, Ojeda S, Solari R, Moss B, Tate EW, Satheshkumar PSet al., 2022, Inhibition of vaccinia virus L1 N-myristoylation by the host N-myristoyltransferase inhibitor IMP-1088 generates non-infectious virions defective in cell entry, PLoS Pathogens, Vol: 18, ISSN: 1553-7366

We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational N-myristoylation of viral proteins by human host cell N-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we examine the importance of N-myristoylation during vaccinia virus (VACV) infection in primate cells and demonstrate the anti-poxviral effects of IMP-1088. N-myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be N-myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of N-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the N-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the N-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of N-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as broad-spectrum poxvirus inhibitors.

Journal article

Couves EC, Gardner S, Voisin TB, Bickel JK, Stansfeld PJ, Tate EW, Bubeck Det al., 2022, Structural basis for membrane attack complex inhibition by CD59

<jats:title>Abstract</jats:title><jats:p>CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryoelectron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming ◻-hairpins of C8 to form an intermolecular ◻-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 ◻-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.</jats:p>

Journal article

Ledger EVK, Lau K, Tate EW, Edwards AMet al., 2022, XerC is required for the repair of antibiotic- and immune-mediated DNA damage in <i>Staphylococcus aureus</i>

<jats:title>Abstract</jats:title><jats:p>To survive in the host environment, pathogenic bacteria need to be able repair DNA damage caused by both antibiotics and the immune system. The SOS response is a key bacterial pathway to repair DNA double strand breaks and may therefore be a good target for novel therapeutics to sensitise bacteria to antibiotics and the immune response. However, the genes required for the SOS response in <jats:italic>Staphylococcus aureus</jats:italic> have not been fully established. Therefore, we carried out a screen of mutants involved in various DNA repair pathways to understand which were required for induction of the SOS response. This led to the identification of 16 genes that may play a role in SOS response induction, and of these, 3 that affected susceptibility of <jats:italic>S. aureus</jats:italic> to ciprofloxacin. Further characterisation revealed that, in addition to ciprofloxacin, loss of the tyrosine recombinase XerC increased the susceptibility of <jats:italic>S. aureus</jats:italic> to various classes of antibiotics, as well as to host immune defences. Therefore, the inhibition of XerC may be a viable therapeutic approach to sensitise <jats:italic>S. aureus</jats:italic> to both antibiotics and the immune response.</jats:p>

Journal article

Zhang Q, Kounde C, Mondal M, Zhang L, Conole D, De Vita E, Fuchter M, Tate Eet al., 2022, Light-mediated multi-target protein degradation using arylazopyrazole photoswitchable PROTACs (AP-PROTACs), Chemical Communications, Vol: 58, Pages: 10933-10936, ISSN: 1359-7345

Light-activable spatiotemporal control of PROTAC-induced protein degradation was achieved with novel arylazopyrazole photoswitchable PROTACs (AP-PROTACs). The use of a promiscuous kinase inhibitor in the design enables this unique photoswitchable PROTAC to selectively degrade four protein kinases together with on/off optical control using different wavelengths of light.

Journal article

Jackson TR, Vuorinen A, Josa-Culleré L, Madden KS, Conole D, Cogswell TJ, Wilkinson IVL, Kettyle LM, Zhang D, O'Mahony A, Gracias D, McCall L, Westwood R, Terstappen GC, Davies SG, Tate EW, Wynne GM, Vyas P, Russell AJ, Milne TAet al., 2022, A tubulin binding molecule drives differentiation of acute myeloid leukemia cells., iScience, Vol: 25

Despite much progress in developing better drugs, many patients with acute myeloid leukemia (AML) still die within a year of diagnosis. This is partly because it is difficult to identify therapeutic targets that are effective across multiple AML subtypes. One common factor across AML subtypes is the presence of a block in differentiation. Overcoming this block should allow for the identification of therapies that are not dependent on a specific mutation for their efficacy. Here, we used a phenotypic screen to identify compounds that stimulate differentiation in genetically diverse AML cell lines. Lead compounds were shown to decrease tumor burden and to increase survival in vivo. Using multiple complementary target deconvolution approaches, these compounds were revealed to be anti-mitotic tubulin disruptors that cause differentiation by inducing a G2-M mitotic arrest. Together, these results reveal a function for tubulin disruptors in causing differentiation of AML cells.

Journal article

Priyamvada L, Kallemeijn WW, Faronato M, Wilkins K, Goldsmith CS, Cotter CA, Ojeda S, Solari R, Moss B, Tate EW, Satheshkumar PSet al., 2022, Inhibition of vaccinia virus L1 <i>N</i>-myristoylation by the host <i>N</i>-myristoyltransferase inhibitor IMP-1088 generates non-infectious virions defective in cell entry

<jats:title>ABSTRACT</jats:title><jats:p>We have recently shown that the replication of rhinovirus, poliovirus and foot-and-mouth disease virus requires the co-translational <jats:italic>N-</jats:italic>myristoylation of viral proteins by human host cell <jats:italic>N</jats:italic>-myristoyltransferases (NMTs), and is inhibited by treatment with IMP-1088, an ultrapotent small molecule NMT inhibitor. Here, we reveal the role of <jats:italic>N</jats:italic>-myristoylation during vaccinia virus (VACV) infection in human host cells and demonstrate the anti-poxviral effects of IMP-1088. <jats:italic>N-</jats:italic>myristoylated proteins from VACV and the host were metabolically labelled with myristic acid alkyne during infection using quantitative chemical proteomics. We identified VACV proteins A16, G9 and L1 to be <jats:italic>N-</jats:italic>myristoylated. Treatment with NMT inhibitor IMP-1088 potently abrogated VACV infection, while VACV gene expression, DNA replication, morphogenesis and EV formation remained unaffected. Importantly, we observed that loss of <jats:italic>N</jats:italic>-myristoylation resulted in greatly reduced infectivity of assembled mature virus particles, characterized by significantly reduced host cell entry and a decline in membrane fusion activity of progeny virus. While the <jats:italic>N</jats:italic>-myristoylation of VACV entry proteins L1, A16 and G9 was inhibited by IMP-1088, mutational and genetic studies demonstrated that the <jats:italic>N</jats:italic>-myristoylation of L1 was the most critical for VACV entry. Given the significant genetic identity between VACV, monkeypox virus and variola virus L1 homologs, our data provides a basis for further investigating the role of <jats:italic>N</jats:italic>-myristoylation in poxviral infections as well as the potential of selective NMT inhibitors like IMP-1088 as

Journal article

Williams D, Mahmoud M, Liu R, Andueza A, Kumar S, Kang D-W, Zhang J, Tamargo I, Villa-Roel N, Baek K-I, Lee H, An Y, Zhang L, Tate EW, Bagchi P, Pohl J, Mosnier LO, Diamandis EP, Mihara K, Hollenberg MD, Dai Z, Jo Het al., 2022, Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis., eLife, Vol: 11, Pages: 1-23, ISSN: 2050-084X

Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (Klk10, a secreted serine protease) as a flow-sensitive gene in mouse arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo& mouse models and in vitro studies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated Klk10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced permeability dysfunction and inflammation in human artery ECs (HAECs), as determined by NFkB activation, expression of vascular cell adhesion molecule 1 (VCAM1) and intracellular adhesion molecule 1 (ICAM1), and monocyte adhesion. Further, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

Journal article

Mondal M, Conole D, Nautiyal J, Tate Eet al., 2022, UCHL1 as a novel target in breast cancer: emerging insights from cell and chemical biology, British Journal of Cancer, Vol: 126, Pages: 24-33, ISSN: 0007-0920

Breast cancer has the highest incidence and death rate among cancers in women worldwide. In particular, metastatic Estrogen Receptor negative (ER–) breast cancer and Triple-Negative Breast Cancer (TNBC) subtypes have very limited treatment options, with low survival rates. Ubiquitin carboxyl terminal hydrolase L1 (UCHL1), a ubiquitin C-terminal hydrolase belonging to the deubiquitinase (DUB) family of enzymes, is highly expressed in these cancer types, and several key reports have revealed emerging and important roles for UCHL1 in breast cancer. However, selective and potent small molecule UCHL1 inhibitors have been disclosed only very recently, alongside chemical biology approaches to detect regulated UHCL1 activity in cancer cells. These tools will enable novel insights into oncogenic mechanisms driven by UCHL1, and identification of substrate proteins deubiquitinated by UCHL1, with the ultimate goal of realizing the potential of UCHL1 as a drug target in breast cancer.

Journal article

Andrei SA, Tate EW, Lanyon-Hogg T, 2022, Evaluating Hedgehog Acyltransferase Activity and Inhibition Using the Acylation-coupled Lipophilic Induction of Polarization (Acyl-cLIP) Assay., Methods Mol Biol, Vol: 2374, Pages: 13-26

Palmitoylation of the Hedgehog family of proteins is a critical step in the Hedgehog signaling pathway and is performed by the membrane-bound O-acyltransferase enzyme Hedgehog acyltransferase (HHAT). Measurement of HHAT activity has traditionally relied on radiolabeled fatty acid substrates, which imposes considerable constraints on throughput, cost, and safety, consequently hindering the efficient identification and development of small-molecule HHAT inhibitors. The Acylation-coupled Lipophilic Induction of Polarisation (Acyl-cLIP) assay was recently developed in our lab as a novel platform to evaluate lipidation of peptides in real time and high throughput. In this chapter, we describe the isolation of active HHAT from HEK293a cells and application of the Acyl-cLIP assay to characterize HHAT inhibitors. Our methodology uses standard chemical biology lab equipment and yields high-quality kinetic data from minimal sample volumes. The assay uses standard 384-well plates and is easily adapted to medium- or high-throughput screening formats.

Journal article

Coupland CE, Andrei SA, Ansell TB, Carrique L, Kumar P, Sefer L, Schwab RA, Byrne EFX, Pardon E, Steyaert J, Magee A, Lanyon-Hogg T, Sansom MSP, Tate EW, Siebold Cet al., 2021, Structure, mechanism, and inhibition of Hedgehog acyltransferase, Molecular Cell, Vol: 81, Pages: 5025-+, ISSN: 1097-2765

The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.

Journal article

Jamshidiha M, Lanyon-Hogg T, Sutherell C, Craven G, Tersa M, De Vita E, Brustur D, Perez-Doraldo I, Hassan S, Petracca R, Morgan R, Sanz-Hernández M, Norman J, Armstrong A, Mann D, Cota E, Tate Eet al., 2021, Identification of the first structurally validated covalent ligands of the small GTPase RAB27A, RSC Medicinal Chemistry, Vol: 13, Pages: 150-155, ISSN: 2632-8682

Rab27A is a small GTPase, which mediates transport and docking of secretory vesicles at the plasma membrane via protein–protein interactions (PPIs) with effector proteins. Rab27A promotes the growth and invasion of multiple cancer types such as breast, lung and pancreatic, by enhancing secretion of chemokines, metalloproteases and exosomes. The significant role of Rab27A in multiple cancer types and the minor role in adults suggest that Rab27A may be a suitable target to disrupt cancer metastasis. Similar to many GTPases, the flat topology of the Rab27A-effector PPI interface and the high affinity for GTP make it a challenging target for inhibition by small molecules. Reported co-crystal structures show that several effectors of Rab27A interact with the Rab27A SF4 pocket (‘WF-binding pocket’) via a conserved tryptophan–phenylalanine (WF) dipeptide motif. To obtain structural insight into the ligandability of this pocket, a novel construct was designed fusing Rab27A to part of an effector protein (fRab27A), allowing crystallisation of Rab27A in high throughput. The paradigm of KRas covalent inhibitor development highlights the challenge presented by GTPase proteins as targets. However, taking advantage of two cysteine residues, C123 and C188, that flank the WF pocket and are unique to Rab27A and Rab27B among the >60 Rab family proteins, we used the quantitative Irreversible Tethering (qIT) assay to identify the first covalent ligands for native Rab27A. The binding modes of two hits were elucidated by co-crystallisation with fRab27A, exemplifying a platform for identifying suitable lead fragments for future development of competitive inhibitors of the Rab27A-effector interaction interface, corroborating the use of covalent libraries to tackle challenging targets.

Journal article

Fedoryshchak RO, Gorelik A, Shen M, Shchepinova MM, Pérez-Dorado I, Tate EWet al., 2021, Discovery of lipid-mediated protein-protein interactions in living cells using metabolic labeling with photoactivatable clickable probes

<jats:title>Abstract</jats:title><jats:p>Protein-protein interactions (PPIs) are essential and pervasive regulatory elements in cell biology. Despite development of a range of techniques to probe PPIs in living systems, there is a dearth of approaches to capture interactions driven by specific post-translational modifications (PTMs). Myristoylation is a lipid PTM added to more than 200 human proteins, where it may regulate membrane localization, stability or activity. Here we report design and synthesis of a panel of novel photocrosslinkable and clickable myristic acid analog probes, and their characterization as efficient substrates for human <jats:italic>N</jats:italic>-myristoyltransferases NMT1 and NMT2, both biochemically and through X-ray co-crystallography. We demonstrate metabolic incorporation of probes to label NMT substrates in cell culture and <jats:italic>in situ</jats:italic> intracellular photoactivation to form a covalent crosslink between modified proteins and their interactors, capturing a snapshot of interactions driven by the presence of the lipid PTM. Proteomic analyses revealed both known and multiple novel interactors of a series of myristoylated proteins, including ferroptosis suppressor protein FSP1 and spliceosome-associated RNA helicase DDX46. The concept exemplified by these probes offers an efficient approach for exploring the PTM-specific interactome, which may prove broadly applicable to other PTMs.</jats:p>

Journal article

Coupland CE, Andrei SA, Ansell TB, Carrique L, Kumar P, Sefer L, Schwab RA, Byrne EFX, Pardon E, Steyaert J, Magee AI, Lanyon-Hogg T, Sansom MSP, Tate EW, Siebold Cet al., 2021, Structure and Mechanism of Hedgehog Acyl Transferase, Molecular Cell, ISSN: 1097-2765

<jats:title>SUMMARY</jats:title><jats:p>The iconic Sonic Hedgehog (SHH) morphogen pathway is a fundamental orchestrator of embryonic development and stem cell maintenance, and is implicated in cancers in various organs. A key step in signalling is transfer of a palmitate group to the N-terminal cysteine residue of SHH, catalysed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT) resident in the endoplasmic reticulum (ER). Here, we present the high-resolution cryo-EM structure of HHAT bound to substrate analogue palmityl-coenzyme A and a SHH mimetic megabody. Surprisingly, we identified a heme group bound to an HHAT cysteine residue and show that this modification is essential for HHAT structure and function. A structure of HHAT bound to potent small molecule inhibitor IMP-1575 revealed conformational changes in the active site which occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the novel mechanism by which HHAT adapts the membrane environment to transfer a long chain fatty acid across the ER membrane from cytosolic acyl-CoA to a luminal protein substrate. This structure of a member of the protein-substrate membrane-bound O-acyltransferase (MBOAT) superfamily provides a blueprint for other protein substrate MBOATs, such as WNT morphogen acyltransferase Porcupine and ghrelin <jats:italic>O</jats:italic>-acyltransferase GOAT, and a template for future drug discovery.</jats:p>

Journal article

Manchanda Y, Ramchunder Z, Shchepinova MM, Rutter GA, Inoue A, Tate EW, Jones B, Tomas Aet al., 2021, Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

<jats:title>Abstract</jats:title><jats:p>Mini-G proteins are engineered thermostable variants of Gα subunits designed to specifically stabilise G protein-coupled receptors (GPCRs) in their active conformation for structural analyses. Due to their smaller size and ease of use, they have become popular tools in recent years to assess specific GPCR behaviours in cells, both as reporters of receptor coupling to each G protein subtype and for in-cell assays designed to quantify compartmentalised receptor signalling from a range of subcellular locations. Here, we describe a previously unappreciated consequence of the co-expression of mini-G proteins with their cognate GPCRs, namely a profound disruption in GPCR trafficking and intracellular signalling caused by the co-expression of the specific mini-G subtype coupled to the affected receptor. We studied the Gαs-coupled pancreatic beta cell class B GPCR glucagon-like peptide-1 receptor (GLP-1R) as a model to describe in detail the molecular consequences derived from this effect, including a complete halt in β-arrestin-2 recruitment and receptor internalisation, despite near-normal levels of receptor GRK2 recruitment and lipid nanodomain segregation, as well as the disruption of endosomal GLP-1R signalling by mini-G<jats:sub>s</jats:sub> co-expression. We also extend our analysis to a range of other prototypical GPCRs covering the spectrum of Gα subtype coupling preferences, to unveil a widely conserved phenomenon of GPCR internalisation blockage by specific mini-G proteins coupled to a particular receptor. Our results have important implications for the design of methods to assess intracellular GPCR signalling. We also present an alternative adapted bystander intracellular signalling assay for the GLP-1R in which we substitute the mini-G<jats:sub>s</jats:sub> by a nanobody, Nb37, with specificity for active Gαs:GPCR complexes and no deleterious effect o

Journal article

Kallemeijn W, Lanyon-Hogg T, Panyain N, Goya Grocin A, Ciepla P, morales-sanfrutos J, Tate Eet al., 2021, Proteome-wide analysis of protein lipidation using chemical probes: in-gel fluorescence visualisation, identification and quantification of N-myristoylation, N- and S-acylation, Ocholesterylation, S-farnesylation and S-geranylgeranylation, Nature Protocols, ISSN: 1750-2799

Journal article

Schlott AC, Knuepfer E, Green JL, Hobson P, Borg AJ, Morales-Sanfrutos J, Perrin AJ, Maclachlan C, Collinson LM, Snijders AP, Tate EW, Holder AAet al., 2021, Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion, PLoS Biology, Vol: 19, ISSN: 1544-9173

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of "pseudoschizonts," which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

Journal article

Panyain N, Godinat A, Thawani AR, Lachiondo-Ortega S, Mason K, Elkhalifa S, Smith LM, Harrigan JA, Tate EWet al., 2021, Activity-based protein profiling reveals deubiquitinase and aldehyde dehydrogenase targets of a cyanopyrrolidine probe, RSC Medicinal Chemistry, Vol: 12, Pages: 1935-1943, ISSN: 2632-8682

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme (DUB), is a potential drug target in various cancers, and liver and lung fibrosis. However, bona fide functions and substrates of UCHL1 remain poorly understood. Herein, we report the characterization of UCHL1 covalent inhibitor MT16-001 based on a thiazole cyanopyrrolidine scaffold. In combination with chemical proteomics, a closely related activity-based probe (MT16-205) was used to generate a comprehensive quantitative profile for on- and off-targets at endogenous cellular abundance. Both compounds are selective for UCHL1 over other DUBs in intact cells but also engage a range of other targets with good selectivity over the wider proteome, including aldehyde dehydrogenases, redox-sensitive Parkinson’s disease related protein PARK7, and glutamine amidotransferase. Taken together, these results underline the importance of robust profiling of activity-based probes as chemical tools and highlight the cyanopyrrolidine warhead as a versatile platform for liganding diverse classes of protein with reactive cysteine residues which can be used for further inhibitor screening, and as a starting point for inhibitor development.

Journal article

De Vita E, Lucy D, Tate EW, 2021, Beyond targeted protein degradation: LD.ATTECs clear cellular lipid droplets, CELL RESEARCH, Vol: 31, Pages: 945-946, ISSN: 1001-0602

Journal article

Goya Grocin A, Kallemeijn W, Tate E, 2021, Targeting methionine aminopeptidase 2 in cancer, obesity and autoimmunity, Trends in Pharmacological Sciences, ISSN: 0165-6147

Journal article

Kennedy C, Goya Grocin A, Kovacic T, Singh R, Tate E, Ward J, Shenoy Aet al., 2021, A probe for NLRP3 inflammasome inhibitor MCC950 identifies carbonic anhydrase 2 as a novel target, ACS Chemical Biology, Vol: 16, Pages: 982-990, ISSN: 1554-8929

Inhibition of inflammasome and pyroptotic pathways are promising strategies for clinical treatment of autoimmune and inflammatory disorders. MCC950, a potent inhibitor of the NLR-family inflammasome pyrin domain-containing 3 (NLRP3) protein, has shown encouraging results in animal models for a range of conditions; however, until now, no off-targets have been identified. Herein, we report the design, synthesis, and application of a novel photoaffinity alkyne-tagged probe for MCC950 (IMP2070) which shows direct engagement with NLRP3 and inhibition of inflammasome activation in macrophages. Affinity-based chemical proteomics in live macrophages identified several potential off-targets, including carbonic anhydrase 2 (CA2) as a specific target of IMP2070, and independent cellular thermal proteomic profiling revealed stabilization of CA2 by MCC950. MCC950 displayed noncompetitive inhibition of CA2 activity, confirming carbonic anhydrase as an off-target class for this compound. These data highlight potential biological mechanisms through which MCC950 and derivatives may exhibit off-target effects in preclinical or clinical studies.

Journal article

Lovell S, Zhang L, Kryza T, Neodo A, Bock N, De Vita E, Williams E, Engelsberger E, Xu C, Bakker A, Maneiro M, Tanaka R, Bevan C, Clements J, Tate Eet al., 2021, A suite of activity-based probes to dissect the KLK activome in drug-resistant prostate cancer, Journal of the American Chemical Society, Vol: 143, Pages: 8911-8924, ISSN: 0002-7863

Kallikrein-related peptidases (KLKs) are a family of secreted serine proteases, which form a network (the KLK activome) with an important role in proteolysis and signaling. In prostate cancer (PCa), increased KLK activity promotes tumor growth and metastasis through multiple biochemical pathways, and specific quantification and tracking of changes in the KLK activome could contribute to validation of KLKs as potential drug targets. Herein we report a technology platform based on novel activity-based probes (ABPs) and inhibitors enabling simultaneous orthogonal analysis of KLK2, KLK3, and KLK14 activity in hormone-responsive PCa cell lines and tumor homogenates. Importantly, we identifed a significant decoupling of KLK activity and abundance and suggest that KLK proteolysis should be considered as an additional parameter, along with the PSA blood test, for accurate PCa diagnosis and monitoring. Using selective inhibitors and multiplexed fluorescent activity-based protein profiling (ABPP), we dissect the KLK activome in PCa cells and show that increased KLK14 activity leads to a migratory phenotype. Furthermore, using biotinylated ABPs, we show that active KLK molecules are secreted into the bone microenvironment by PCa cells following stimulation by osteoblasts suggesting KLK-mediated signaling mechanisms could contribute to PCa metastasis to bone. Together our findings show that ABPP is a powerful approach to dissect dysregulation of the KLK activome as a promising and previously underappreciated therapeutic target in advanced PCa.

Journal article

LanyonHogg T, Ritzefeld M, Zhang L, Andrei SA, Pogranyi B, Mondal M, Sefer L, Johnston CD, Coupland CE, Greenfield JL, Newington J, Fuchter MJ, Magee AI, Siebold C, Tate EWet al., 2021, Photochemical Probe Identification of a Small‐Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT)**, Angewandte Chemie, Vol: 133, Pages: 13654-13659, ISSN: 0044-8249

Journal article

Lanyon-Hogg T, Ritzefeld M, Zhang L, Pogranyi B, Mondal M, Sefer L, Johnston CD, Coupland CE, Andrei SA, Newington J, Magee AI, Siebold C, Tate EWet al., 2021, Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase, Angewandte Chemie International Edition, Vol: 60, Pages: 13542-13547, ISSN: 1433-7851

The mammalian membrane‐bound O ‐acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small‐molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small‐molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure‐activity relationship investigation identified single enantiomer IMP‐1575 , the most potent HHAT inhibitor reported to‐date, and guided design of photocrosslinking probes that maintained HHAT‐inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small‐molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed via kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP‐1575 ( K i = 38 nM) and a strategy for mapping small molecule interaction sites in MBOATs.

Journal article

Tate E, 2021, PROTACs, molecular glues and bifunctionals from bench to bedside: Unlocking the clinical potential of catalytic drugs, Progress in medicinal chemistry, ISSN: 0079-6468

Journal article

Kryza T, Khan T, Lovell S, Harrington BS, Yin J, Porazinski S, Pajic M, Koistinen H, Rantala JK, Dreyer T, Magdolen V, Reuning U, He Y, Tate EW, Hooper JDet al., 2021, Substrate-biased activity-based probes identify proteases that cleave receptor CDCP1, Nature Chemical Biology, Vol: 17, Pages: 776-783, ISSN: 1552-4450

CUB domain-containing protein 1 (CDCP1) is an oncogenic orphan transmembrane receptor and a promising target for the detection and treatment of cancer. Extracellular proteolysis of CDCP1 by poorly defined mechanisms induces pro-metastatic signaling. We describe a new approach for the rapid identification of proteases responsible for key proteolytic events using a substrate-biased activity-based probe (sbABP) that incorporates a substrate cleavage motif grafted onto a peptidyl diphenyl phosphonate warhead for specific target protease capture, isolation and identification. Using a CDCP1-biased probe, we identify urokinase (uPA) as the master regulator of CDCP1 proteolysis, which acts both by directly cleaving CDCP1 and by activating CDCP1-cleaving plasmin. We show that coexpression of uPA and CDCP1 is strongly predictive of poor disease outcome across multiple cancers and demonstrate that uPA-mediated CDCP1 proteolysis promotes metastasis in disease-relevant preclinical in vivo models. These results highlight CDCP1 cleavage as a potential target to disrupt cancer and establish sbABP technology as a new approach to identify disease-relevant proteases.

Journal article

Benns HJ, Storch M, Falco J, Fisher FR, Alves E, Wincott CJ, Baum J, Baldwin GS, Weerapana E, Tate EW, Child MAet al., 2021, Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

<jats:title>Summary</jats:title><jats:p>Nucleophilic amino acids are important in covalent drug development yet underutilized as antimicrobial targets. Over recent years, several chemoproteomic technologies have been developed to mine chemically-accessible residues via their intrinsic reactivity toward electrophilic probes. However, these approaches cannot discern which reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based Oligo Recombineering (CORe) platform to systematically prioritize reactive amino acids according to their contribution to protein function. Our approach directly couples protein sequence and function with biological fitness. Here, we profile the reactivity of &gt;1,000 cysteines on ~700 proteins in the eukaryotic pathogen <jats:italic>Toxoplasma gondii</jats:italic> and prioritize functional sites using CORe. We competitively compared the fitness effect of 370 codon switches at 74 cysteines and identify functional sites in a diverse range of proteins. In our proof of concept, CORe performed &gt;800 times faster than a standard genetic workflow. Reactive cysteines decorating the ribosome were found to be critical for parasite growth, with subsequent target-based screening validating the apicomplexan translation machinery as a target for covalent ligand development. CORe is system-agnostic, and supports expedient identification, functional prioritization, and rational targeting of reactive sites in a wide range of organisms and diseases.</jats:p>

Journal article

Losada de la Lastra A, Hassan S, Tate EW, 2021, Deconvoluting the biology and druggability of protein lipidation using chemical proteomics, Current Opinion in Chemical Biology, Vol: 60, Pages: 97-112, ISSN: 1367-5931

Lipids are indispensable cellular building blocks, and their post-translational attachment to proteins makes them important regulators of many biological processes. Dysfunction of protein lipidation is also implicated in many pathological states, yet its systematic analysis presents significant challenges. Thanks to innovations in chemical proteomics, lipidation can now be readily studied by metabolic tagging using functionalized lipid analogs, enabling global profiling of lipidated substrates using mass spectrometry. This has spearheaded the first deconvolution of their full scope in a range of contexts, from cells to pathogens and multicellular organisms. Protein N-myristoylation, S-acylation, and S-prenylation are the most well-studied lipid post-translational modifications because of their extensive contribution to the regulation of diverse cellular processes. In this review, we focus on recent advances in the study of these post-translational modifications, with an emphasis on how novel mass spectrometry methods have elucidated their roles in fundamental biological processes.

Journal article

Bickel JK, Voisin TB, Tate EW, Bubeck Det al., 2021, How Structures of Complement Complexes Guide Therapeutic Design., Subcell Biochem, Vol: 96, Pages: 273-295, ISSN: 0306-0225

The complement system is essential for immune defence against infection and modulation of proinflammatory responses. Activation of the terminal pathway of complement triggers formation of the membrane attack complex (MAC), a multi-protein pore that punctures membranes. Recent advances in structural biology, specifically cryo-electron microscopy (cryoEM), have provided atomic resolution snapshots along the pore formation pathway. These structures have revealed dramatic conformational rearrangements that enable assembly and membrane rupture. Here we review the structural basis for MAC formation and show how soluble proteins transition into a giant β-barrel pore. We also discuss regulatory complexes of the terminal pathway and their impact on structure-guided drug discovery of complement therapeutics.

Journal article

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