Author: "Richard Bayliss" - Searchworks@Jio Institute Digital Library Search Results

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1. Targeting ERBB3 and AKT to overcome adaptive resistance in EML4-ALK-driven non-small cell lung cancer

Author: Josephina Sampson, Hyun-min Ju, Nan Zhang, Sharon Yeoh, Jene Choi, and Richard Bayliss
Subjects: Cytology, QH573-671
Abstract: Abstract The fusion event between EML4 and ALK drives a significant oncogenic activity in 5% of non-small cell lung cancer (NSCLC). Even though potent ALK-tyrosine kinase inhibitors (ALK-TKIs) are successfully used for the treatment of EML4-ALK-positive NSCLC patients, a subset of those patients eventually acquire resistance during their therapy. Here, we investigate the kinase responses in EML4-ALK V1 and V3-harbouring NSCLC cancer cells after acute inhibition with ALK TKI, lorlatinib (LOR). Using phosphopeptide chip array and upstream kinase prediction analysis, we identified a group of phosphorylated tyrosine peptides including ERBB and AKT proteins that are upregulated upon ALK-TKI treatment in EML4-ALK-positive NSCLC cell lines. Dual inhibition of ALK and ERBB receptors or AKT disrupts RAS/MAPK and AKT/PI3K signalling pathways, and enhances apoptosis in EML4-ALK + NSCLC cancer cells. Heregulin, an ERBB3 ligand, differentially modulates the sensitivity of EML4-ALK cell lines to ALK inhibitors. We found that EML4-ALK cells made resistant to LOR are sensitive to inhibition of ERBB and AKT. These findings emphasize the important roles of AKT and ERBB3 to regulate signalling after acute LOR treatment, identifying them as potential targets that may be beneficial to prevent adaptive resistance to EML4-ALK-targeted therapies in NSCLC.
Published: 2024
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2. Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

Author: Raphael Vidal, Eoin Leen, Steffi Herold, Mareike Müller, Daniel Fleischhauer, Christina Schülein-Völk, Dimitrios Papadopoulos, Isabelle Röschert, Leonie Uhl, Carsten P Ade, Peter Gallant, Richard Bayliss, Martin Eilers, and Gabriele Büchel
Subjects: MYCN, TFIIIC, neuroblastoma, promoter hub, nuclear exosome, Medicine, Science, Biology (General), QH301-705.5
Abstract: MYC family oncoproteins regulate the expression of a large number of genes and broadly stimulate elongation by RNA polymerase II (RNAPII). While the factors that control the chromatin association of MYC proteins are well understood, much less is known about how interacting proteins mediate MYC’s effects on transcription. Here, we show that TFIIIC, an architectural protein complex that controls the three-dimensional chromatin organisation at its target sites, binds directly to the amino-terminal transcriptional regulatory domain of MYCN. Surprisingly, TFIIIC has no discernible role in MYCN-dependent gene expression and transcription elongation. Instead, MYCN and TFIIIC preferentially bind to promoters with paused RNAPII and globally limit the accumulation of non-phosphorylated RNAPII at promoters. Consistent with its ubiquitous role in transcription, MYCN broadly participates in hubs of active promoters. Depletion of TFIIIC further increases MYCN localisation to these hubs. This increase correlates with a failure of the nuclear exosome and BRCA1, both of which are involved in nascent RNA degradation, to localise to active promoters. Our data suggest that MYCN and TFIIIC exert an censoring function in early transcription that limits promoter accumulation of inactive RNAPII and facilitates promoter-proximal degradation of nascent RNA.
Published: 2024
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3. A nanobody inhibitor of Fascin-1 actin-bundling activity and filopodia formation

Author: Selena G. Burgess, Nikki R. Paul, Mark W. Richards, James R. Ault, Laurie Askenatzis, Sophie G. Claydon, Ryan Corbyn, Laura M. Machesky, and Richard Bayliss
Subjects: nanobody, Fascin-1, actin, cell migration, actin-bundling inhibitor, Biology (General), QH301-705.5
Abstract: Fascin-1-mediated actin-bundling activity is central to the generation of plasma membrane protrusions required for cell migration. Dysregulated formation of cellular protrusions is observed in metastatic cancers, where they are required for increased invasiveness, and is often correlated with increased Fascin-1 abundance. Therefore, there is interest in generating therapeutic Fascin-1 inhibitors. We present the identification of Nb 3E11, a nanobody inhibitor of Fascin-1 actin-bundling activity and filopodia formation. The crystal structure of the Fascin-1/Nb 3E11 complex reveals the structural mechanism of inhibition. Nb 3E11 occludes an actin-binding site on the third β-trefoil domain of Fascin-1 that is currently not targeted by chemical inhibitors. Binding of Nb 3E11 to Fascin-1 induces a conformational change in the adjacent domains to stabilize Fascin-1 in an inhibitory state similar to that adopted in the presence of small-molecule inhibitors. Nb 3E11 could be used as a tool inhibitor molecule to aid in the development of Fascin-1 targeted therapeutics.
Published: 2024
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4. EML4‐ALK biology and drug resistance in non‐small cell lung cancer: a new phase of discoveries

Author: Mariam Elshatlawy, Josephina Sampson, Katy Clarke, and Richard Bayliss
Subjects: cancer, drug resistance, NSCLC, signalling, tyrosine kinase inhibitors, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract: Anaplastic lymphoma kinase (ALK) can be driven to oncogenic activity by different types of mutational events such as point‐mutations, for example F1174L in neuroblastoma, and gene fusions, for example with echinoderm microtubule‐associated protein‐like 4 (EML4) in non‐small cell lung cancer (NSCLC). EML4‐ALK variants result from different breakpoints, generating fusions of different sizes and properties. The most common variants (Variant 1 and Variant 3) form cellular compartments with distinct physical properties. The presence of a partial, probably misfolded beta‐propeller domain in variant 1 confers solid‐like properties to the compartments it forms, greater dependence on Hsp90 for protein stability and higher cell sensitivity to ALK tyrosine kinase inhibitors (TKIs). These differences translate to the clinic because variant 3, on average, worsens patient prognosis and increases metastatic risk. Latest generation ALK‐TKIs are beneficial for most patients with EML4‐ALK fusions. However, resistance to ALK inhibitors can occur via point‐mutations within the kinase domain of the EML4‐ALK fusion, for example G1202R, reducing inhibitor effectiveness. Here, we discuss the biology of EML4‐ALK variants, their impact on treatment response, ALK‐TKI drug resistance mechanisms and potential combination therapies.
Published: 2023
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5. ALKing the flames of lung cancer immunosensitivity

Author: Richard Bayliss, Elżbieta Sarnowska, Sharon Yeoh, and Josephina Sampson
Subjects: cancer vaccine, combination therapy, immunotherapy, lung cancer, tyrosine kinase inhibitors, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract: Immune checkpoint inhibitors (ICIs) are utilised in treating non‐small cell lung cancer (NSCLC) by enhancing the immune response against cancer cells. However, they are not effective against cancers with certain genetic alterations. A recent study by Mota et al. focussed on understanding why ALK+ NSCLC cancers are immune cold and making them more receptive to ICIs using a vaccine‐based approach. The study highlighted cell‐specific differences in the presentation of immunogenic peptides and the location of tumours as factors in the poor immune response. Vaccines based on ALK peptides improved immune response, and when combined with ICIs, this led to a striking improvement in survival in a mouse model of ALK+ NSCLC.
Published: 2023
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6. Mapping of a N-terminal α-helix domain required for human PINK1 stabilization, Serine228 autophosphorylation and activation in cells

Author: Poonam Kakade, Hina Ojha, Olawale G. Raimi, Andrew Shaw, Andrew D. Waddell, James R. Ault, Sophie Burel, Kathrin Brockmann, Atul Kumar, Mohd Syed Ahangar, Ewelina M. Krysztofinska, Thomas Macartney, Richard Bayliss, Julia C. Fitzgerald, and Miratul M. K. Muqit
Subjects: PINK1, kinase, mitochondria, translocase, phosphorylation, Parkinson's disease, Biology (General), QH301-705.5
Abstract: Autosomal recessive mutations in the PINK1 gene are causal for Parkinson's disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE : CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.
Published: 2022
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7. Association with Aurora-A Controls N-MYC-Dependent Promoter Escape and Pause Release of RNA Polymerase II during the Cell Cycle

Author: Gabriele Büchel, Anne Carstensen, Ka-Yan Mak, Isabelle Roeschert, Eoin Leen, Olga Sumara, Julia Hofstetter, Steffi Herold, Jacqueline Kalb, Apoorva Baluapuri, Evon Poon, Colin Kwok, Louis Chesler, Hans Michael Maric, David S. Rickman, Elmar Wolf, Richard Bayliss, Susanne Walz, and Martin Eilers
Subjects: Biology (General), QH301-705.5
Abstract: Summary: MYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle. : Büchel et al. demonstrate that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21. Aurora-A competes with TFIIIC and RAD21 for binding to N-MYC, and Aurora-A displaces the three proteins from N-MYC during S phase. As consequence, N-MYC-dependent pause release is inhibited during S phase, preventing activation of the ATR checkpoint kinase. Keywords: N-MYC, MYC, Aurora-A, TFIIIC, RAD21, pause release, neuroblastoma
Published: 2017
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8. Covalent Aurora A regulation by the metabolic integrator coenzyme A

Author: Yugo Tsuchiya, Dominic P. Byrne, Selena G. Burgess, Jenny Bormann, Jovana Baković, Yueyang Huang, Alexander Zhyvoloup, Bess Yi Kun Yu, Sew Peak-Chew, Trang Tran, Fiona Bellany, Alethea B. Tabor, AW Edith Chan, Lalitha Guruprasad, Oleg Garifulin, Valeriy Filonenko, Matthias Vonderach, Samantha Ferries, Claire E. Eyers, John Carroll, Mark Skehel, Richard Bayliss, Patrick A. Eyers, and Ivan Gout
Subjects: Medicine (General), R5-920, Biology (General), QH301-705.5
Abstract: Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition.Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a “dual anchor” mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues.
Published: 2020
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9. TACC3–ch-TOG track the growing tips of microtubules independently of clathrin and Aurora-A phosphorylation

Author: Cristina Gutiérrez-Caballero, Selena G. Burgess, Richard Bayliss, and Stephen J. Royle
Subjects: TACC3, +TIP, ch-TOG, XMAP215, Microtubule dynamics, Science, Biology (General), QH301-705.5
Abstract: The interaction between TACC3 (transforming acidic coiled coil protein 3) and the microtubule polymerase ch-TOG (colonic, hepatic tumor overexpressed gene) is evolutionarily conserved. Loading of TACC3–ch-TOG onto mitotic spindle microtubules requires the phosphorylation of TACC3 by Aurora-A kinase and the subsequent interaction of TACC3 with clathrin to form a microtubule-binding surface. Recent work indicates that TACC3 can track the plus-ends of microtubules and modulate microtubule dynamics in non-dividing cells via its interaction with ch-TOG. Whether there is a pool of TACC3–ch-TOG that is independent of clathrin in human cells, and what is the function of this pool, are open questions. Here, we describe the molecular interaction between TACC3 and ch-TOG that permits TACC3 recruitment to the plus-ends of microtubules. This TACC3–ch-TOG pool is independent of EB1, EB3, Aurora-A phosphorylation and binding to clathrin. We also describe the distinct combinatorial subcellular pools of TACC3, ch-TOG and clathrin. TACC3 is often described as a centrosomal protein, but we show that there is no significant population of TACC3 at centrosomes. The delineation of distinct protein pools reveals a simplified view of how these proteins are organized and controlled by post-translational modification.
Published: 2015
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10. Mitotic Regulation by NEK Kinase Networks

Author: Andrew M. Fry, Richard Bayliss, and Joan Roig
Subjects: protein kinase, mitosis, microtubule, centrosome, cilia, Biology (General), QH301-705.5
Abstract: Genetic studies in yeast and Drosophila led to identification of cyclin-dependent kinases (CDKs), Polo-like kinases (PLKs) and Aurora kinases as essential regulators of mitosis. These enzymes have since been found in the majority of eukaryotes and their cell cycle-related functions characterized in great detail. However, genetic studies in another fungal species, Aspergillus nidulans, identified a distinct family of protein kinases, the NEKs, that are also widely conserved and have key roles in the cell cycle, but which remain less well studied. Nevertheless, it is now clear that multiple NEK family members act in networks to regulate specific events of mitosis, including centrosome separation, spindle assembly and cytokinesis. Here, we describe our current understanding of how the NEK kinases contribute to these processes, particularly through targeted phosphorylation of proteins associated with the microtubule cytoskeleton. We also present the latest findings on molecular events that control the activation state of the NEKs and how these are revealing novel modes of enzymatic regulation relevant not only to other kinases but also to pathological mechanisms of disease.
Published: 2017
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11. Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain

Author: Selena G. Burgess, Arkadiusz Oleksy, Tommaso Cavazza, Mark W. Richards, Isabelle Vernos, David Matthews, and Richard Bayliss
Subjects: antibody-assisted drug discovery, structural biology, biochemistry, protein kinase, Biology (General), QH301-705.5
Abstract: The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors.
Published: 2016
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12. A closed conformation of the Caenorhabditis elegans separase–securin complex

Author: Gudrun Bachmann, Mark W. Richards, Anja Winter, Fabienne Beuron, Edward Morris, and Richard Bayliss
Subjects: chromosome segregation, mitosis, protein purification, electron microscopy, Biology (General), QH301-705.5
Abstract: The protease separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans separase–securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21–24 Å that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of separase, whereas the second lobe readily accommodates homology models of the separase C-terminal death and caspase-like domains. The globular structure of the C. elegans separase–securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of separase activity through conformational change.
Published: 2016
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13. Structural Insights into Separase Architecture and Substrate Recognition through Computational Modelling of Caspase-Like and Death Domains.

Author: Anja Winter, Ralf Schmid, and Richard Bayliss
Subjects: Biology (General), QH301-705.5
Abstract: Separases are large proteins that mediate sister chromatid disjunction in all eukaryotes. They belong to clan CD of cysteine peptidases and contain a well-conserved C-terminal catalytic protease domain similar to caspases and gingipains. However, unlike other well-characterized groups of clan CD peptidases, there are no high-resolution structures of separases and the details of their regulation and substrate recognition are poorly understood. Here we undertook an in-depth bioinformatical analysis of separases from different species with respect to their similarity in amino acid sequence and protein fold in comparison to caspases, MALT-1 proteins (mucosa-associated lymphoidtissue lymphoma translocation protein 1) and gingipain-R. A comparative model of the single C-terminal caspase-like domain in separase from C. elegans suggests similar binding modes of substrate peptides between these protein subfamilies, and enables differences in substrate specificity of separase proteins to be rationalised. We also modelled a newly identified putative death domain, located N-terminal to the caspase-like domain. The surface features of this domain identify potential sites of protein-protein interactions. Notably, we identified a novel conserved region with the consensus sequence WWxxRxxLD predicted to be exposed on the surface of the death domain, which we termed the WR motif. We envisage that findings from our study will guide structural and functional studies of this important protein family.
Published: 2015
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14. Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly.

Author: Selena G Burgess, Isabel Peset, Nimesh Joseph, Tommaso Cavazza, Isabelle Vernos, Mark Pfuhl, Fanni Gergely, and Richard Bayliss
Subjects: Genetics, QH426-470
Abstract: The essential mammalian gene TACC3 is frequently mutated and amplified in cancers and its fusion products exhibit oncogenic activity in glioblastomas. TACC3 functions in mitotic spindle assembly and chromosome segregation. In particular, phosphorylation on S558 by the mitotic kinase, Aurora-A, promotes spindle recruitment of TACC3 and triggers the formation of a complex with ch-TOG-clathrin that crosslinks and stabilises kinetochore microtubules. Here we map the Aurora-A-binding interface in TACC3 and show that TACC3 potently activates Aurora-A through a domain centered on F525. Vertebrate cells carrying homozygous F525A mutation in the endogenous TACC3 loci exhibit defects in TACC3 function, namely perturbed localization, reduced phosphorylation and weakened interaction with clathrin. The most striking feature of the F525A cells however is a marked shortening of mitosis, at least in part due to rapid spindle assembly. F525A cells do not exhibit chromosome missegregation, indicating that they undergo fast yet apparently faithful mitosis. By contrast, mutating the phosphorylation site S558 to alanine in TACC3 causes aneuploidy without a significant change in mitotic duration. Our work has therefore defined a regulatory role for the Aurora-A-TACC3 interaction beyond the act of phosphorylation at S558. We propose that the regulatory relationship between Aurora-A and TACC3 enables the transition from the microtubule-polymerase activity of TACC3-ch-TOG to the microtubule-crosslinking activity of TACC3-ch-TOG-clathrin complexes as mitosis progresses. Aurora-A-dependent control of TACC3 could determine the balance between these activities, thereby influencing not only spindle length and stability but also the speed of spindle formation with vital consequences for chromosome alignment and segregation.
Published: 2015
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15. Diverse functionalization of Aurora-A kinase at specified surface and buried sites by native chemical modification.

Author: Fiona Rowan, Meirion Richards, Marcella Widya, Richard Bayliss, and Julian Blagg
Subjects: Medicine, Science
Abstract: The ability to obtain a homogeneous sample of protein is invaluable when studying the effect of alterations such as post-translational modifications (PTMs). Selective functionalization of a protein to investigate the effect of PTMs on its structure or activity can be achieved by chemical modification of cysteine residues. We demonstrate here that one such technique, which involves conversion of cysteine to dehydroalanine followed by thiol nucleophile addition, is suitable for the site-specific installation of a wide range of chemical mimics of PTMs, including acetylated and dimethylated lysine, and other unnatural amino acids. These reactions, optimized for the clinically relevant kinase Aurora-A, readily proceed to completion as revealed by intact protein mass spectrometry. Moreover, these reactions proceed under non-denaturing conditions, which is desirable when working with large protein substrates. We have determined reactivity trends for a diverse range of thiol nucleophile addition reactions at two separate sites on Aurora-A, and we also highlight limitations when using thiol nucleophiles that contain basic functional groups. We show that chemical modification of cysteine residues is possible not only on a flexible surface-exposed loop, but also within a deep active site pocket at the conserved DFG motif, which reveals the potential use of this method in exploring enzyme function through modification of catalytic site residues.
Published: 2014
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16. On the molecular mechanisms of mitotic kinase activation

Author: Richard Bayliss, Andrew Fry, Tamanna Haq, and Sharon Yeoh
Subjects: mitosis, protein kinases, regulation, protein structures, Biology (General), QH301-705.5
Abstract: During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell's shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein–protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients.
Published: 2012
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17. Supplementary Figure from EML4-ALK Variant 3 Promotes Mitotic Errors and Spindle Assembly Checkpoint Deficiency Leading to Increased Microtubule Poison Sensitivity

Author: Andrew M. Fry, Sam Khan, Richard Bayliss, Sarah L. Pashley, Josephina Sampson, Jene Choi, Laura O'Regan, and Kellie Lucken
Abstract: Supplementary Figure from EML4-ALK Variant 3 Promotes Mitotic Errors and Spindle Assembly Checkpoint Deficiency Leading to Increased Microtubule Poison Sensitivity
Published: 2023
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18. Regulation of Human PINK1 ubiquitin kinase by Serine167, Serine228 and Cysteine412 phosphorylation

Author: Andrew D. Waddell, Hina Ojha, Shalini Agarwal, Christopher J. Clarke, Ana Terriente-Felix, Houjiang Zhou, Poonam Kakade, Axel Knebel, Andrew M. Shaw, Robert Gourlay, Joby Varghese, Renata F. Soares, Rachel Toth, Thomas Macartney, Patrick A. Eyers, Nick Morrice, Richard Bayliss, Alexander J. Whitworth, Claire E. Eyers, and Miratul M. K. Muqit
Abstract: Loss-of-function mutations in the human PINK1 kinase (hPINK1) are causative of early-onset Parkinson’s disease (PD). Activation of hPINK1 induces phosphorylated ubiquitin to initiate removal of damaged mitochondria by autophagy. Previously we solved the structure of the insect PINK1 orthologue, Tribolium castaneum PINK1, and showed that autophosphorylation of Ser205 was critical for ubiquitin interaction and phosphorylation (Kumar, Tamjar, Waddell et al., 2017). In this advance, we report new findings on the regulation of hPINK1 by phosphorylation. We reconstitute E. coli expressed hPINK1 activity in vitro by direct incorporation of phosphoserine at the equivalent site Serine 228 (pSer228), providing direct evidence for a role for Ser228 phosphorylation in hPINK1 activation. Furthermore, using mass spectrometry, we identify six novel Ser/Thr autophosphorylation sites including regulatory Serine167 phosphorylation (pSer167), which in addition to pSer228 is required for ubiquitin recognition and phosphorylation. Strikingly, we also detect phosphorylation of a conserved Cysteine412 (pCys412) residue in the hPINK1 activation segment. Structural modelling suggests that pCys412 inhibits ubiquitin recognition and we demonstrate that mutation of Cys412 to Ala renders hPINK1 more active towards ubiquitin when expressed in human cells. These results outline new insights into hPINK1 activation by pSer167 and pSer228 and a novel inhibitory mechanism mediated by pCys412. These findings will aid in the development of small molecule activators of hPINK1. Description of files: Figure 1 Figure 1c OPA1 Coomassie: First gel: +Phos Tag Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1 Second gel: -Phos Tag Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1 Figure 1c blot: Lane 1-2: WT, Lane 3-4 pS228 recombinant PINK1 Figure 1d Coomassie: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1d autorad: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1f FLAG: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f HSP60: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f OPA1: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f pS228: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1f PINK1 pS228 FLAG merge: Lane 1: Marker, Lane 2: empty, Lane 3-4: FLAG-empty (-CCCP), Lane 5-6: FLAG-empty (+CCCP), Lane 7-8: hPINK1 WT (-CCCP), Lane 9-10: hPINK1 WT (+CCCP), Lane 11-12: hPINK1 KI (-CCCP), Lane 13-14: hPINK1 KI (+CCCP), Lane 15-16: hPINK1 S228A (-CCCP), Lane 17-18: hPINK1 S228A (+CCCP) Figure 1g PINK1 pS228 PINK1 multiplex: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1g pS228: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1g PINK1: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1g Tom20: Lane 1: Marker, Lane 2-3: WT SK-OV-3 (-O/A), Lane 4-5: WT SK-OV-3 (+O/A), Lane 6-7: KO PINK1 SK-OV-3 (-O/A), Lane 8-9: KO PINK1 SK-OV-3 (+O/A) Figure 1h HSP60: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP), Rest of the lanes: Extra samples not included in the paper. Figure 1h OPA1: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP) Figure 1h pS228 PINK1: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP) Figure 1h pUb: Upper blot Lane 1: Marker, Lane 2-3: emp FLAG (-CCCP), Lane 4-5: emp FLAG (+CCCP), Lane 6-7: WT PINK1-FLAG (-CCCP), Lane 8-9: WT PINK1-FLAG (+CCCP), Lane10-11: KI PINK1-FLAG (-CCCP), Lane 12-13: KI PINK1-FLAG (+CCCP), Lane14-15: S228A PINK1-FLAG (-CCCP), Lane 16-17: S228A PINK1-FLAG (+CCCP) Lower blot Lane 1: Marker, Lane 2-3: S228N PINK1 FLAG (-CCCP), Lane 4-5: S228N PINK1 FLAG (+CCCP), Lane 6-7: S230A PINK1-FLAG (-CCCP), Lane 8-9: S230A PINK1-FLAG (+CCCP), Lane10-11: DIns3 PINK1-FLAG (-CCCP), Lane 12-13: DIns3 PINK1-FLAG (+CCCP) Figure 1- Figure Supplement 2b: Recombinant jason chin post-purification. 2 lanes loaded per construct, one for amylose purification and another for the subsequent 6His purification. Lane1 and Lane5 are included in the paper. Lane 1 - WT PINK1, amylose (included in paper), Lane 2 - WT PINK1, 6His, Lane 3 - KI PINK1, amylose, Lane 4 - KI PINK1, 6His, Lane 5 - pSer228 PINK1, amylose (included in paper), Lane 6 - pSer228 PINK1, 6His, Lane 7 - pSer284 PINK1, amylose, Lane 8 - pSer284 PINK1, 6His, Lane 9 - pSer402 PINK1, amylose, Lane 10 - pSer402 PINK1, 6His Figure 1- Figure Supplement 2c: Lane10 and Lane11 are included in the paper. Lane 1 - 2mg/ml BSA, Lane 2 - 1mg/ml BSA, Lane 3 - 0.5mg/ml BSA, Lane 4 - 0.25mg/ml BSA, Lane 5 - 0.125 mg/ml BSA, Lane 6 – ladder, Lane 7 - unrelated PINK1 construct (Ser228A), Lane 8 - unrelated PINK1 construct (Ser284A), Lane 9 - KI PINK1, Lane 10 - WT PINK1, Lane 11 - pSer228 PINK1, 6His, Lane 12 - pSer284 PINK1, 6His Figure 1- Figure Supplement 2d (+ phos tag): Lane 1-2: WT recombinant PINK1, Lane 3-4: KI recombinant PINK1, Lane 5-6: pS228 recombinant PINK1, Lane 7-8: pSer284 recombinant PINK1 (Not included in the paper) Figure 1- Figure Supplement 2d (no phos tag): Lane 1-2: WT recombinant PINK1, Lane 3-4: KI recombinant PINK1, Lane 5-6: pS228 recombinant PINK1, Lane 7-8: pSer284 recombinant PINK1 (Not included in the paper) Figure 1- Figure Supplement 2f Coomassie: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1 - Figure Supplement 2f autorad: Recombinant PINK1: Lane 1-2: WT, Lane 3-4: KI, Lane 5-6 pS228, Lane 7-8: pS228DIns3, Lane 9-10: pS228G309D Figure 1 - Figure Supplement 3: Immunofluorescence images as mentioned in the paper Figure 2 Figure 2a FLAG: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP) Figure 2a HSP60: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP), rest of the lanes: Extra samples not included in the paper Figure 2a OPA1: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP) Figure 2a Phostag: Lane 1: Marker, Lane3-4: WT PINK1 3FLAG (-CCCP), Lane 5-6: WT PINK1 3FLAG (+CCCP), Lane 7-8: KI PINK1 3FLAG (-CCCP), Lane 9-10: KI PINK1 3FLAG (+CCCP) Figure 2- Figure Supplement 1a: Lane 1: Marker, Lane 3: emp 3-FLAG (-CCCP), Lane 4: emp 3-FLAG (+CCCP), Lane 5: KI PINK1 3-FLAG (-CCCP), Lane 6: KI PINK1 3-FLAG (+CCCP), Lane 7: WT PINK1 3-FLAG (-CCCP), Lane 8: WT PINK1 3-FLAG (+CCCP), Rest of the lanes: Not included in the paper Figure 2- Figure Supplement 1b: Lane 1: Marker, Lane3-5: 1% TX-100 (Total, soluble, insoluble), Lane 6-8: 1% SDS (Total, soluble, insoluble), Lane 10-12: 2% SDS (Total, soluble, insoluble) Figure 2- Figure Supplement 1c: Lane 1: Marker, Lane3-5: WT DMSO (Input, SN, IP), Lane 6-8: WT CCCP (Input, SN, IP), Lane 10-12: KI DMSO (Input, SN, IP), Lane 14-16: KI CCCP (Input, SN, IP) Figure 2- Figure Supplement 1d DCP: Upper blot Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Lower blot Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d ps228: Upper blot Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Lower blot Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d mix: Upper blot Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Lower blot Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d HSP60 KO: Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d HSP60 WT: Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d ACSL1 HSP60 KO: HSP60 blot (Lower band) re-probed for ACSL1 (Upper band); Lane 1: Marker, Lane3-4: SK-OV-3 PINK1 KO SDS (+CCCP), Lane 5-6: PINK1 KO Triton (+CCCP), Lane 7-8: PINK1 KO SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1d ACSL1 HSP60 WT: HSP60 blot (Lower band) re-probed for ACSL1 (Upper band); Lane 1: Marker, Lane3-4: SK-OV-3 WT SDS (-CCCP), Lane 5-6: SK-OV-3 WT Triton (-CCCP), Lane 7-8: SK-OV-3 WT SDS of leftover pellet (-CCCP), Lane9-10: SK-OV-3 WT SDS (+CCCP), Lane 11-12: SK-OV-3 WT Triton (+CCCP), Lane 13-14: SK-OV-3 WT SDS of leftover pellet (+CCCP) Figure 2- Figure Supplement 1e: Lane 1: Marker, Lane 3: WT Trypsin (-CCCP), Lane 5: WT chymotrypsin (-CCCP), Lane 7: WT Trypsin (+CCCP), Lane 9: WT chymotrypsin (+CCCP), Lane 11: KI Trypsin (-CCCP), Lane 13: KI chymotrypsin (-CCCP), Lane 15: KI Trypsin (+CCCP), Lane 17: KI chymotrypsin (+CCCP) Figure 3 Figure 3c OPA1 PINK1: Upper blot: OPA1 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Lower blot: PINK1 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Figure 3c ps228 PINK1: Upper blot: pS228 PINK1 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Lower blot: HSP60 Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Figure 3c pUb: Blot: pS65 Ub Lane 1: Marker, Lane 2: empty, Lane 3-4: hPINK1 WT (-O/A), Lane 5-6: hPINK1 WT (+O/A), Lane 7-8: hPINK1 KI (-O/A), Lane 9-10: hPINK1 KI (+O/A), Lane 11-12: hPINK1 S228A (-O/A), Lane 13-14: hPINK1 S228A (+O/A), Lane 15-16: hPINK1 S167A (-O/A), Lane 17-18: hPINK1 S167A (+O/A), Lane 19-20: hPINK1 S228A+S167A (-O/A), Lane 21-22: hPINK1 S228A+S167A (+O/A), Lane 23-24: hPINK1 multi-mutant (-O/A), Lane 25-26: hPINK1 multi-mutant (+O/A) Figure 4 Figure 4e PINK1: Full blot (cut according to kDa before probing with antibody); Lane 1: Marker, Lane 3: emp 3FLAG (-CCCP), Lane4: emp 3FLAG (+CCCP for 180 min), Lane 5: KI PINK 3FLAG (-CCCP), Lane6: KI PINK 3FLAG (+CCCP for 180 min), Lane 7: WT PINK 3FLAG (-CCCP), Lane 8: WT PINK 3FLAG (+CCCP for 15 min), Lane 9: WT PINK 3FLAG (+CCCP for 30 min), Lane 10: WT PINK 3FLAG (+CCCP for 45 min), Lane 11: WT PINK 3FLAG (+CCCP for 60 min), Lane 12: WT PINK 3FLAG (+CCCP for 120 min), Lane 13: WT PINK 3FLAG (+CCCP for 180 min), Lane 14: C412A PINK 3FLAG (-CCCP), Lane 15: C412A PINK 3FLAG (+CCCP for 15 min), Lane 16: C412A PINK 3FLAG (+CCCP for 30 min), Lane 17: C412A PINK 3FLAG (+CCCP for 45 min), Lane 18: C412A PINK 3FLAG (+CCCP for 60 min), Lane 19: C412A PINK 3FLAG (+CCCP for 120 min), Lane 20: C412A PINK 3FLAG (+CCCP for 180 min) Figure 4e GAPDH OPA: Upper bands: OPA1, Lower bands: GAPDH; Lane 1: Marker, Lane 3: emp 3FLAG (-CCCP), Lane4: emp 3FLAG (+CCCP for 180 min), Lane 5: KI PINK 3FLAG (-CCCP), Lane6: KI PINK 3FLAG (+CCCP for 180 min), Lane 7: WT PINK 3FLAG (-CCCP), Lane 8: WT PINK 3FLAG (+CCCP for 15 min), Lane 9: WT PINK 3FLAG (+CCCP for 30 min), Lane 10: WT PINK 3FLAG (+CCCP for 45 min), Lane 11: WT PINK 3FLAG (+CCCP for 60 min), Lane 12: WT PINK 3FLAG (+CCCP for 120 min), Lane 13: WT PINK 3FLAG (+C
Published: 2023
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19. Figure S2 from Hsp72 and Nek6 Cooperate to Cluster Amplified Centrosomes in Cancer Cells

Author: Andrew M. Fry, Richard Bayliss, Martin J.S. Dyer, Laura O'Regan, and Josephina Sampson
Abstract: Figure S2 shows Hsp70 inhibition does not block clustering of acentrosomal spindle poles.
Published: 2023
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20. Supplementary Figure Legends from Hsp72 and Nek6 Cooperate to Cluster Amplified Centrosomes in Cancer Cells

Author: Andrew M. Fry, Richard Bayliss, Martin J.S. Dyer, Laura O'Regan, and Josephina Sampson
Abstract: Supplementary figure legends
Published: 2023
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21. Data from Hsp72 and Nek6 Cooperate to Cluster Amplified Centrosomes in Cancer Cells

Author: Andrew M. Fry, Richard Bayliss, Martin J.S. Dyer, Laura O'Regan, and Josephina Sampson
Abstract: Cancer cells frequently possess extra amplified centrosomes clustered into two poles whose pseudo-bipolar spindles exhibit reduced fidelity of chromosome segregation and promote genetic instability. Inhibition of centrosome clustering triggers multipolar spindle formation and mitotic catastrophe, offering an attractive therapeutic approach to selectively kill cells with amplified centrosomes. However, mechanisms of centrosome clustering remain poorly understood. Here, we identify a new pathway that acts through NIMA-related kinase 6 (Nek6) and Hsp72 to promote centrosome clustering. Nek6, as well as its upstream activators polo-like kinase 1 and Aurora-A, targeted Hsp72 to the poles of cells with amplified centrosomes. Unlike some centrosome declustering agents, blocking Hsp72 or Nek6 function did not induce formation of acentrosomal poles, meaning that multipolar spindles were observable only in cells with amplified centrosomes. Inhibition of Hsp72 in acute lymphoblastic leukemia cells resulted in increased multipolar spindle frequency that correlated with centrosome amplification, while loss of Hsp72 or Nek6 function in noncancer-derived cells disturbs neither spindle formation nor mitotic progression. Hence, the Nek6–Hsp72 module represents a novel actionable pathway for selective targeting of cancer cells with amplified centrosomes. Cancer Res; 77(18); 4785–96. ©2017 AACR.
Published: 2023
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22. EML4-ALK Variant 3 Promotes Mitotic Errors and Spindle Assembly Checkpoint Deficiency Leading to Increased Microtubule Poison Sensitivity

Author: Kellie Lucken, Laura O'Regan, Jene Choi, Josephina Sampson, Sarah L. Pashley, Richard Bayliss, Sam Khan, and Andrew M. Fry
Subjects: Cancer Research, Lung Neoplasms, Oncogene Proteins, Fusion, Paclitaxel, Oncology, Carcinoma, Non-Small-Cell Lung, Humans, M Phase Cell Cycle Checkpoints, Receptor Protein-Tyrosine Kinases, Microtubules, Protein Kinase Inhibitors, Molecular Biology, Article
Abstract: EML4-ALK is an oncogenic fusion protein present in approximately 5% of non–small cell lung cancers (NSCLC). Alternative breakpoints in the gene encoding EML4 result in distinct variants that are linked to markedly different patient outcomes. Patients with EML4-ALK variant 3 (V3) respond poorly to ALK inhibitors and have lower survival rates compared with patients with other common variants, such as V1. Here, we use isogenic Beas-2B bronchial epithelial cell lines expressing EML4-ALK V1 or V3, as well as ALK-positive NSCLC patient cells that express V1 (H3122 cells) or V3 (H2228 cells), to show that EML4-ALK V3 but not V1 leads to hyperstabilized K-fibers in mitosis, as well as errors in chromosome congression and segregation. This is consistent with our observation that EML4-ALK V3 but not V1 localizes to spindle microtubules and that wild-type EML4 is a microtubule stabilizing protein. In addition, cells expressing EML4-ALK V3 exhibit loss of spindle assembly checkpoint control that is at least in part dependent on ALK catalytic activity. Finally, we demonstrate that cells expressing EML4-ALK V3 have increased sensitivity to microtubule poisons that interfere with mitotic spindle assembly, whereas combination treatment with paclitaxel and clinically approved ALK inhibitors leads to a synergistic response in terms of reduced survival of H2228 cells. Implications: This study suggests that combining the microtubule poison, paclitaxel, with targeted ALK inhibitors may provide an effective new treatment option for patients with NSCLC with tumors that express the EML4-ALK V3 oncogenic fusion.
Published: 2022
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23. Affimer-Mediated Locking of a PAK5 Intermediate Activation State Reveals a Novel Mechanism of Kinase Inhibition

Author: Heather Louise Martin, Amy L. Turner, Julie Higgins, Anna A. Tang, Christian Tiede, Thomas Taylor, Thomas L. Adams, Sandra M. Bell, Ewan E. Morrison, Jacquelyn Bond, Chi H. Trinh, Carolyn D. Hurst, Margaret Knowles, Richard Bayliss, and Darren C. Tomlinson
Published: 2023
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24. Safer by design: Building a collaborative, integrated and evidence-based framework to inform the regulation and mitigation of gambling product risk

Author: Richard Bayliss, Jonathan Parke, Chris Percy, Paul Delfabbro, and Simo Dragecvic
Subjects: Psychiatry and Mental health, Clinical Psychology, Evidence-based practice, Harm, Product design, Risk analysis (engineering), Strategic alignment, Information sharing, Stakeholder, Position paper, Product (category theory), Business
Abstract: Evidence suggests that harms may result from gambling participation as a result of a complex interaction between individual differences among consumers, environmental factors, and the characteristics of the gambling product. The latter of these factors, broadly referred to in this paper as product risk, has received increased policy attention in recent years. Product-focussed approaches to harm reduction, however, are under-developed relative to other forms of player protection and likely reflects the limitations of existing evidence and relative complexity of the topic. In this position paper, we define and explain the concept of product risk and consider what is currently known regarding the link between gambling products and harm. The paper describes the present barriers to develop effective product risk regulation and harm mitigation strategies. These include the competing interests of stakeholders, limited collaboration and information sharing, clear roles, responsibilities and leadership and a lack of integrated evidence-informed approaches. In response to these challenges, we propose adopting a framework comprised of a series of principles to progress this contested area of policy. The framework encourages better collaboration and communication between stakeholders; the accelerated production of valid and reliable evidence; a strategic alignment of stakeholder activity; and, more effective and efficient approaches to assessing and mitigating product risk.
Published: 2021
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25. Major incidents

Author: Richard Bayliss and Tom Hurst
Subjects: Anesthesiology and Pain Medicine, Critical Care and Intensive Care Medicine
Published: 2020
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26. Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine

Author: Chitra Bhatia, Katherine H. Carr, Sharon Yeoh, Corine Mas-Droux, Richard Bayliss, Rory F. Cunnison, Matthew J. Byrne, Nazia Nasir, Celine Cano, and Christine Basmadjian
Subjects: Protein Conformation, Stereochemistry, Mutant, Substituent, Biochemistry, Catalysis, Serine, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Structural Biology, Chemical Biology, Humans, NIMA-Related Kinases, Enzyme Inhibitors, Molecular Biology, Research Articles, 030304 developmental biology, 0303 health sciences, Chemistry, Kinase, protein engineering, Cell Biology, Protein engineering, Small molecule, Kinetics, small molecules, kinases, 030220 oncology & carcinogenesis, Mutation, Multipolar spindles, Cytokinesis, Protein Binding
Abstract: Nek7 is a serine/threonine-protein kinase required for proper spindle formation and cytokinesis. Elevated Nek7 levels have been observed in several cancers, and inhibition of Nek7 might provide a route to the development of cancer therapeutics. To date, no selective and potent Nek7 inhibitors have been identified. Nek7 crystal structures exhibit an improperly formed regulatory-spine (R-spine), characteristic of an inactive kinase. We reasoned that the preference of Nek7 to crystallise in this inactive conformation might hinder attempts to capture Nek7 in complex with Type I inhibitors. Here, we have introduced aromatic residues into the R-spine of Nek7 with the aim to stabilise the active conformation of the kinase through R-spine stacking. The strong R-spine mutant Nek7SRS retained catalytic activity and was crystallised in complex with compound 51, an ATP-competitive inhibitor of Nek2 and Nek7. Subsequently, we obtained the same crystal form for wild-type Nek7WT in apo form and bound to compound 51. The R-spines of the three well-ordered Nek7WT molecules exhibit variable conformations while the R-spines of the Nek7SRS molecules all have the same, partially stacked configuration. Compound 51 bound to Nek2 and Nek7 in similar modes, but differences in the precise orientation of a substituent highlights features that could be exploited in designing inhibitors that are selective for particular Nek family members. Although the SRS mutations are not required to obtain a Nek7–inhibitor structure, we conclude that it is a useful strategy for restraining the conformation of a kinase in order to promote crystallogenesis.
Published: 2020
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27. Exploring the Conformational Landscape and Stability of Aurora A Using Ion-Mobility Mass Spectrometry and Molecular Modeling

Author: Claire E. Eyers, Lauren J. Tomlinson, Dominic P. Byrne, Joscelyn Sarsby, Matthew Bachelor, Richard Bayliss, Philip Brownridge, and Patrick A. Eyers
Subjects: chemistry.chemical_classification, Models, Molecular, Molecular model, Chemistry, Kinase, Protein Conformation, Protein Stability, Protein Data Bank (RCSB PDB), Small molecule, Mass Spectrometry, Molecular dynamics, Enzyme, Structural Biology, Ion Mobility Spectrometry, Biophysics, Humans, Protein kinase A, Conformational isomerism, Spectroscopy, Aurora Kinase A
Abstract: Protein kinase inhibitors are proving highly effective in helping treat a number of non-communicable diseases driven by aberrant kinase signaling. They are also extremely valuable as chemical tools to help delineate cellular roles of kinase signaling complexes. The binding of small molecule inhibitors induces conformational effects on kinase dynamics; evaluating the effect of such interactions can assist in developing specific inhibitors and is deemed imperative to understand both inhibition and resistance mechanisms. Using gas-phase ion mobility-mass spectrometry (IM-MS) we characterized changes in the conformational landscape and stability of the protein kinase Aurora A (Aur A) driven by binding of the physiological activator TPX2 or small molecule inhibition. Aided by molecular modeling, we establish three major conformations: one highly-populated compact conformer similar to that observed in most crystal structures, a second highly-populated conformer possessing a more open structure that is infrequently found in crystal structures, and an additional low-abundance conformer not currently represented in the protein databank. Comparison of active (phosphorylated) and inactive (non-phosphorylated) forms of Aur A revealed that the active enzyme has different conformer weightings and is less stable than the inactive enzyme. Notably, inhibitor binding shifts conformer balance towards the more compact configurations adopted by the unbound enzyme, with both IM-MS and modelling revealing inhibitor-mediated stabilisation of active Aur A. These data highlight the power of IM-MS in combination with molecular dynamics simulations to probe and compare protein kinase structural dynamics that arise due to differences in activity and as a result of compound binding.
Published: 2022

28. Phase‐separated foci of EML4‐ALK facilitate signalling and depend upon an active kinase conformation

Author: Richard Bayliss, Andrew M. Fry, Mark W. Richards, Josephina Sampson, and Jene Choi
Subjects: MAPK/ERK pathway, Lung Neoplasms, Oncogene Proteins, Fusion, Protein Conformation, Mutant, NSCLC, Biochemistry, Article, Structural Biology, Microtubule, Carcinoma, Non-Small-Cell Lung, hemic and lymphatic diseases, tyrosine kinase inhibitors, Genetics, cancer, Humans, Anaplastic Lymphoma Kinase, signalling, Protein Kinase Inhibitors, Molecular Biology, Chemistry, Kinase, Articles, Fusion protein, Lorlatinib, Cell biology, Protein kinase domain, Salt bridge, phase separation, Signal Transduction
Abstract: Variants of the oncogenic EML4‐ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4‐ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4‐ALK V3 but not V1 protein re‐localises to microtubules, an effect recapitulated in a catalytically inactive EML4‐ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild‐type and catalytically inactive EML4‐ALK V3 proteins, but not a Lys‐Glu salt bridge mutant. We propose that EML4‐ALK foci formation occurs as a result of transient association of stable EML4‐ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4‐ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors., EML4‐ALK fusion proteins phase separate into foci that contain oncogenic signaling factors, through a mechanism that depends on an active ALK kinase conformation. Variants have distinct dynamics determined by a specific region of EML4.
Published: 2021
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29. Discovery and Optimization of wt-RET/KDR-Selective Inhibitors of RETV804M Kinase

Author: Bohdan Waszkowycz, Habiba Begum, Chitra Seewooruthun, Aude Echalier, Amanda J. Watson, Stuart Donald Jones, Richard Bayliss, Mark W. Richards, Rebecca Newton, Li-Ying Lin, Daniel Burschowsky, Allan M. Jordan, Donald J. Ogilvie, Niall M. Hamilton, Samantha Hitchin, Eleanor French, and Ian D. Waddell
Subjects: Gene isoform, congenital, hereditary, and neonatal diseases and abnormalities, endocrine system, Virtual screening, endocrine system diseases, 010405 organic chemistry, Kinase, Organic Chemistry, Mutant, Wild type, Rational design, Biology, 01 natural sciences, Biochemistry, 0104 chemical sciences, 010404 medicinal & biomolecular chemistry, Drug Discovery, Cancer research, neoplasms, Clinical evaluation
Abstract: [Image: see text] A combination of focused library and virtual screening, hit expansion, and rational design has resulted in the development of a series of inhibitors of RET(V804M) kinase, the anticipated drug-resistant mutant of RET kinase. These agents do not inhibit the wild type (wt) isoforms of RET or KDR and therefore offer a potential adjunct to RET inhibitors currently undergoing clinical evaluation.
Published: 2020
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30. A point mutation in the kinase domain of CRK10 leads to xylem vessel collapse and activates defence responses

Author: Johnathan A. Napier, Piovesana M, Michael J. Deery, Richard Bayliss, Smith Dp, Machado Wood Ak, Esther Carrera, Smita Kurup, and Matthes Mc
Subjects: Protein kinase domain, Kinase, Mutant, Wild type, Arabidopsis thaliana, Phosphorylation, Biology, Threonine, biology.organism_classification, Gene, Cell biology
Abstract: Cysteine-rich receptor-like kinases (CRKs) are a large family of plasma membrane-bound receptors ubiquitous in higher plants. They are transcriptionally regulated by a wide variety of environmental cues and stresses, however their precise biological roles remain largely unknown. Here we report a novel mutant isolated for the CYSTEINE-RICH RECEPTOR-LIKE KINASE 10 (CRK10) of Arabidopsis thaliana which harbours the substitution of alanine 397 by a threonine in the αC-helix of its kinase domain and which we registered as crk10-A397T in the community database. In situ phosphorylation assays with the His-tagged wild type (WT) and crk10-A397T versions of the CRK10 kinase domain revealed that both alleles are active kinases capable of auto-phosphorylation with the newly introduced threonine acting as an additional phosphorylation site in crk10-A397T. Phenotypically the mutant is a dwarf and the analysis of thin cross sections with light and transmission electron microscopy revealed that collapsed xylem vessels in roots and hypocotyls are very likely the cause for this reduction in stature. Transcriptomic analysis of WT and mutant hypocotyls revealed that predominantly biotic and abiotic stress-responsive genes are constitutively up-regulated in the mutant. Root-infection assays with the vascular pathogen Fusarium oxysporum demonstrated that the crk10-A397T mutant has enhanced resistance to this pathogen compared to WT plants. Taken together our results suggest that crk10-A397T is a gain-of-function allele of CRK10 and open up new avenues for the investigation of this elusive receptor-like kinase family.
Published: 2021
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31. Combined inhibition of Aurora-A and ATR kinase results in regression of

Author: Isabelle, Roeschert, Evon, Poon, Anton G, Henssen, Heathcliff Dorado, Garcia, Marco, Gatti, Celeste, Giansanti, Yann, Jamin, Carsten P, Ade, Peter, Gallant, Christina, Schülein-Völk, Petra, Beli, Mark, Richards, Mathias, Rosenfeldt, Matthias, Altmeyer, John, Anderson, Angelika, Eggert, Matthias, Dobbelstein, Richard, Bayliss, Louis, Chesler, Gabriele, Büchel, and Martin, Eilers
Subjects: Mice, N-Myc Proto-Oncogene Protein, Neuroblastoma, Cell Line, Tumor, Animals, Apoptosis, macromolecular substances, biological phenomena, cell phenomena, and immunity, neoplasms, Article, Aurora Kinase A
Abstract: Amplification of MYCN is the driving oncogene in a subset of high-risk neuroblastoma. The MYCN protein and the Aurora-A kinase form a complex during S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription-replication conflicts and activates the Ataxia telangiectasia and Rad3 related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription-replication conflicts is an effective therapy for MYCN-driven neuroblastoma (141 words).
Published: 2021

32. Combined inhibition of Aurora-A and ATR kinases results in regression of MYCN-amplified neuroblastoma

Author: Martin Eilers, Peter Gallant, Celeste Giansanti, Anton G. Henssen, Angelika Eggert, Carsten P. Ade, Marco Gatti, Yann Jamin, John Anderson, Gabriele Büchel, Matthias Altmeyer, Petra Beli, Isabelle Roeschert, Heathcliff Dorado Garcia, Evon Poon, Louis Chesler, Mathias T. Rosenfeldt, Richard Bayliss, Matthias Dobbelstein, Christina Schülein-Völk, Mark W. Richards, University of Zurich, Chesler, Louis, Büchel, Gabriele, and Eilers, Martin
Subjects: Cancer Research, macromolecular substances, 03 medical and health sciences, Histone H3, 0302 clinical medicine, Neuroblastoma, medicine, 1306 Cancer Research, neoplasms, 030304 developmental biology, 0303 health sciences, biology, Chemistry, Kinase, medicine.disease, 10226 Department of Molecular Mechanisms of Disease, 3. Good health, Chromatin, enzymes and coenzymes (carbohydrates), Histone, Oncology, 030220 oncology & carcinogenesis, embryonic structures, Ataxia-telangiectasia, biology.protein, Cancer research, 570 Life sciences, Phosphorylation, 2730 Oncology, biological phenomena, cell phenomena, and immunity, N-Myc
Abstract: Amplification of MYCN is the driving oncogenic change in a subset of high-risk neuroblastomas. The MYCN protein and the Aurora-A kinase form a complex during the S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in the S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription–replication conflicts and activates ataxia telangiectasia and Rad3-related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR kinases induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription–replication conflicts is an effective therapy for MYCN-driven neuroblastoma. Eilers and colleagues report that Aurora-A suppresses transcription–replication conflicts in MYCN-driven neuroblastoma, a vulnerability that can be targeted with a combination of Aurora-A and ATR kinase inhibitors.
Published: 2021

33. Structural features of the protein kinase domain and targeted binding by small-molecule inhibitors

Author: Chris Arter, Luke Trask, Sarah Ward, Sharon Yeoh, and Richard Bayliss
Subjects: Adenosine Triphosphate, Drug Discovery, Humans, Cell Biology, Phosphorylation, Protein Kinase Inhibitors, Protein Kinases, Molecular Biology, Biochemistry, Protein Binding
Abstract: Protein kinases are key components in cellular signaling pathways as they carry out the phosphorylation of proteins, primarily on Ser, Thr, and Tyr residues. The catalytic activity of protein kinases is regulated, and they can be thought of as molecular switches that are controlled through protein-protein interactions and post-translational modifications. Protein kinases exhibit diverse structural mechanisms of regulation and have been fascinating subjects for structural biologists from the first crystal structure of a protein kinase over 30 years ago, to recent insights into kinase assemblies enabled by the breakthroughs in cryo-EM. Protein kinases are high-priority targets for drug discovery in oncology and other disease settings, and kinase inhibitors have transformed the outcomes of specific groups of patients. Most kinase inhibitors are ATP competitive, deriving potency by occupying the deep hydrophobic pocket at the heart of the kinase domain. Selectivity of inhibitors depends on exploiting differences between the amino acids that line the ATP site and exploring the surrounding pockets that are present in inactive states of the kinase. More recently, allosteric pockets outside the ATP site are being targeted to achieve high selectivity and to overcome resistance to current therapeutics. Here, we review the key regulatory features of the protein kinase family, describe the different types of kinase inhibitors, and highlight examples where the understanding of kinase regulatory mechanisms has gone hand in hand with the development of inhibitors.
Published: 2022
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34. Alternative Treatment Options to ALK Inhibitor Monotherapy for EML4-ALK-Driven Lung Cancer

Author: Savvas Papageorgiou, Sarah L. Pashley, Laura O’Regan, Sam Khan, Richard Bayliss, and Andrew M. Fry
Subjects: Cancer Research, Oncology
Abstract: EML4-ALK is an oncogenic fusion protein that accounts for approximately 5% of NSCLC cases. Targeted inhibitors of ALK are the standard of care treatment, often leading to a good initial response. Sadly, some patients do not respond well, and most will develop resistance over time, emphasizing the need for alternative treatments. This review discusses recent advances in our understanding of the mechanisms behind EML4-ALK-driven NSCLC progression and the opportunities they present for alternative treatment options to ALK inhibitor monotherapy. Targeting ALK-dependent signalling pathways can overcome resistance that has developed due to mutations in the ALK catalytic domain, as well as through activation of bypass mechanisms that utilise the same pathways. We also consider evidence for polytherapy approaches that combine targeted inhibition of these pathways with ALK inhibitors. Lastly, we review combination approaches that use targeted inhibitors of ALK together with chemotherapy, radiotherapy or immunotherapy. Throughout this article, we highlight the importance of alternative breakpoints in the EML4 gene that result in the generation of distinct EML4-ALK variants with different biological and pathological properties and consider monotherapy and polytherapy approaches that may be selective to particular variants.
Published: 2022
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35. Defining endogenous TACC3–chTOG–clathrin–GTSE1 interactions at the mitotic spindle using induced relocalization

Author: Richard Bayliss, James Shelford, Ellis L. Ryan, Stephen J. Royle, and Teresa Massam-Wu
Subjects: Multiprotein complex, Mitosis, Context (language use), Spindle Apparatus, Mitochondrion, Clathrin, Microtubules, 03 medical and health sciences, 0302 clinical medicine, Microtubule, GTSE1, CRISPR, Humans, 030304 developmental biology, 0303 health sciences, biology, Cas9, Chemistry, Knocksideways, Mitotic spindle, Cell Biology, Spindle apparatus, Cell biology, TACC3, biology.protein, Microtubule-Associated Proteins, 030217 neurology & neurosurgery, Research Article, HeLa Cells
Abstract: A multiprotein complex containing TACC3, clathrin and other proteins has been implicated in mitotic spindle stability. To disrupt this complex in an anti-cancer context, we need to understand its composition and how it interacts with microtubules. Induced relocalization of proteins in cells is a powerful way to analyze protein–protein interactions and, additionally, monitor where and when these interactions occur. We used CRISPR/Cas9 gene editing to add tandem FKBP–GFP tags to each complex member. The relocalization of endogenous tagged protein from the mitotic spindle to mitochondria and assessment of the effect on other proteins allowed us to establish that TACC3 and clathrin are core complex members and that chTOG (also known as CKAP5) and GTSE1 are ancillary to the complex, binding respectively to TACC3 and clathrin, but not each other. We also show that PIK3C2A, a clathrin-binding protein that was proposed to stabilize the TACC3–chTOG–clathrin–GTSE1 complex during mitosis, is not a member of the complex. This work establishes that targeting the TACC3–clathrin interface or their microtubule-binding sites are the two strategies most likely to disrupt spindle stability mediated by this multiprotein complex., Summary: The composition of the mitotic TACC3–chTOG–clathrin–GTSE1 complex and its interaction with microtubules is determined using induced relocalization of endogenous proteins from the spindle to mitochondria.
Published: 2021

36. Drugging the 'Undruggable' MYCN Oncogenic Transcription Factor: Overcoming Previous Obstacles to Impact Childhood Cancers

Author: Yael P. Mosse, Mark A. Lemmon, David Levens, Gabriele Büchel, Chi V. Dang, William A. Weiss, Richard Bayliss, Stefan Knapp, Linda Z. Penn, Adam J. Wolpaw, Ronen Marmorstein, Michael Rape, Kevan M. Shokat, W. Clay Gustafson, Julie R. Park, Martin Eilers, Seychelle M. Vos, Elmar Wolf, Martine F. Roussel, Steven J. Metallo, John M. Maris, Gwenn H. Hansen, Kliment A. Verba, Natalia Jura, William P. Tansey, and Massachusetts Institute of Technology. Department of Biology
Subjects: 0301 basic medicine, History, Cancer Research, Investigational, Drug Resistance, Drug Screening Assays, Neuroblastoma, 0302 clinical medicine, Neoplasms, Drug Discovery, Medicine, Age of Onset, Child, Cancer, Pediatric, Regulation of gene expression, N-Myc Proto-Oncogene Protein, Therapies, Investigational, 21st Century, Gene Expression Regulation, Neoplastic, 20th Century, Oncology, 5.1 Pharmaceuticals, Novel agents, 030220 oncology & carcinogenesis, Development of treatments and therapeutic interventions, Pediatric Research Initiative, Pediatric Cancer, Oncology and Carcinogenesis, Antineoplastic Agents, Computational biology, History, 21st Century, Article, 03 medical and health sciences, Rare Diseases, Genetics, Humans, Effective treatment, Oncology & Carcinogenesis, Transcription factor, neoplasms, Medulloblastoma, Neoplastic, business.industry, Neurosciences, Antitumor, History, 20th Century, medicine.disease, Pediatric cancer, 030104 developmental biology, Gene Expression Regulation, Drug Resistance, Neoplasm, Therapies, Neoplasm, Drug Screening Assays, Antitumor, business, N-Myc
Abstract: Effective treatment of pediatric solid tumors has been hampered by the predominance of currently “undruggable” driver transcription factors. Improving outcomes while decreasing the toxicity of treatment necessitates the development of novel agents that can directly inhibit or degrade these elusive targets. MYCN in pediatric neural-derived tumors, including neuroblastoma and medulloblastoma, is a paradigmatic example of this problem. Attempts to directly and specifically target MYCN have failed due to its similarity to MYC, the unstructured nature of MYC family proteins in their monomeric form, the lack of an understanding of MYCN-interacting proteins and ability to test their relevance in vivo, the inability to obtain structural information on MYCN protein complexes, and the challenges of using traditional small molecules to inhibit protein–protein or protein–DNA interactions. However, there is now promise for directly targeting MYCN based on scientific and technological advances on all of these fronts. Here, we discuss prior challenges and the reasons for renewed optimism in directly targeting this “undruggable” transcription factor, which we hope will lead to improved outcomes for patients with pediatric cancer and create a framework for targeting driver oncoproteins regulating gene transcription.
Published: 2021

37. Author Reply to Peer Reviews of Defining endogenous TACC3–chTOG–clathrin–GTSE1 interactions at the mitotic spindle using induced relocalization

Author: Stephen J. Royle, Richard Bayliss, Teresa Massam-Wu, James Shelford, and Ellis L. Ryan
Published: 2020
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38. Decision letter: Breakage of the oligomeric CaMKII hub by the regulatory segment of the kinase

Author: Richard Bayliss and Dorothee Kern
Subjects: Breakage, Kinase, Chemistry, Ca2+/calmodulin-dependent protein kinase, Cell biology
Published: 2020
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39. Phase-separation of EML4-ALK variant 3 is dependent upon an active ALK conformation

Author: Mark W. Richards, Jene Choi, Richard Bayliss, Andrew M. Fry, and Josephina Sampson
Subjects: Alectinib, Ceritinib, biology, Chemistry, Context (language use), Lorlatinib, Cell biology, Protein kinase domain, hemic and lymphatic diseases, medicine, biology.protein, GRB2, Kinase activity, Tyrosine kinase, medicine.drug
Abstract: Oncogenic fusions involving tyrosine kinases are common drivers of non-small cell lung cancer (NSCLC). There are at least 15 different variants of the EML4-ALK fusion, all of which have a similar portion of ALK that includes the kinase domain, but different portions of EML4. Targeted treatment with ALK tyrosine kinase inhibitors (TKIs) has proven effective but patient outcomes are variable. Here, we focus on one common variant, EML4-ALK V3, which drives an aggressive form of the disease. EML4-ALK V3 protein forms cytoplasmic liquid droplets that contain the signalling proteins GRB2 and SOS1. The TKIs ceritinib and lorlatinib dissolve these droplets and the EML4-ALK V3 protein re-localises to microtubules, an effect recapitulated by an inactivating mutation in the ALK catalytic site. Mutations that promote a constitutively active ALK stabilise the liquid droplets even in the presence of TKIs, indicating that droplets do not depend on kinase activity per se. Uniquely, the TKI alectinib promotes droplet formation of both the wild-type and catalytically inactive EML4-ALK V3 mutant, but not in a mutant that disrupts a hallmark of the kinase activity, the Lys-Glu salt-bridge. We propose that EML4-ALK V3 liquid droplet formation occurs through transient dimerization of the ALK kinase domain in its active conformation in the context of stable EML4-ALK trimers. Our results provide insights into the relationship between ALK activity, conformational state and the sub-cellular localisation of EML4-ALK V3 protein, and reveal the different effects of structurally divergent ALK TKIs on these properties.
Published: 2020
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40. 2-Arylamino-6-ethynylpurines are cysteine-targeting irreversible inhibitors of Nek2 kinase

Author: Kathy Boxall, Mangaleswaran Sivaprakasam, David P. Turner, Roger J. Griffin, Corine Mas-Droux, Benoit Carbain, Bernard T. Golding, Richard Bayliss, Ian R. Hardcastle, Mark W. Richards, Andrew M. Fry, Christopher R. Coxon, David R. Newell, Celine Cano, Suzannah J. Harnor, and Christopher J. Matheson
Subjects: Pharmacology, Alanine, 0303 health sciences, 010405 organic chemistry, Stereochemistry, Organic Chemistry, Substituent, Pharmaceutical Science, macromolecular substances, 01 natural sciences, Biochemistry, 0104 chemical sciences, Chemistry, 03 medical and health sciences, chemistry.chemical_compound, chemistry, Covalent bond, Drug Discovery, Alkoxy group, Molecular Medicine, Structure–activity relationship, Growth inhibition, Acetamide, 030304 developmental biology, Cysteine
Abstract: Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers., Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-cyclohexylmethoxy-7H-purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl-N-phenyl-7H-purin-2-amine [IC50 0.15 μM (Nek2)] and 4-((6-ethynyl-7H-purin-2-yl)amino)benzenesulfonamide (IC50 0.14 μM) were selected for determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group by ethyl or cyano abrogated activity. Variation of substituents on the N-phenyl moiety for 6-ethynylpurines gave further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent, indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide [IC50 0.06 μM (Nek2); GI50 (SKBR3) 2.2 μM] which exhibited >5–10-fold selectivity for Nek2 over other kinases; it also showed > 50% growth inhibition at 10 μM concentration against selected breast and leukaemia cell lines. X-ray crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9H-purin-2-yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no inhibition of hERG at 25 μM and a good stability profile in human liver microsomes. It is concluded that 6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2.
Published: 2020

41. Solution NMR assignment of the C-terminal domain of human chTOG

Author: Selena G. Burgess, Mark Pfuhl, Richard Bayliss, and Elena Rostkova
Subjects: 0301 basic medicine, CKAP5, Mitosis, Cell cycle, Biochemistry, Clathrin, Article, Domain (software engineering), ChTOG, 03 medical and health sciences, Protein Domains, Structural Biology, Microtubule, Humans, Nuclear Magnetic Resonance, Biomolecular, Ternary complex, biology, Chemistry, Kinetochore, C-terminus, Cell biology, Solutions, TACC3, 030104 developmental biology, biology.protein, Microtubule-Associated Proteins, Heteronuclear single quantum coherence spectroscopy
Abstract: The microtubule regulatory protein colonic and hepatic tumor overexpressed gene (chTOG), also known as cytoskeleleton associated protein 5 (CKAP5) plays an important role in organizing the cytoskeleton and in particular in the assembly of k-fibres in mitosis. Recently, we dissected the hitherto poorly understood C-terminus of this protein by discovering two new domains—a cryptic TOG domain (TOG6) and a smaller, helical domain at the very C-terminus. It was shown that the C-terminal domain is important for the interaction with the TACC domain in TACC3 during the assembly of k-fibres in a ternary complex that also includes clathrin. Here we now present the solution NMR assignment of the chTOG C-terminal domain which confirms our earlier prediction that it is mainly made of α-helices. However, the appearance of the 1H–15N HSQC spectrum is indicative of the presence of a considerable amount of unstructured and possibly flexible portions of protein in the domain.
Published: 2018
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42. Hsp72 and Nek6 Cooperate to Cluster Amplified Centrosomes in Cancer Cells

Author: Martin J. S. Dyer, Laura O'Regan, Andrew M. Fry, Richard Bayliss, and Josephina Sampson
Subjects: 0301 basic medicine, Cancer Research, Mitosis, Apoptosis, Breast Neoplasms, Cell Cycle Proteins, HSP72 Heat-Shock Proteins, Centrosome cycle, Spindle Apparatus, Protein Serine-Threonine Kinases, Biology, PLK1, Chromosome segregation, Mice, Neuroblastoma, 03 medical and health sciences, Proto-Oncogene Proteins, Tumor Cells, Cultured, Animals, Humans, NIMA-Related Kinases, Mitotic catastrophe, Aurora Kinase A, Cell Proliferation, Centrosome, Cell growth, Cell biology, 030104 developmental biology, Oncology, Cancer cell, Female, Multipolar spindles
Abstract: Cancer cells frequently possess extra amplified centrosomes clustered into two poles whose pseudo-bipolar spindles exhibit reduced fidelity of chromosome segregation and promote genetic instability. Inhibition of centrosome clustering triggers multipolar spindle formation and mitotic catastrophe, offering an attractive therapeutic approach to selectively kill cells with amplified centrosomes. However, mechanisms of centrosome clustering remain poorly understood. Here, we identify a new pathway that acts through NIMA-related kinase 6 (Nek6) and Hsp72 to promote centrosome clustering. Nek6, as well as its upstream activators polo-like kinase 1 and Aurora-A, targeted Hsp72 to the poles of cells with amplified centrosomes. Unlike some centrosome declustering agents, blocking Hsp72 or Nek6 function did not induce formation of acentrosomal poles, meaning that multipolar spindles were observable only in cells with amplified centrosomes. Inhibition of Hsp72 in acute lymphoblastic leukemia cells resulted in increased multipolar spindle frequency that correlated with centrosome amplification, while loss of Hsp72 or Nek6 function in noncancer-derived cells disturbs neither spindle formation nor mitotic progression. Hence, the Nek6–Hsp72 module represents a novel actionable pathway for selective targeting of cancer cells with amplified centrosomes. Cancer Res; 77(18); 4785–96. ©2017 AACR.
Published: 2017
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43. Switching Aurora-A kinase on and off at an allosteric site

Author: Richard Bayliss, Patrick J. McIntyre, and Selena G. Burgess
Subjects: Models, Molecular, Protein Conformation, alpha-Helical, 0301 basic medicine, Amino Acid Motifs, Allosteric regulation, Cell Cycle Proteins, Crystallography, X-Ray, Biochemistry, SH3 domain, Substrate Specificity, MAP2K7, 03 medical and health sciences, 0302 clinical medicine, Allosteric Regulation, Humans, Protein Interaction Domains and Motifs, c-Raf, Phosphorylation, Protein kinase A, Molecular Biology, Aurora Kinase A, Binding Sites, biology, Cyclin-dependent kinase 2, Nuclear Proteins, Cell Biology, Single-Domain Antibodies, Protein Structure, Tertiary, Cell biology, Kinetics, 030104 developmental biology, biology.protein, Cyclin-dependent kinase complex, Protein Conformation, beta-Strand, Casein kinase 2, Peptides, Microtubule-Associated Proteins, Protein Processing, Post-Translational, Allosteric Site, 030217 neurology & neurosurgery, Protein Binding
Abstract: Protein kinases are central players in the regulation of cell cycle and signalling pathways. Their catalytic activities are strictly regulated through post-translational modifications and protein-protein interactions that control switching between inactive and active states. These states have been studied extensively using protein crystallography, although the dynamic nature of protein kinases makes it difficult to capture all relevant states. Here, we describe two recent structures of Aurora-A kinase that trap its active and inactive states. In both cases, Aurora-A is trapped through interaction with a synthetic protein, either a single-domain antibody that inhibits the kinase or a hydrocarbon-stapled peptide that activates the kinase. These structures show how the distinct synthetic proteins target the same allosteric pocket with opposing effects on activity. These studies pave the way for the development of tools to probe these allosteric mechanisms in cells.
Published: 2017
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44. Differential protein stability and clinical responses ofEML4-ALK fusion variants to various ALK inhibitors in advancedALK-rearranged non-small cell lung cancer

Author: K.S. Park, C.G. Woo, Sun Whe Kim, Mark W. Richards, J.Y. Song, Richard Bayliss, S.J. Jang, Jene Choi, B. Lee, D.H. Lee, and S. Seo
Subjects: Male, 0301 basic medicine, Alectinib, Lung Neoplasms, Oncogene Proteins, Fusion, Pyridines, Kaplan-Meier Estimate, Bioinformatics, 0302 clinical medicine, Piperidines, Carcinoma, Non-Small-Cell Lung, hemic and lymphatic diseases, Genotype, Anaplastic lymphoma kinase, Sulfones, In Situ Hybridization, Fluorescence, Protein Stability, Hematology, Middle Aged, Oncology, 030220 oncology & carcinogenesis, Female, medicine.drug, Adult, medicine.drug_class, Carbazoles, Antineoplastic Agents, Disease-Free Survival, Article, 03 medical and health sciences, Crizotinib, medicine, Humans, Progression-free survival, Lung cancer, Protein Kinase Inhibitors, Aged, Retrospective Studies, Ceritinib, business.industry, medicine.disease, ALK inhibitor, Pyrimidines, 030104 developmental biology, Drug Resistance, Neoplasm, Cancer research, Pyrazoles, business, Multiplex Polymerase Chain Reaction
Abstract: Background Anaplastic lymphoma kinase (ALK) inhibition using crizotinib has become the standard of care in advancedALK-rearranged non-small cell lung cancer (NSCLC), but the treatment outcomes and duration of response vary widely. Echinoderm microtubule-associated protein-like 4 (EML4)-ALK is the most common translocation, and the fusion variants show different sensitivity to crizotinibin vitro. However, there are only limited data on the specificEML4-ALK variants and clinical responses of patients to various ALK inhibitors. Patients and methods By multiplex reverse-transcriptase PCR, which detects 12 variants of knownEML4-ALK rearrangements, we retrospectively determinedALK fusion variants in 54 advancedALK rearrangement-positive NSCLCs. We subdivided the patients into two groups (variants 1/2/others and variants 3a/b) by protein stability and evaluated correlations of the variant status with clinical responses to crizotinib, alectinib, or ceritinib. Moreover, we established the EML4-ALK variant-expressing system and analyzed patterns of sensitivity of the variants to ALK inhibitors. Results Of the 54 tumors analyzed,EML4-ALK variants 3a/b (44.4%) was the most common type, followed by variants 1 (33.3%) and 2 (11.1%). The 2-year progression-free survival (PFS) rate was 76.0% [95% confidence interval (CI) 56.8–100] in groupEML4-ALK variants 1/2/others versus 26.4% (95% CI 10.5–66.6) in group variants 3a/b (P = 0.034) among crizotinib-treated patients. Meanwhile, the 2-year PFS rate was 69.0% (95% CI 49.9–95.4) in group variants 1/2/others versus 32.7% (95% CI 15.6–68.4) in group variants 3a/b (P = 0.108) among all crizotinib-, alectinib-, and ceritinib-treated patients. Variant 3a- or 5a-harboring cells were resistant to ALK inhibitors with >10-fold higher half maximal inhibitory concentrationin vitro. Conclusion Our findings show that groupEML4-ALK variants 3a/b may be a major source of ALK inhibitor resistance in the clinic. The variant-specific genotype of theEML4-ALK fusion allows for more precise stratification of patients with advanced NSCLC.
Published: 2017
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45. New tools for evaluating protein tyrosine sulfation and carbohydrate sulfation

Author: Sharon Yeoh and Richard Bayliss
Subjects: 0301 basic medicine, Tyrosine sulfation, Glycan, enzymology, Carbohydrates, Biochemistry, 03 medical and health sciences, Sulfation, chemical probes, Commentaries, Animals, Humans, Tyrosine, Molecular Biology, Nuclear Magnetic Resonance, Biomolecular, chemistry.chemical_classification, biology, Chemistry, Kinase, sulfation, Cell Biology, Carbohydrate, 030104 developmental biology, Enzyme, biology.protein, Commentary, Sulfotransferases, Signalling pathways
Abstract: Sulfation is a common modification of extracelluar glycans and tyrosine residues on proteins, which is important in many signalling pathways and interactions. Existing methods for studying sulfotransferases, the enzymes that catalyse sulfation, are cumbersome and low-throughput. Recent studies published in the Biochemical Journal have repurposed established biochemical assays from the kinase field and applied them to the characterisation of sulfotransferases. Biochemical screening of a library of kinase inhibitors revealed that compounds that target RAF kinases may also be repurposed to inhibit sulfotransferases. Together with the available structures of sulfotransferases, these studies open the door to the development of chemical tools to probe the biological functions of these important enzymes.
Published: 2018

46. EML4-ALK V3 oncogenic fusion proteins promote microtubule stabilization and accelerated migration through NEK9 and NEK7

Author: Chang Gok Woo, Hui Jeong Jeong, Richard Bayliss, Giancarlo Barone, Jene Choi, Spencer J. Collis, Patricia A.J. Muller, Dean A. Fennell, Emily L. Richardson, Rozita Adib, Laura O'Regan, Mark W. Richards, and Andrew M. Fry
Subjects: Lung Neoplasms, Oncogene Proteins, Fusion, Cell, Biology, Cell morphology, Microtubules, Metastasis, 03 medical and health sciences, 0302 clinical medicine, Microtubule, hemic and lymphatic diseases, Carcinoma, Non-Small-Cell Lung, medicine, Humans, NIMA-Related Kinases, 030304 developmental biology, 0303 health sciences, Kinase, Receptor Protein-Tyrosine Kinases, Cell migration, Cell Biology, medicine.disease, Fusion protein, Cell biology, medicine.anatomical_structure, Cytoplasm, 030220 oncology & carcinogenesis, Research Article
Abstract: EML4–ALK is an oncogenic fusion present in ∼5% of non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants of EML4–ALK with different patient outcomes. Here, we show that, in cell models, EML4–ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. EML4–ALK V3 also recruits the NEK9 and NEK7 kinases to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but does not require ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4–ALK patients. Hence, we propose that EML4–ALK V3 promotes microtubule stabilization through NEK9 and NEK7, leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4–ALK lung cancer.
Published: 2019

47. The journey of Zika to the developing brain

Author: Francesca Rombi, Richard Bayliss, Sharon Yeoh, and Andrew Tuplin
Subjects: 0301 basic medicine, medicine.medical_specialty, Microcephaly, Brain development, Virus, Zika virus, Disease Outbreaks, 03 medical and health sciences, 0302 clinical medicine, Pregnancy, Genetics, medicine, Humans, Molecular Biology, biology, Transmission (medicine), business.industry, Zika Virus Infection, Public health, Outbreak, Brain, Infant, General Medicine, Zika Virus, biology.organism_classification, medicine.disease, Virology, Flavivirus, 030104 developmental biology, Female, Public Health, business, 030217 neurology & neurosurgery, Brazil
Abstract: Zika virus is a mosquito-borneFlavivirusoriginally isolated from humans in 1952. Following its re-emergence in Brazil in 2015, an increase in the number of babies born with microcephaly to infected mothers was observed. Microcephaly is a neurodevelopmental disorder, characterised phenotypically by a smaller than average head size, and is usually developed in utero.The 2015 outbreak in the Americas led to the World Health Organisation declaring Zika a Public Health Emergency of International Concern. Since then, much research into the effects of Zika has been carried out. Studies have investigated the structure of the virus, its effects on and evasion of the immune response, cellular entry including target receptors, its transmission from infected mother to foetus and its cellular targets. This review discusses current knowledge and novel research into these areas, in hope of developing a further understanding of how exposure of pregnant women to the Zika virus can lead to impaired brain development of their foetus. Although no longer considered an epidemic in the Americas, the mechanism by which Zika acts is still not comprehensively and wholly understood, and this understanding will be crucial in developing effective vaccines and treatments.
Published: 2019

48. Construction of a shape-diverse fragment set: design, synthesis and screen against aurora-A kinase

Author: Rong Zhang, Patrick J. McIntyre, Patrick M. Collins, Daniel J. Foley, Christopher Arter, Frank von Delft, Richard Bayliss, Stuart Warriner, and Adam Nelson
Subjects: Binding Sites, Organic Chemistry, General Chemistry, respiratory system, Crystallography, X-Ray, Catalysis, Protein Structure, Tertiary, Molecular Docking Simulation, Allosteric Regulation, Drug Design, Protein Kinase Inhibitors, human activities, Databases, Chemical, Aurora Kinase A, Protein Binding
Abstract: Historically, chemists have explored chemical space in a highly uneven and unsystematic manner. As an example, the shape diversity of existing fragment sets does not generally reflect that of all theoretically possible fragments. To assess experimentally the added value of increased three dimensionality, a shape-diverse fragment set was designed and collated. The set was assembled by both using commercially available fragments and harnessing unified synthetic approaches to sp3 -rich molecular scaffolds. The resulting set of 80 fragments was highly three-dimensional, and its shape diversity was significantly enriched by twenty synthesised fragments. The fragment set was screened by high-throughput protein crystallography against Aurora-A kinase, revealing four hits that targeted the binding site of allosteric regulators. In the longer term, it is envisaged that the fragment set could be screened against a range of functionally diverse proteins, allowing the added value of more shape-diverse screening collections to be more fully assessed.
Published: 2019

49. Structural basis of N-Myc binding by Aurora-A and its destabilization by kinase inhibitors

Author: Selena G. Burgess, Mark W. Richards, Anne Carstensen, Evon Poon, Martin Eilers, Richard Bayliss, and Louis Chesler
Subjects: 0301 basic medicine, macromolecular substances, Crystallography, X-Ray, 03 medical and health sciences, chemistry.chemical_compound, Ubiquitin, Catalytic Domain, Neoplasms, Humans, Phosphorylation, Polyubiquitin, Protein kinase A, Protein Kinase Inhibitors, Aurora Kinase A, N-Myc Proto-Oncogene Protein, Binding Sites, SKP Cullin F-Box Protein Ligases, Multidisciplinary, biology, Kinase, Phenylurea Compounds, Azepines, Biological Sciences, Molecular biology, 3. Good health, Ubiquitin ligase, Cell biology, enzymes and coenzymes (carbohydrates), A-site, Pyrimidines, 030104 developmental biology, chemistry, Alisertib, biology.protein, N-Myc, Protein Binding
Abstract: Myc family proteins promote cancer by inducing widespread changes in gene expression. Their rapid turnover by the ubiquitin–proteasome pathway is regulated through phosphorylation of Myc Box I and ubiquitination by the E3 ubiquitin ligase SCFFbxW7. However, N-Myc protein (the product of the MYCN oncogene) is stabilized in neuroblastoma by the protein kinase Aurora-A in a manner that is sensitive to certain Aurora-A–selective inhibitors. Here we identify a direct interaction between the catalytic domain of Aurora-A and a site flanking Myc Box I that also binds SCFFbxW7. We determined the crystal structure of the complex between Aurora-A and this region of N-Myc to 1.72-A resolution. The structure indicates that the conformation of Aurora-A induced by compounds such as alisertib and CD532 is not compatible with the binding of N-Myc, explaining the activity of these compounds in neuroblastoma cells and providing a rational basis for the design of cancer therapeutics optimized for destabilization of the complex. We also propose a model for the stabilization mechanism in which binding to Aurora-A alters how N-Myc interacts with SCFFbxW7 to disfavor the generation of Lys48-linked polyubiquitin chains.
Published: 2016
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50. Structure of a Potential Therapeutic Antibody Bound to Interleukin-16 (IL-16)

Author: Kovilen Sawmynaden, Mark D. Carr, Gareth Hall, Debra L. Taylor, Eilish Cullen, Richard Cowan, Richard Bayliss, Simon Fox, Catherine A. Kettleborough, Frederick W. Muskett, William W. Cruikshank, David Matthews, Andrew Merritt, and Joanne Arnold
Subjects: 0301 basic medicine, biology, medicine.drug_class, Protein domain, PDZ domain, Cell Biology, Ligand (biochemistry), Monoclonal antibody, Biochemistry, Cell biology, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, Mechanism of action, biology.protein, medicine, Antibody, medicine.symptom, Binding site, Receptor, Molecular Biology, 030215 immunology
Abstract: Interleukin-16 (IL-16) is reported to be a chemoattractant cytokine and modulator of T-cell activation, and has been proposed as a ligand for the co-receptor CD4. The secreted active form of IL-16 has been detected at sites of TH1-mediated inflammation, such as those seen in autoimmune diseases, ischemic reperfusion injury (IRI), and tissue transplant rejection. Neutralization of IL-16 recruitment to its receptor, using an anti-IL16 antibody, has been shown to significantly attenuate inflammation and disease pathology in IRI, as well as in some autoimmune diseases. The 14.1 antibody is a monoclonal anti-IL-16 antibody, which when incubated with CD4+ cells is reported to cause a reduction in the TH1-type inflammatory response. Secreted IL-16 contains a characteristic PDZ domain. PDZ domains are typically characterized by a defined globular structure, along with a peptide-binding site located in a groove between the αB and βB structural elements and a highly conserved carboxylate-binding loop. In contrast to other reported PDZ domains, the solution structure previously reported for IL-16 reveals a tryptophan residue obscuring the recognition groove. We have solved the structure of the 14.1Fab fragment in complex with IL-16, revealing that binding of the antibody requires a conformational change in the IL-16 PDZ domain. This involves the rotation of the αB-helix, accompanied movement of the peptide groove obscuring tryptophan residue, and consequent opening up of the binding site for interaction. Our study reveals a surprising mechanism of action for the antibody and identifies new opportunities for the development of IL-16-targeted therapeutics, including small molecules that mimic the interaction of the antibody.
Published: 2016
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