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Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength)	Free energy perturbation in the design of EED ligands as inhibitors of polycomb repressive complex 2 (PRC2) methyltransferase Daniel H. O' Donovan , Clare Gregson , Martin J. Packer , Ryan Greenwood , Kurt G. Pike , Sameer Kawatkar , Andrew Bloecher , James Robinson , Jon Read , Erin Code , Jessie Hao-Ru Hsu , Minhui Shen , Haley Woods , Peter Barton , Shaun Fillery , Beth Williamson , Philip B. Rawlins , Sharan K. Bagal Bioorganic & Medicinal Chemistry Letters 39 DOI: 10.1016/j.bmcl.2021.127904 Show Abstract ▼ Abstract: Free Energy Perturbation (FEP) calculations can provide high-confidence predictions of the interaction strength between a ligand and its protein target. We sought to explore a series of triazolopyrimidines which bind to the EED subunit of the PRC2 complex as potential anticancer therapeutics, using FEP calculations to inform compound design. Combining FEP predictions with a late-stage functionalisation (LSF) inspired synthetic approach allowed us to rapidly evaluate structural modifications in a previously unexplored region of the EED binding site. This approach generated a series of novel triazolopyrimidine EED ligands with improved physicochemical properties and which inhibit PRC2 methyltransferase activity in a cancer-relevant G401 cell line.	May 2021
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Generating selective leads for Mer kinase inhibitors - example of a comprehensive lead-generation strategy J. Willem M. Nissink , Sana Bazzaz , Carolyn Blackett , Matthew A. Clark , Olga Collingwood , Jeremy S. Disch , Diana Gikunju , Kristin Goldberg , John P. Guilinger , Elizabeth Hardaker , Edward J. Hennessy , Rachael Jetson , Anthony D. Keefe , William Mccoull , Lindsay Mcmurray , Allison Olszewski , Ross Overman , Alexander Pflug , Marian Preston , Philip B. Rawlins , Emma Rivers , Marianne Schimpl , Paul Smith , Caroline Truman , Elizabeth Underwood , Juli Warwicker , Jon Winter-Holt , Simon Woodcock , Ying Zhang Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.0c01904 Show Abstract ▼ Abstract: Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.	Mar 2021
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Dynamics of the HD regulatory subdomain of PARP-1; substrate access and allostery in PARP activation and inhibition Tom E. H. Ogden , Ji-Chun Yang , Marianne Schimpl , Laura E. Easton , Elizabeth Underwood , Philip b. Rawlins , Michael m Mccauley , Marie-France Langelier , John m. Pascal , Kevin j. Embrey , David Neuhaus Nucleic Acids Research 25 DOI: 10.1093/nar/gkab020 Diamond Proposal Number(s): [17180, 20015] Open Access Show Abstract ▼ Abstract: PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1’s F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.	Jan 2021
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-Macromolecular Crystallography	A-loop interactions in Mer tyrosine kinase give rise to inhibitors with two-step mechanism and long residence time of binding Alexander Pflug , Marianne Schimpl , J. Willem M. Nissink , Ross C. Overman , Philip B. Rawlins , Caroline Truman , Elizabeth Underwood , Juli Warwicker , Jon J. Winter-Holt , William Mccoull Biochemical Journal DOI: 10.1042/BCJ20200735 Diamond Proposal Number(s): [14631, 17180, 20015] Open Access Show Abstract ▼ Abstract: The activation loop (A-loop) plays a key role in regulating the catalytic activity of protein kinases. Phosphorylation in this region enhances the phosphoryl transfer rate of the kinase domain and increases its affinity for ATP. Furthermore, the A-loop possesses autoinhibitory functions in some kinases, where it collapses onto the protein surface and blocks substrate binding when unphosphorylated. Due to its flexible nature, the A-loop is usually disordered and untraceable in kinase domain crystal structures. The resulting lack of structural information is regrettable as it impedes the design of drug A-loop contacts, which have proven favourable in multiple cases. Here we characterize the binding with A-loop engagement between type 1.5 kinase inhibitor ‘example 172’ (EX172) and Mer tyrosine kinase (MerTK). With the help of crystal structures and binding kinetics we portray how the recruitment of the A-loop elicits a two-step binding mechanism which results in a drug-target complex characterized by high affinity and long residence time. In addition, the type 1.5 compound possesses excellent kinome selectivity and a remarkable preference for the phosphorylated over the dephosphorylated form of MerTK. We discuss these unique characteristics in the context of known type 1 and type 2 inhibitors and highlight opportunities for future kinase inhibitor design.	Oct 2020
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Antibody fragments structurally enable a drug-discovery campaign on the cancer target Mcl-1 Jakub Luptak , Michal Bista , David Fisher , Liz Flavell , Ning Gao , Kate Wickson , Steven L. Kazmirski , Tina Howard , Philip B. Rawlins , David Hargreaves Acta Crystallographica Section D Structural Biology 75, 1003 - 1014 DOI: 10.1107/S2059798319014116 Open Access Show Abstract ▼ Abstract: Apoptosis is a crucial process by which multicellular organisms control tissue growth, removal and inflammation. Disruption of the normal apoptotic function is often observed in cancer, where cell death is avoided by the overexpression of anti-apoptotic proteins of the Bcl-2 (B-cell lymphoma 2) family, including Mcl-1 (myeloid cell leukaemia 1). This makes Mcl-1 a potential target for drug therapy, through which normal apoptosis may be restored by inhibiting the protective function of Mcl-1. Here, the discovery and biophysical properties of an anti-Mcl-1 antibody fragment are described and the utility of both the scFv and Fab are demonstrated in generating an Mcl-1 crystal system amenable to iterative structure-guided drug design.	Nov 2019
I04-Macromolecular Crystallography	Discovery of a potent and selective oral inhibitor of ERK1/2 (AZD0364) that is efficacious in both monotherapy and combination therapy in models of non-small cell lung cancer (NSCLC) Richard A Ward , Mark J Anderton , Paul A. Bethel , Jason Breed , Calum Cook , Emma Davies , Andrew Dobson , Zhiqiang Dong , Gary Fairley , Paul Farrington , Lyman J. Feron , Vikki Flemington , Francis D Gibbons , Mark A. Graham , Ryan David Robert Greenwood , Lyndsey Hanson , Philip Hopcroft , Rachel Howells , Julian Hudson , Michael James , Clifford D. Jones , Christopher Jones , Yongchao Li , Scott Lamont , Richard James Lewis , Nicola A. Lindsay , James Francis Mccabe , Thomas M. Mcguire , Philip B. Rawlins , Karen Roberts , Linda Sandin , Iain Simpson , Steve Swallow , Jia Tang , Gary Tomkinson , Michael Tonge , Zhenhua Wang , Baochang Zhai Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.9b01295 Diamond Proposal Number(s): [20015, 17180] Show Abstract ▼ Abstract: The RAS/MAPK pathway is a major driver of oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in the BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the clinical responses observed through the use of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma, however resistance frequently develops. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. We describe our structure-based design approach leading to the discovery of AZD0364, a potent and selective inhibitor of ERK1 and ERK2. AZD0364 exhibits high cellular potency (IC50 = 6 nM) and excellent physico-chemical and absorption, distribution, metabolism, and excretion (ADME) properties and has demonstrated encouraging anti-tumour activity in pre-clinical models.	Nov 2019
I04-1-Macromolecular Crystallography (fixed wavelength)	Rapid identification of novel allosteric PRC2 inhibitors Jon A. Read , Jonathan Tart , Philip B. Rawlins , Clare Gregson , Karen Jones , Ning Gao , Xiahui Zhu , Ron Tomlinson , Erin Code , Tony Cheung , Huawei Chen , Sameer P. Kawatkar , Andy Bloecher , Sharan Bagal , Daniel H. O’donovan , James Robinson Acs Chemical Biology DOI: 10.1021/acschembio.9b00468 Show Abstract ▼ Abstract: Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), regulates chromatin state and gene expression by methylating histone H3 lysine 27. EZH2 is overexpressed or mutated in various hematological malignancies and solid cancers. Our previous efforts to identify inhibitors of PRC2 methyltransferase activity by high-throughput screening (HTS) resulted in large numbers of false positives and thus a significant hit deconvolution challenge. More recently, others have reported compounds that bind to another PRC2 core subunit, EED, and allosterically inhibit EZH2 activity. This mechanism is particularly appealing as it appears to retain potency in cell lines that have acquired resistance to orthosteric EZH2 inhibition. By designing a fluorescence polarization probe based on the reported EED binding compounds, we were able to quickly and cleanly re-triage our previously challenging HTS hit list and identify novel allosteric PRC2 inhibitors.	Sep 2019
I03-Macromolecular Crystallography	Structure based design of non-natural peptidic macrocyclic Mcl-1 inhibitors Jeffrey W. Johannes , Stephanie Bates , Carl Beigie , Matthew A. Belmonte , John Breen , Shenggen Cao , Paolo A. Centrella , Matthew A. Clark , John W. Cuozzo , Christoph E. Dumelin , Andrew D. Ferguson , Sevan Habeshian , David Hargreaves , Camil Joubran , Steven Kazmirski , Anthony D. Keefe , Michelle L. Lamb , Haiye Lan , Yunxia Li , Hao Ma , Scott Mlynarski , Martin J. Packer , Philip B. Rawlins , Daniel W. Robbins , Haidong Shen , Eric A. Sigel , Holly H. Soutter , Nancy Su , Dawn M. Troast , Haiyun Wang , Kate F. Wickson , Chengyan Wu , Ying Zhang , Qiuying Zhao , Xiaolan Zheng , Alexander W. Hird Acs Medicinal Chemistry Letters 8, 239 - 244 DOI: 10.1021/acsmedchemlett.6b00464 Show Abstract ▼ Abstract: Mcl-1 is a pro-apoptotic BH3 protein family member similar to Bcl-2 and Bcl-xL. Overexpression of Mcl-1 is often seen in various tumors and allows cancer cells to evade apoptosis. Here we report the discovery and optimization of a series of non-natural peptide Mcl-1 inhibitors. Screening of DNA-encoded libraries resulted in hit compound 1, a 1.5 μM Mcl-1 inhibitor. A subsequent crystal structure demonstrated that compound 1 bound to Mcl-1 in a β-turn conformation, such that the two ends of the peptide were close together. This proximity allowed for the linking of the two ends of the peptide to form a macrocycle. Macrocyclization resulted in an approximately 10-fold improvement in binding potency. Further exploration of a key hydrophobic interaction with Mcl-1 protein and also with the moiety that engages Arg256 led to additional potency improvements. The use of protein–ligand crystal structures and binding kinetics contributed to the design and understanding of the potency gains. Optimized compound 26 is a <3 nM Mcl-1 inhibitor, while inhibiting Bcl-2 at only 5 μM and Bcl-xL at >99 μM, and induces cleaved caspase-3 in MV4–11 cells with an IC50 of 3 μM after 6 h.	Feb 2017

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