I24-Microfocus Macromolecular Crystallography
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Andrew D.
Cansfield
,
Mark A.
Ator
,
Joydeep
Banerjee
,
Michael
Bestwick
,
Andrea
Bortolato
,
Giles A.
Brown
,
Jason
Brown
,
Kristina
Butkovic
,
Julie E.
Cansfield
,
John A.
Christopher
,
Miles
Congreve
,
Gabriella
Cseke
,
Francesca
Deflorian
,
Benjamin
Dugan
,
Martina Petrovic
Hunjadi
,
Antun
Hutinec
,
Trinadh Kumar
Inturi
,
Goran
Landek
,
Jonathan
Mason
,
Alistair
O’brien
,
Gregory R.
Ott
,
Renata
Rupcic
,
Gordon
Saxty
,
Stacey M.
Southall
,
Rahela
Zadravec
,
Stephen P.
Watson
Abstract: A series of macrocyclic calcitonin gene-related peptide (CGRP) receptor antagonists identified using structure-based design principles, exemplified by HTL0028016 (1) and HTL0028125 (2), is described. Structural characterization by X-ray crystallography of the interaction of two of the macrocycle antagonists with the CGRP receptor ectodomain is described, along with structure–activity relationships associated with point changes to the macrocyclic antagonists. The identification of non-peptidic/natural product-derived, macrocyclic ligands for a G protein coupled receptor (GPCR) is noteworthy.
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Mar 2022
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I24-Microfocus Macromolecular Crystallography
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Alastair J. H.
Brown
,
Sophie J.
Bradley
,
Fiona H.
Marshall
,
Giles A.
Brown
,
Kirstie A.
Bennett
,
Jason
Brown
,
Julie E.
Cansfield
,
David M.
Cross
,
Chris
De Graaf
,
Brian D.
Hudson
,
Louis
Dwomoh
,
João M.
Dias
,
James C.
Errey
,
Edward
Hurrell
,
Jan
Liptrot
,
Giulio
Mattedi
,
Colin
Molloy
,
Pradeep J.
Nathan
,
Krzysztof
Okrasa
,
Greg
Osborne
,
Jayesh C.
Patel
,
Mark
Pickworth
,
Nathan
Robertson
,
Shahram
Shahabi
,
Christoffer
Bundgaard
,
Keith
Phillips
,
Lisa M.
Broad
,
Anushka V.
Goonawardena
,
Stephen R.
Morairty
,
Michael
Browning
,
Francesca
Perini
,
Gerard R.
Dawson
,
John F. W.
Deakin
,
Robert T.
Smith
,
Patrick M.
Sexton
,
Julie
Warneck
,
Mary
Vinson
,
Tim
Tasker
,
Benjamin G.
Tehan
,
Barry
Teobald
,
Arthur
Christopoulos
,
Christopher J.
Langmead
,
Ali
Jazayeri
,
Robert M.
Cooke
,
Prakash
Rucktooa
,
Miles S.
Congreve
,
Malcolm
Weir
,
Andrew B.
Tobin
Abstract: Current therapies for Alzheimer’s disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936—a potential candidate for the treatment of memory loss in Alzheimer’s disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.
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Nov 2021
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I24-Microfocus Macromolecular Crystallography
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Alexandra
Borodovsky
,
Christine M
Barbon
,
Yanjun
Wang
,
Minwei
Ye
,
Laura
Prickett
,
Dinesh
Chandra
,
Joseph
Shaw
,
Nanhua
Deng
,
Kris
Sachsenmeier
,
James D.
Clarke
,
Bolan
Linghu
,
Giles A
Brown
,
James
Brown
,
Miles
Congreve
,
Robert K. Y.
Cheng
,
Andrew
Dore
,
Edward
Hurrell
,
Wenlin
Shao
,
Richard
Woessner
,
Corinne
Reimer
,
Lisa
Drew
,
Stephen
Fawell
,
Alwin G
Schuller
,
Deanna A
Mele
Open Access
Abstract: Background: Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated.
Methods: We report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition.
Results: We provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies.
Conclusion: We provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.
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Jul 2020
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I24-Microfocus Macromolecular Crystallography
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Willem
Jespers
,
Gregory
Verdon
,
Jhonny
Azuaje
,
Maria
Majellaro
,
Henrik
Keränen
,
Xerardo
García‐mera
,
Miles
Congreve
,
Francesca
Deflorian
,
Chris
De Graaf
,
Andrei
Zhukov
,
Andrew S.
Dore
,
Jonathan S.
Mason
,
Johan
Åqvist
,
Robert M.
Cooke
,
Eddy
Sotelo
,
Hugo
Gutiérrez‐de‐terán
Diamond Proposal Number(s):
[17185]
Open Access
Abstract: We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X‐ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2AAR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2AAR were experimentally determined and investigated through a cycle of ligand‐FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X‐ray crystallography of the A2AAR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2AAR, an emerging target in immuno‐oncology.
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Jul 2020
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I24-Microfocus Macromolecular Crystallography
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Sarah J.
Bucknell
,
Mark A.
Ator
,
Alastair
Brown
,
Jason
Brown
,
Andrew
Cansfield
,
Julie
Cansfield
,
John
Christopher
,
Miles
Congreve
,
Gabriella
Cseke
,
Francesca
Deflorian
,
Christopher
Jones
,
Jonathan S.
Mason
,
Alistair
O'Brien
,
Gregory R.
Ott
,
Mark
Pickworth
,
Stacey
Southall
Diamond Proposal Number(s):
[14637, 23280]
Abstract: Structure-based drug design enabled the discovery of 8, HTL22562, a CGRP receptor antagonist. The structure of 8 complexed with the CGRP receptor was determined at 1.6 Å resolution. Compound 8 is a highly potent, selective, metabolically stable and soluble compound suitable for a range of administration routes that have the potential to provide rapid systemic exposures with resultant high levels of receptor coverage (e.g. subcutaneous). The low lipophilicity coupled with a low anticipated clinically efficacious plasma exposure for migraine also suggests a reduced potential for hepatotoxicity. These properties have led to 8 being taken forward as a clinical candidate for acute treatment of migraine.
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Jun 2020
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I24-Microfocus Macromolecular Crystallography
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Mathieu
Rappas
,
Ammar
Ali
,
Kirstie A.
Bennett
,
Jason D.
Brown
,
Sarah J.
Bucknell
,
Miles
Congreve
,
Robert M.
Cooke
,
Gabriella
Cseke
,
Chris
De Graaf
,
Andrew S.
Dore
,
James C.
Errey
,
Ali
Jazayeri
,
Fiona H.
Marshall
,
Jonathan S.
Mason
,
Richard
Mould
,
Jayesh C.
Patel
,
Benjamin G.
Tehan
,
Malcolm
Weir
,
John A.
Christopher
Diamond Proposal Number(s):
[5788, 20025, 12425, 5999, 5788, 17185, 20025]
Abstract: The orexin system, which consists of the two G protein-coupled receptors OX1 and OX2, activated by the neuropeptides OX-A and OX-B, is firmly established as a key regulator of behavioural arousal, sleep and wakefulness, and has been an area of intense research effort over the past two decades. X-ray structures of the receptors in complex with ten new antagonist ligands from diverse chemotypes are presented, which complement the existing structural information for the system and highlight the critical importance of lipophilic hotspots and water molecules for these peptidergic GPCR targets. Learnings from the structural information regarding the utility of pharmacophore models and how selectivity between OX1 and OX2 can be achieved are discussed.
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Dec 2019
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I24-Microfocus Macromolecular Crystallography
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John A.
Christopher
,
Zoltán
Orgován
,
Miles
Congreve
,
Andrew S.
Dore
,
James C.
Errey
,
Fiona H.
Marshall
,
Jonathan S.
Mason
,
Krzysztof
Okrasa
,
Prakash
Rucktooa
,
Maria J.
Serrano-Vega
,
György G.
Ferenczy
,
Gyorgy M.
Keseru
Abstract: Two interesting new X-ray structures of negative allosteric modulator (NAM) ligands for the mGlu5 receptor, M-MPEP (3) and fenobam (4), are reported. The new structures show how the binding of the ligands induces different receptor water channel conformations to previously published structures. The structure of fenobam, where a urea replaces the acetylenic linker in M-MPEP and mavoglurant, reveals a binding mode where the ligand is rotated by 180º compared to a previously proposed docking model. The need for multiple ligand structures for accurate GPCR structure-based drug design is demonstrated by the different growing vectors identified for the head groups of M-MPEP and mavoglurant and by the unexpected water mediated receptor interactions of a new chemotype represented by fenobam. The implications of the new structures for ligand design are discussed, with extensive analysis of the energetics of the water networks of both pseudo-apo and bound structures providing a new design strategy for allosteric modulators.
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Feb 2018
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I24-Microfocus Macromolecular Crystallography
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Ali
Jazayeri
,
Mathieu
Rappas
,
Alastair J. H.
Brown
,
James
Kean
,
James C.
Errey
,
Nathan J.
Robertson
,
Cédric
Fiez-Vandal
,
Stephen P.
Andrews
,
Miles
Congreve
,
Andrea
Bortolato
,
Jonathan S.
Mason
,
Asma H.
Baig
,
Iryna
Teobald
,
Andrew S.
Dore
,
Malcolm
Weir
,
Robert M.
Cooke
,
Fiona H.
Marshall
Diamond Proposal Number(s):
[12425]
Abstract: Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To gain insight into the molecular mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helices reveals hallmarks of an active conformation similar to that observed in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.
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May 2017
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I24-Microfocus Macromolecular Crystallography
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Christine
Oswald
,
Mathieu
Rappas
,
James
Kean
,
Andrew
Dore
,
James C.
Errey
,
Kirstie
Bennett
,
Francesca
Deflorian
,
John A.
Christopher
,
Ali
Jazayeri
,
Jonathan S.
Mason
,
Miles
Congreve
,
Robert M.
Cooke
,
Fiona H.
Marshall
Diamond Proposal Number(s):
[14637]
Abstract: Chemokines and their G-protein-coupled receptors play a diverse role in immune defence by controlling the migration, activation and survival of immune cells. They are also involved in viral entry, tumour growth and metastasis and hence are important drug targets in a wide range of diseases. Despite very significant efforts by the pharmaceutical industry to develop drugs, with over 50 small-molecule drugs directed at the family entering clinical development, only two compounds have reached the market: maraviroc (CCR5) for HIV infection and plerixafor (CXCR4) for stem-cell mobilization4. The high failure rate may in part be due to limited understanding of the mechanism of action of chemokine antagonists and an inability to optimize compounds in the absence of structural information. CC chemokine receptor type 9 (CCR9) activation by CCL25 plays a key role in leukocyte recruitment to the gut and represents a therapeutic target in inflammatory bowel disease6. The selective CCR9 antagonist vercirnon progressed to phase 3 clinical trials in Crohn’s disease but efficacy was limited, with the need for very high doses to block receptor activation6. Here we report the crystal structure of the CCR9 receptor in complex with vercirnon at 2.8 Å resolution. Remarkably, vercirnon binds to the intracellular side of the receptor, exerting allosteric antagonism and preventing G-protein coupling. This binding site explains the need for relatively lipophilic ligands and describes another example of an allosteric site on G-protein-coupled receptors that can be targeted for drug design, not only at CCR9, but potentially extending to other chemokine receptors.
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Dec 2016
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I24-Microfocus Macromolecular Crystallography
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Abstract: Comparisons between structures of the β1-adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser5.46, coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tert-butylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of β1AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both β1AR and β2AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey β1AR and an inverse agonist of human β2AR. The structure of 7-methylcyanopindolol–bound β1AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound β1AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol–bound β1AR and the hydroxyl group of Ser5.46 is positioned 0.8 Å further from the ligand, with respect to the position of the Ser5.46 side chain in cyanopindolol-bound β1AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists.
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Oct 2015
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