I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Chagas disease is a neglected tropical disease (NTD) caused by Trypanosoma cruzi, whilst leishmaniasis, which is caused by over 20 species of Leishmania, represents a group of NTDs endemic to most countries in the tropical and subtropical belt of the planet. These diseases remain a significant health problem both in endemic countries and globally. These parasites and other trypanosomatids, including T. theileri, a bovine pathogen, rely on cysteine biosynthesis for the production of trypanothione, which is essential for parasite survival in hosts. The de novo pathway of cysteine biosynthesis requires the conversion of O-acetyl-L-serine into L-cysteine, which is catalysed by cysteine synthase (CS). These enzymes present potential for drug development against T. cruzi, Leishmania spp. and T. theileri. To enable these possibilities, biochemical and crystallographic studies of CS from T. cruzi (TcCS), L. infantum (LiCS) and T. theileri (TthCS) were conducted. Crystal structures of the three enzymes were determined at resolutions of 1.80 Å for TcCS, 1.75 Å for LiCS and 2.75 Å for TthCS. These three homodimeric structures show the same overall fold and demonstrate that the active-site geometry is conserved, supporting a common reaction mechanism. Detailed structural analysis revealed reaction intermediates of the de novo pathway ranging from an apo structure of LiCS and holo structures of both TcCS and TthCS to the substrate-bound structure of TcCS. These structures will allow exploration of the active site for the design of novel inhibitors. Additionally, unexpected binding sites discovered at the dimer interface represent new potential for the development of protein–protein inhibitors.
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Jun 2023
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I03-Macromolecular Crystallography
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David
Hargreaves
,
Rodrigo J.
Carbajo
,
Michael S.
Bodnarchuk
,
Kevin
Embrey
,
Philip B.
Rawlins
,
Martin
Packer
,
Sébastien L.
Degorce
,
Alexander W.
Hird
,
Jeffrey W.
Johannes
,
Elisabetta
Chiarparin
,
Markus
Schade
Abstract: The structure-based design of small-molecule inhibitors targeting protein–protein interactions (PPIs) remains a huge challenge as the drug must bind typically wide and shallow protein sites. A PPI target of high interest for hematological cancer therapy is myeloid cell leukemia 1 (Mcl-1), a prosurvival guardian protein from the Bcl-2 family. Despite being previously considered undruggable, seven small-molecule Mcl-1 inhibitors have recently entered clinical trials. Here, we report the crystal structure of the clinical-stage inhibitor AMG-176 bound to Mcl-1 and analyze its interaction along with clinical inhibitors AZD5991 and S64315. Our X-ray data reveal high plasticity of Mcl-1 and a remarkable ligand-induced pocket deepening. Nuclear Magnetic Resonance (NMR)-based free ligand conformer analysis demonstrates that such unprecedented induced fit is uniquely achieved by designing highly rigid inhibitors, preorganized in their bioactive conformation. By elucidating key chemistry design principles, this work provides a roadmap for targeting the largely untapped PPI class more successfully.
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May 2023
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Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[21809]
Open Access
Abstract: Lysine acetylation in histone tails is a key post-translational modification that controls transcription activation. Histone deacetylase complexes remove histone acetylation, thereby repressing transcription and regulating the transcriptional output of each gene. Although these complexes are drug targets and crucial regulators of organismal physiology, their structure and mechanisms of action are largely unclear. Here, we present the structure of a complete human SIN3B histone deacetylase holo-complex with and without a substrate mimic. Remarkably, SIN3B encircles the deacetylase and contacts its allosteric basic patch thereby stimulating catalysis. A SIN3B loop inserts into the catalytic tunnel, rearranges to accommodate the acetyl-lysine moiety, and stabilises the substrate for specific deacetylation, which is guided by a substrate receptor subunit. Our findings provide a model of specificity for a main transcriptional regulator conserved from yeast to human and a resource of protein-protein interactions for future drug designs.
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May 2023
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Krios V-Titan Krios V at Diamond
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Shahid
Rehan
,
Dale
Tranter
,
Phillip P.
Sharp
,
Gregory B.
Craven
,
Eric
Lowe
,
Janet L.
Anderl
,
Tony
Muchamuel
,
Vahid
Abrishami
,
Suvi
Kuivanen
,
Nicole A.
Wenzell
,
Andy
Jennings
,
Chakrapani
Kalyanaraman
,
Tomas
Strandin
,
Matti
Javanainen
,
Olli
Vapalahti
,
Matthew P.
Jacobson
,
Dustin
Mcminn
,
Christopher J.
Kirk
,
Juha T.
Huiskonen
,
Jack
Taunton
,
Ville O.
Paavilainen
Open Access
Abstract: Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.
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May 2023
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I03-Macromolecular Crystallography
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Takashi
Miura
,
Tika R.
Malla
,
C. David
Owen
,
Anthony
Tumber
,
Lennart
Brewitz
,
Michael A.
Mcdonough
,
Eidarus
Salah
,
Naohiro
Terasaka
,
Takayuki
Katoh
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Halina
Mikolajek
,
Martin A.
Walsh
,
Akane
Kawamura
,
Christopher J.
Schofield
,
Hiroaki
Suga
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cyclic γ2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ1) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ2), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent Mpro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ1 at the fourth position, manifests a 5.2 nM dissociation constant. An Mpro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ1 interacts with the S1′ catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and Mpro enabled production of a variant with a 5-fold increase in potency.
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May 2023
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[26855]
Open Access
Abstract: Monkeypox virus (MPXV) is a double-stranded DNA virus from the family Poxviridae, which is endemic in West and Central Africa. Various human outbreaks occurred in the 1980s, resulting from a cessation of smallpox vaccination. Recently, MPXV cases have reemerged in non-endemic nations, and the 2022 outbreak has been declared a public health emergency. Treatment optionsare limited, and many countries lack the infrastructure to provide symptomatic treatments. The development of cost-effective antivirals could ease severe health outcomes. G-quadruplexes have been a target of interest in treating viral infections with different chemicals. In the present work, a genomic-scale mapping of different MPXV isolates highlighted two conserved putative quadruplex-forming sequences MPXV-exclusive in 590 isolates. Subsequently, we assessed the G-quadruplex formation using circular dichroism spectroscopy and solution small-angle X-ray scattering. Furthermore, biochemical assays indicated the ability of MPXV quadruplexes to be recognized by two specific G4-binding partners—Thioflavin T and DHX36. Additionally, our work also suggests that a quadruplex binding small-molecule with previously reported antiviral activity, TMPyP4, interacts with MPXV G-quadruplexes with nanomolar affinity in the presence and absence of DHX36. Finally, cell biology experiments suggests that TMPyP4 treatment substantially reduced gene expression of MPXV proteins. In summary, our work provides insights into the G-quadruplexes from the MPXV genome that can be further exploited to develop therapeutics.
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May 2023
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Jack
Scantlebury
,
Lucy
Vost
,
Anna
Carbery
,
Thomas E.
Hadfield
,
Oliver M.
Turnbull
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Nathan
Brown
,
Vijil
Chenthamarakshan
,
Payel
Das
,
Harold
Grosjean
,
Frank
Von Delft
,
Charlotte M.
Deane
Open Access
Abstract: Over the past few years, many machine learning-based scoring functions for predicting the binding of small molecules to proteins have been developed. Their objective is to approximate the distribution which takes two molecules as input and outputs the energy of their interaction. Only a scoring function that accounts for the interatomic interactions involved in binding can accurately predict binding affinity on unseen molecules. However, many scoring functions make predictions based on data set biases rather than an understanding of the physics of binding. These scoring functions perform well when tested on similar targets to those in the training set but fail to generalize to dissimilar targets. To test what a machine learning-based scoring function has learned, input attribution, a technique for learning which features are important to a model when making a prediction on a particular data point, can be applied. If a model successfully learns something beyond data set biases, attribution should give insight into the important binding interactions that are taking place. We built a machine learning-based scoring function that aimed to avoid the influence of bias via thorough train and test data set filtering and show that it achieves comparable performance on the Comparative Assessment of Scoring Functions, 2016 (CASF-2016) benchmark to other leading methods. We then use the CASF-2016 test set to perform attribution and find that the bonds identified as important by PointVS, unlike those extracted from other scoring functions, have a high correlation with those found by a distance-based interaction profiler. We then show that attribution can be used to extract important binding pharmacophores from a given protein target when supplied with a number of bound structures. We use this information to perform fragment elaboration and see improvements in docking scores compared to using structural information from a traditional, data-based approach. This not only provides definitive proof that the scoring function has learned to identify some important binding interactions but also constitutes the first deep learning-based method for extracting structural information from a target for molecule design.
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May 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Grace Q.
Gong
,
Benoit
Bilanges
,
Ben
Allsop
,
Glenn
Masson
,
Victoria
Roberton
,
Trevor
Askwith
,
Sally
Oxenford
,
Ralitsa R.
Madsen
,
Sarah E.
Conduit
,
Dom
Bellini
,
Martina
Fitzek
,
Matt
Collier
,
Osman
Najam
,
Zhenhe
He
,
Ben
Wahab
,
Stephen H.
Mclaughlin
,
A. W. Edith
Chan
,
Isabella
Feierberg
,
Andrew
Madin
,
Daniele
Morelli
,
Amandeep
Bhamra
,
Vanesa
Vinciauskaite
,
Karen E.
Anderson
,
Silvia
Surinova
,
Nikos
Pinotsis
,
Elena
Lopez-Guadamillas
,
Matthew
Wilcox
,
Alice
Hooper
,
Chandni
Patel
,
Maria A.
Whitehead
,
Tom D.
Bunney
,
Len R.
Stephens
,
Phillip T.
Hawkins
,
Matilda
Katan
,
Derek M.
Yellon
,
Sean M.
Davidson
,
David M.
Smith
,
James B.
Phillips
,
Richard
Angell
,
Roger L.
Williams
,
Bart
Vanhaesebroeck
Diamond Proposal Number(s):
[28677]
Abstract: Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia–reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
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May 2023
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Open Access
Abstract: Fragment merging is a promising approach to progressing fragments directly to on-scale potency: each designed compound incorporates the structural motifs of overlapping fragments in a way that ensures compounds recapitulate multiple high-quality interactions. Searching commercial catalogues provides one useful way to quickly and cheaply identify such merges and circumvents the challenge of synthetic accessibility, provided they can be readily identified. Here, we demonstrate that the Fragment Network, a graph database that provides a novel way to explore the chemical space surrounding fragment hits, is well-suited to this challenge. We use an iteration of the database containing >120 million catalogue compounds to find fragment merges for four crystallographic screening campaigns and contrast the results with a traditional fingerprint-based similarity search. The two approaches identify complementary sets of merges that recapitulate the observed fragment–protein interactions but lie in different regions of chemical space. We further show our methodology is an effective route to achieving on-scale potency by retrospective analyses for two different targets; in analyses of public COVID Moonshot and Mycobacterium tuberculosis EthR inhibitors, potential inhibitors with micromolar IC50 values were identified. This work demonstrates the use of the Fragment Network to increase the yield of fragment merges beyond that of a classical catalogue search.
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May 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10619]
Open Access
Abstract: KEAP1 promotes the ubiquitin-dependent degradation of NRF2 by assembling into a CUL3-dependent ubiquitin ligase complex. Oxidative and electrophilic stress inhibit KEAP1 allowing NRF2 to accumulate for the transactivation of stress response genes. To date there are no structures of the KEAP1-CUL3 interaction nor binding data to show the contributions of different domains to their binding affinity. We determined a crystal structure of the BTB and 3-box domains of human KEAP1 in complex with the CUL3 N-terminal domain that showed a heterotetrameric assembly with 2:2 stoichiometry. To support the structural data, we developed a versatile TR-FRET-based assay system to profile the binding of BTB-domain-containing proteins to CUL3 and determine the contribution of distinct protein features, revealing the importance of the CUL3 N-terminal extension for high affinity binding. We further provide direct evidence that the investigational drug CDDO does not disrupt the KEAP1-CUL3 interaction, even at high concentrations, but reduces the affinity of KEAP1-CUL3 binding. The TR-FRET-based assay system offers a generalizable platform for profiling this protein class and may form a suitable screening platform for ligands that disrupt these interactions by targeting the BTB or 3-box domains to block E3 ligase function.
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May 2023
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