I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13587]
Open Access
Abstract: Isoelectronic metal fluoride transition state analogue (TSA) complexes, MgF3– and AlF4–, have proven to be immensely useful in understanding mechanisms of biological motors utilizing phosphoryl transfer. Here we report a previously unobserved octahedral TSA complex, MgF3(H2O)−, in a 1.5 Å resolution Zika virus NS3 helicase crystal structure. 19F NMR provided independent validation and also the direct observation of conformational tightening resulting from ssRNA binding in solution. The TSA stabilizes the two conformations of motif V of the helicase that link ATP hydrolysis with mechanical work. DFT analysis further validated the MgF3(H2O)− species, indicating the significance of this TSA for studies of biological motors.
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Feb 2021
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I02-Macromolecular Crystallography
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Open Access
Abstract: Structure-guided vaccine design provides a route to elicit a focused immune response against the most functionally important regions of a pathogen surface. This can be achieved by identifying epitopes for neutralizing antibodies through structural methods and recapitulating these epitopes by grafting their core structural features onto smaller scaffolds. In this study, we conducted a modified version of this protocol. We focused on the PfEMP1 protein family found on the surfaces of erythrocytes infected with Plasmodium falciparum. A subset of PfEMP1 proteins bind to endothelial protein C receptor (EPCR), and their expression correlates with development of the symptoms of severe malaria. Structural studies revealed that PfEMP1 molecules present a helix-kinked-helix motif that forms the core of the EPCR-binding site. Using Rosetta-based design, we successfully grafted this motif onto a three-helical bundle scaffold. We show that this synthetic binder interacts with EPCR with nanomolar affinity and adopts the expected structure. We also assessed its ability to bind to antibodies found in immunized animals and in humans from malaria-endemic regions. Finally, we tested the capacity of the synthetic binder to effectively elicit antibodies that prevent EPCR binding and analyzed the degree of cross-reactivity of these antibodies across a diverse repertoire of EPCR-binding PfEMP1 proteins. Despite our synthetic binder adopting the correct structure, we find that it is not as effective as the CIDRα domain on which it is based for inducing adhesion-inhibitory antibodies. This cautions against the rational design of focused immunogens that contain the core features of a ligand-binding site of a protein family, rather than those of a neutralizing antibody epitope.
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Feb 2021
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I02-Macromolecular Crystallography
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Abstract: Anisotropic environments can drastically alter the spectroscopy and photochemistry of molecules, leading to complex structure‐function relationships. We examined this using fluorescent proteins as easy‐to‐modify model systems. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, including the determination of 43 crystal structures. Correlation and principal component analysis confirmed the complex relationship between structure and spectroscopy, but also allowed us to identify consistent trends and to relate these to the spatial organization. We find that changes in spectroscopic properties can come about through multiple underlying mechanisms, of which polarity, hydrogen bonding and presence of water molecules are key modulators. We anticipate that our findings and rich structure/spectroscopy dataset can open opportunities for the development and evaluation of new and existing protein engineering methods.
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Feb 2021
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Vitor
Mendes
,
Simon R.
Green
,
Joanna C.
Evans
,
Jeannine
Hess
,
Michael
Blaszczyk
,
Christina
Spry
,
Owain
Bryant
,
James
Cory-wright
,
Daniel S-h.
Chan
,
Pedro H. M.
Torres
,
Zhe
Wang
,
Navid
Nahiyaan
,
Sandra
O’neill
,
Sebastian
Damerow
,
John
Post
,
Tracy
Bayliss
,
Sasha L.
Lynch
,
Anthony G.
Coyne
,
Peter C.
Ray
,
Chris
Abell
,
Kyu Y.
Rhee
,
Helena I. M.
Boshoff
,
Clifton E.
Barry
,
Valerie
Mizrahi
,
Paul G.
Wyatt
,
Tom L.
Blundell
Diamond Proposal Number(s):
[9537, 14043, 18548]
Open Access
Abstract: Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.
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Jan 2021
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12346]
Open Access
Abstract: Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein–protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.
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Dec 2020
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I02-Macromolecular Crystallography
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Open Access
Abstract: In modern societies, biodegradation of hydrophobic pollutants generated by industry is important for environmental and human health. In Gram-negative bacteria, biodegradation depends on facilitated diffusion of the pollutant substrates into the cell, mediated by specialised outer membrane (OM) channels. Here we show, via a combined experimental and computational approach, that the uptake of monoaromatic hydrocarbons such as toluene in Pseudomonas putida F1 (PpF1) occurs via lateral diffusion through FadL channels. Contrary to classical diffusion channels via which polar substrates move directly into the periplasmic space, PpF1 TodX and CymD direct their hydrophobic substrates into the OM via a lateral opening in the channel wall, bypassing the polar barrier formed by the lipopolysaccharide leaflet on the cell surface. Our study suggests that lateral diffusion of hydrophobic molecules is the modus operandi of all FadL channels, with potential implications for diverse areas such as biodegradation, quorum sensing and gut biology.
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Dec 2020
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I02-Macromolecular Crystallography
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Hengmiao
Cheng
,
Suvi T. M.
Orr
,
Simon
Bailey
,
Alexei
Brooun
,
Ping
Chen
,
Judith G.
Deal
,
Yali L.
Deng
,
Martin P.
Edwards
,
Gary M.
Gallego
,
Neil
Grodsky
,
Buwen
Huang
,
Mehran
Jalaie
,
Stephen
Kaiser
,
Robert S.
Kania
,
Susan E.
Kephart
,
Jennifer
Lafontaine
,
Martha A.
Ornelas
,
Mason
Pairish
,
Simon
Planken
,
Hong
Shen
,
Scott
Sutton
,
Luke
Zehnder
,
Chau D.
Almaden
,
Shubha
Bagrodia
,
Matthew D.
Falk
,
Hovhannes J.
Gukasyan
,
Caroline
Ho
,
Xiaolin
Kang
,
Rachel E.
Kosa
,
Ling
Liu
,
Mary E.
Spilker
,
Sergei
Timofeevski
,
Ravi
Visswanathan
,
Zhenxiong
Wang
,
Fanxiu
Meng
,
Shijian
Ren
,
Li
Shao
,
Feng
Xu
,
John C.
Kath
Abstract: The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway is a frequently dysregulated pathway in human cancer, and PI3Kα is one of the most frequently mutated kinases in human cancer. A PI3Kα-selective inhibitor may provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family. Here, we describe our efforts to discover a PI3Kα-selective inhibitor by applying structure-based drug design (SBDD) and computational analysis. A novel series of compounds, exemplified by 2,2-difluoroethyl (3S)-3-{[2′-amino-5-fluoro-2-(morpholin-4-yl)-4,5′-bipyrimidin-6-yl]amino}-3-(hydroxymethyl)pyrrolidine-1-carboxylate (1) (PF-06843195), with high PI3Kα potency and unique PI3K isoform and mTOR selectivity were discovered. We describe here the details of the design and synthesis program that lead to the discovery of 1.
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Dec 2020
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I02-Macromolecular Crystallography
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Alfredo
Picado
,
Apirat
Chaikuad
,
Carrow I.
Wells
,
Safal
Shrestha
,
William J.
Zuercher
,
Julie E.
Pickett
,
Frank E.
Kwarcinski
,
Parvathi
Sinha
,
Chandi S.
De Silva
,
Reena
Zutshi
,
Shubin
Liu
,
Natarajan
Kannan
,
Stefan
Knapp
,
David H.
Drewry
,
Timothy M.
Willson
Diamond Proposal Number(s):
[442]
Abstract: STK17B is a member of the death-associated protein kinase family and has been genetically linked to the development of diverse diseases. However, the role of STK17B in normal and disease pathology is poorly defined. Here, we present the discovery of thieno[3,2-d] pyrimidine SGC-STK17B-1 (11s), a high-quality chemical probe for this understudied “dark” kinase. 11s is an ATP-competitive inhibitor that showed remarkable selectivity over other kinases including the closely related STK17A. X-ray crystallography of 11s and related thieno[3,2-d]pyrimidines bound to STK17B revealed a unique P-loop conformation characterized by a salt bridge between R41 and the carboxylic acid of the inhibitor. Molecular dynamic simulations of STK17B revealed the flexibility of the P-loop and a wide range of R41 conformations available to the apo-protein. The isomeric thieno[2,3-d]pyrimidine SGC-STK17B-1N (19g) was identified as a negative control compound. The >100-fold lower activity of 19g on STK17B was attributed to the reduced basicity of its pyrimidine N1.
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Nov 2020
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Yousuke
Yamada
,
Hajime
Takashima
,
David Lee
Walmsley
,
Fumihito
Ushiyama
,
Yohei
Matsuda
,
Harumi
Kanazawa
,
Toru
Yamaguchi-sasaki
,
Nozomi
Tanaka-yamamoto
,
Junya
Yamagishi
,
Risa
Kurimoto-tsuruta
,
Yuya
Ogata
,
Norikazu
Ohtake
,
Hayley
Angove
,
Lisa
Baker
,
Richard
Harris
,
Alba
Macias
,
Alan
Robertson
,
Allan
Surgenor
,
Hayato
Watanabe
,
Koichiro
Nakano
,
Masashi
Mima
,
Kunihiko
Iwamoto
,
Atsushi
Okada
,
Iichiro
Takata
,
Kosuke
Hitaka
,
Akihiro
Tanaka
,
Kiyoko
Fujita
,
Hiroyuki
Sugiyama
,
Roderick E.
Hubbard
Diamond Proposal Number(s):
[12428]
Abstract: UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of Lipid A, an essential component of the cell envelope of Gram-negative bacteria. The most advanced, disclosed LpxC inhibitors showing antibacterial activity coordinate zinc through a hydroxamate moiety with concerns about binding to other metalloenzymes. Here, we describe the discovery, optimization, and efficacy of two series of compounds derived from fragments with differing modes of zinc chelation. A series was evolved from a fragment where a glycine moiety complexes zinc, which achieved low nanomolar potency in an enzyme functional assay but poor antibacterial activity on cell cultures. A second series was based on a fragment that chelated zinc through an imidazole moiety. Structure-guided design led to a 2-(1S-hydroxyethyl)-imidazole derivative exhibiting low nanomolar inhibition of LpxC and a minimum inhibitory concentration (MIC) of 4 μg/mL against Pseudomonas aeruginosa, which is little affected by the presence of albumin.
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Nov 2020
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I02-Macromolecular Crystallography
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Zoltan
Szlavik
,
Marton
Csekei
,
Attila
Paczal
,
Zoltan B.
Szabo
,
Szabolcs
Sipos
,
Gabor
Radics
,
Agnes
Proszenyak
,
Balazs
Balint
,
James
Murray
,
James
Davidson
,
Ijen
Chen
,
Pawel
Dokurno
,
Allan E
Surgenor
,
Zoe Marie
Daniels
,
Roderick E.
Hubbard
,
Gaëtane
Le Toumelin-braizat
,
Audrey
Claperon
,
Gaëlle
Lysiak-auvity
,
Anne-marie
Girard
,
Alain
Bruno
,
Maia
Chanrion
,
Frédéric
Colland
,
Ana-leticia
Maragno
,
Didier
Demarles
,
Olivier
Geneste
,
Andras
Kotschy
Diamond Proposal Number(s):
[2103]
Abstract: Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1. Starting from a fragment derived lead compound, we have conducted structure guided optimization that has led to a significant (3 log) improvement of target affinity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compounds against other members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both efficient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has drug-like properties that have enabled its development and entry into clinical trials.
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Nov 2020
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