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Abstract: This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA–DNA processing, and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 mL of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures.
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Oct 2020
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Abstract: In Chapter 3, we described the Structural Genomics Consortium (SGC) process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or structural studies (e.g., crystallization or cryo-EM experiments). Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.
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Oct 2020
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Abstract: Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the Structural Genomics Consortium, we opted for the ligation-independent cloning (LIC) method which provides the throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).
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Oct 2020
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Tika R.
Malla
,
Anthony
Tumber
,
Tobias
John
,
Lennart
Brewitz
,
Claire
Strain-Damerell
,
C. David
Owen
,
Petra
Lukacik
,
H. T. Henry
Chan
,
Pratheesh
Maheswaran
,
Eidarus
Salah
,
Fernanda
Duarte
,
Haitao
Yang
,
Zihe
Rao
,
Martin A.
Walsh
,
Christopher J.
Schofield
Open Access
Abstract: The main viral protease (Mpro) of SARS-CoV-2 is a nucleophilic cysteine hydrolase and a current target for anti-viral chemotherapy. We describe a high-throughput solid phase extraction coupled to mass spectrometry Mpro assay. The results reveal some β-lactams, including penicillin esters, are active site reacting Mpro inhibitors, thus highlighting the potential of acylating agents for Mpro inhibition.
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Jan 2021
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Abstract: The two SARS-CoV-2 proteases, i.e. the main protease (M pro ) and the papain-like protease (PL pro ), which hydrolyze the viral polypeptide chain giving functional non-structural proteins, are essential for viral replication and are medicinal chemistry targets. We report a high-throughput mass spectrometry (MS)-based assay which directly monitors PL pro catalysis in vitro . The assay was applied to investigate the effect of reported small-molecule PL pro inhibitors and selected M pro inhibitors on PL pro catalysis. The results reveal that some, but not all, PL pro inhibitor potencies differ substantially from those obtained using fluorescence-based assays. Some substrate-competing M pro inhibitors, notably PF-07321332 (nirmatrelvir) which is in clinical development, do not inhibit PL pro . Less selective M pro inhibitors, e.g. auranofin, inhibit PL pro , highlighting the potential for dual PL pro /M pro inhibition. MS-based PL pro assays, which are orthogonal to widely employed fluorescence-based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non-covalent mechanisms.
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Jan 2022
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Luiz Carlos
Saramago
,
Marcos V.
Santana
,
Bárbara Figueira
Gomes
,
Rafael Ferreira
Dantas
,
Mario R.
Senger
,
Pedro Henrique
Oliveira Borges
,
Vivian Neuza
Dos Santos Ferreira
,
Alice
Dos Santos Rosa
,
Amanda Resende
Tucci
,
Milene
Dias Miranda
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
C. David
Owen
,
Martin A.
Walsh
,
Sabrina
Baptista Ferreira
,
Floriano Paes
Silva-Junior
Abstract: SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global pandemic. The viral genome contains 5 major open reading frames of which the largest ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into 16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp’s, making it essential for viral replication and has been successfully targeted for the development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid. Increasing the arsenal of antivirals and development of protease inhibitors and other antivirals with a varied mode of action remains a priority to reduce the likelihood for resistance emerging. Here, we report results from an artificial intelligence-driven approach followed by in vitro validation, allowing the identification of five fragment-like Mpro inhibitors with IC50 values ranging from 1.5 to 241 μM. The three most potent molecules (compounds 818, 737, and 183) were tested against SARS-CoV-2 by in vitro replication in Vero E6 and Calu-3 cells. Compound 818 was active in both cell models with an EC50 value comparable to its measured IC50 value. On the other hand, compounds 737 and 183 were only active in Calu-3, a preclinical model of respiratory cells, showing selective indexes twice as high as those for compound 818. We also show that our in silico methodology was successful in identifying both reversible and covalent inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of 8-methyl-γ-carboline, while compound 737 is an N-pyridyl-isatin that covalently inhibits Mpro. Given the small molecular weights of these fragments, their high binding efficiency in vitro and efficacy in blocking viral replication, these compounds represent good starting points for the development of potent lead molecules targeting the Mpro of SARS-CoV-2.
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Apr 2023
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[21035, 10619]
Open Access
Abstract: The core machinery for de novo biosynthesis of iron-sulfur clusters (ISC), located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich’s ataxia (FRDA). Here the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates the interactions of FXN with other component proteins of the complex. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure reveals how FXN facilitates ISC production through stabilizing key loop conformations of NFS1 and ISCU at the protein–protein interfaces, and suggests how FRDA clinical mutations affect complex formation and FXN activation.
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May 2019
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Catrine
Johansson
,
Velupillai
Srikannathasan
,
Anthony
Tumber
,
Aleksandra
Szykowska
,
Edward S.
Hookway
,
Radoslaw
Nowak
,
Claire
Strain-Damerell
,
Carina
Gileadi
,
Martin
Philpott
,
Nicola
Burgess-Brown
,
Na
Wu
,
Jolanta
Kopec
,
Andrea
Nuzzi
,
Holger
Steuber
,
Ursula
Egner
,
Volker
Badock
,
Shonagh
Munro
,
Nicholas B
Lathangue
,
Sue
Westaway
,
Jack
Brown
,
Nick
Athanasou
,
Rab
Prinjha
,
Paul E
Brennan
,
Udo
Oppermann
Diamond Proposal Number(s):
[10619]
Abstract: Members of the KDM5 (also known as JARID1) family are 2-oxoglutarate- and Fe2+-dependent oxygenases that act as histone H3K4 demethylases, thereby regulating cell proliferation and stem cell self-renewal and differentiation. Here we report crystal structures of the catalytic core of the human KDM5B enzyme in complex with three inhibitor chemotypes. These scaffolds exploit several aspects of the KDM5 active site, and their selectivity profiles reflect their hybrid features with respect to the KDM4 and KDM6 families. Whereas GSK-J1, a previously identified KDM6 inhibitor, showed about sevenfold less inhibitory activity toward KDM5B than toward KDM6 proteins, KDM5-C49 displayed 25–100-fold selectivity between KDM5B and KDM6B. The cell-permeable derivative KDM5-C70 had an antiproliferative effect in myeloma cells, leading to genome-wide elevation of H3K4me3 levels. The selective inhibitor GSK467 exploited unique binding modes, but it lacked cellular potency in the myeloma system. Taken together, these structural leads deliver multiple starting points for further rational and selective inhibitor design.
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May 2016
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I02-Macromolecular Crystallography
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Anthony
Tumber
,
Andrea
Nuzzi
,
Edward S.
Hookway
,
Stephanie B.
Hatch
,
Srikannathasan
Velupillai
,
Catrine
Johansson
,
Akane
Kawamura
,
Pavel
Savitsky
,
Clarence
Yapp
,
Aleksandra
Szykowska
,
Na
Wu
,
Chas
Bountra
,
Claire
Strain-Damerell
,
Nicola A.
Burgess-Brown
,
Gian Filippo
Ruda
,
Oleg
Fedorov
,
Shonagh
Munro
,
Katherine S.
England
,
Radoslaw P.
Nowak
,
Christopher J.
Schofield
,
Nicholas B.
La Thangue
,
Charlotte
Pawlyn
,
Faith
Davies
,
Gareth
Morgan
,
Nick
Athanasou
,
Susanne
Müller
,
Udo
Oppermann
,
Paul E.
Brennan
Open Access
Abstract: Methylation of lysine residues on histone tail is a dynamic epigenetic modification that plays a key role in chromatin structure and gene regulation. Members of the KDM5 (also known as JARID1) sub-family are 2-oxoglutarate (2-OG) and Fe2+-dependent oxygenases acting as histone 3 lysine 4 trimethyl (H3K4me3) demethylases, regulating proliferation, stem cell self-renewal, and differentiation. Here we present the characterization of KDOAM-25, an inhibitor of KDM5 enzymes. KDOAM-25 shows biochemical half maximal inhibitory concentration values of <100 nM for KDM5A-D in vitro, high selectivity toward other 2-OG oxygenases sub-families, and no off-target activity on a panel of 55 receptors and enzymes. In human cell assay systems, KDOAM-25 has a half maximal effective concentration of ∼50 μM and good selectivity toward other demethylases. KDM5B is overexpressed in multiple myeloma and negatively correlated with the overall survival. Multiple myeloma MM1S cells treated with KDOAM-25 show increased global H3K4 methylation at transcriptional start sites and impaired proliferation.
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Mar 2017
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Thomas
Mccorvie
,
Jolanta
Kopec
,
S J
Hyung
,
Fiona
Fitzpatrick
,
X
Feng
,
D.
Termine
,
C.
Strain-Damerell
,
Melanie
Vollmar
,
J.
Fleming
,
J. M.
Janz
,
C.
Bulawa
,
Wyatt
Yue
Diamond Proposal Number(s):
[8421, 10619]
Open Access
Abstract: Cystathionine β-synthase (CBS) is a key enzyme in sulfur metabolism, and its inherited deficiency causes homocystinuria. Mammalian CBS is modulated by the binding of S-adenosyl-l-methionine (AdoMet) to its regulatory domain, which activates its catalytic domain. To investigate the underlying mechanism, we performed x-ray crystallography, mutagenesis, and mass spectrometry (MS) on human CBS. The 1.7 Å structure of a AdoMet-bound CBS regulatory domain shows one AdoMet molecule per monomer, at the interface between two constituent modules (CBS-1, CBS-2). AdoMet binding is accompanied by a reorientation between the two modules, relative to the AdoMet-free basal state, to form interactions with AdoMet via residues verified by mutagenesis to be important for AdoMet binding (Phe443, Asp444, Gln445, and Asp538) and for AdoMet-driven inter-domain communication (Phe443, Asp538). The observed structural change is further supported by ion mobility MS, showing that as-purified CBS exists in two conformational populations, which converged to one in the presence of AdoMet. We therefore propose that AdoMet-induced conformational change alters the interface and arrangement between the catalytic and regulatory domains within the CBS oligomer, thereby increasing the accessibility of the enzyme active site for catalysis.
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Dec 2014
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