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Open Access
Abstract: The SARS-CoV-2 papain-like protease (PLpro) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, so releasing the non-structural proteins (nsps) 1–3 that are essential for the coronavirus lifecycle. The LXGG↓X motif in pp1a/1ab is crucial for recognition and cleavage by PLpro. We describe molecular dynamics, docking, and quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how oligopeptide substrates derived from the viral polyprotein bind to PLpro. The results reveal how the substrate sequence affects the efficiency of PLpro-catalyzed hydrolysis. In particular, a proline at the P2′ position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PLpro catalyzed hydrolysis of LXGG motif-containing oligopeptides derived from human proteins suggests that factors beyond the LXGG motif and the presence of a proline residue at P2′ contribute to catalytic efficiency, possibly reflecting the promiscuity of PLpro. The results will help in identifying PLpro substrates and guiding inhibitor design.
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Oct 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Epoxides are an established class of electrophilic alkylating agents that react with nucleophilic protein residues. We report αβ,α′β′-diepoxyketones (DEKs) as a new type of mechanism-based inhibitors of nucleophilic cysteine enzymes. Studies with the L,D-transpeptidase LdtMt2 from Mycobacterium tuberculosis and the main protease from SARS-CoV-2 (Mpro) reveal that following epoxide ring opening by a nucleophilic cysteine, further reactions can occur, leading to irreversible alkylation.
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Oct 2023
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I03-Macromolecular Crystallography
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Thomas P.
Corner
,
Ryan Z. R.
Teo
,
Yue
Wu
,
Eidarus
Salah
,
Yu
Nakashima
,
Giorgia
Fiorini
,
Anthony
Tumber
,
Amelia
Brasnett
,
James P.
Holt-Martyn
,
William D.
Figg
,
Xiaojin
Zhang
,
Lennart
Brewitz
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: The human 2-oxoglutarate (2OG)- and Fe(II)-dependent oxygenases factor inhibiting hypoxia-inducible factor-α (FIH) and HIF-α prolyl residue hydroxylases 1–3 (PHD1–3) regulate the response to hypoxia in humans via catalysing hydroxylation of the α-subunits of the hypoxia-inducible factors (HIFs). Small-molecule PHD inhibitors are used for anaemia treatment; by contrast, few selective inhibitors of FIH have been reported, despite their potential to regulate the hypoxic response, either alone or in combination with PHD inhibition. We report molecular, biophysical, and cellular evidence that the N-hydroxythiazole scaffold, reported to inhibit PHD2, is a useful broad spectrum 2OG oxygenase inhibitor scaffold, the inhibition potential of which can be tuned to achieve selective FIH inhibition. Structure-guided optimisation resulted in the discovery of N-hydroxythiazole derivatives that manifest substantially improved selectivity for FIH inhibition over PHD2 and other 2OG oxygenases, including Jumonji-C domain-containing protein 5 (∼25-fold), aspartate/asparagine-β-hydroxylase (>100-fold) and histone Nε-lysine demethylase 4A (>300-fold). The optimised N-hydroxythiazole-based FIH inhibitors modulate the expression of FIH-dependent HIF target genes and, consistent with reports that FIH regulates cellular metabolism, suppressed lipid accumulation in adipocytes. Crystallographic studies reveal that the N-hydroxythiazole derivatives compete with both 2OG and the substrate for binding to the FIH active site. Derivatisation of the N-hydroxythiazole scaffold has the potential to afford selective inhibitors for 2OG oxygenases other than FIH.
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Oct 2023
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Lennart
Brewitz
,
Yu
Nakashima
,
Sonia K.
Piasecka
,
Eidarus
Salah
,
Sally C.
Fletcher
,
Anthony
Tumber
,
Thomas P.
Corner
,
Tristan J.
Kennedy
,
Giorgia
Fiorini
,
Armin
Thalhammer
,
Kirsten E.
Christensen
,
Mathew L.
Coleman
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Jumonji-C domain-containing protein 5 (JMJD5) is a 2-oxoglutarate (2OG)-dependent oxygenase that plays important roles in development, circadian rhythm, and cancer through unclear mechanisms. JMJD5 has been reported to have activity as a histone protease, as an Nε-methyl lysine demethylase, and as an arginine residue hydroxylase. Small-molecule JMJD5-selective inhibitors will be useful for investigating its (patho)physiological roles. Following the observation that the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylic acid (2,4-PDCA) is a 2OG-competing JMJD5 inhibitor, we report that 5-aminoalkyl-substituted 2,4-PDCA derivatives are potent JMJD5 inhibitors manifesting selectivity for JMJD5 over other human 2OG oxygenases. Crystallographic analyses with five inhibitors imply induced fit binding and reveal that the 2,4-PDCA C5 substituent orients into the JMJD5 substrate-binding pocket. Cellular studies indicate that the lead compounds display similar phenotypes as reported for clinically observed JMJD5 variants, which have a reduced catalytic activity compared to wild-type JMJD5.
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Aug 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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VMXm-Versatile Macromolecular Crystallography microfocus
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Lennart
Brewitz
,
Leo
Dumjahn
,
Yilin
Zhao
,
C. David
Owen
,
Stephen M.
Laidlaw
,
Tika R.
Malla
,
Dung
Nguyen
,
Petra
Lukacik
,
Eidarus
Salah
,
Adam D.
Crawshaw
,
Anna J.
Warren
,
Jose
Trincao
,
Claire
Strain-Damerell
,
Miles W.
Carroll
,
Martin A.
Walsh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (Mpro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated Mpro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit Mpro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of Mpro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.
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Feb 2023
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I03-Macromolecular Crystallography
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Xiao
Liu
,
Raphael
Reinbold
,
Shuang
Liu
,
Ryan A.
Herold
,
Patrick
Rabe
,
Stéphanie
Duclos
,
Rahul B.
Yadav
,
Martine I.
Abboud
,
Sandrine
Thieffine
,
Fraser A.
Armstrong
,
Lennart
Brewitz
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[23459]
Open Access
Abstract: Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutate (2OG) to (2R)-hydroxyglutarate (2HG). However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3 and C4 alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of wildtype IDH1/2. Absorbance-based, NMR and electrochemical assays were employed to monitor wildtype IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of wildtype IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates compared to 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl-, 3-butyl-, and 3-benzyl-substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts.
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Jan 2023
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I03-Macromolecular Crystallography
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Tika R.
Malla
,
Lennart
Brewitz
,
Dorian-Gabriel
Muntean
,
Hiba
Aslam
,
C. David
Owen
,
Eidarus
Salah
,
Anthony
Tumber
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Halina
Mikolajek
,
Martin
Walsh
,
Christopher J.
Schofield
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: The SARS-CoV-2 main protease (Mpro) is a medicinal chemistry target for COVID-19 treatment. Given the clinical efficacy of β-lactams as inhibitors of bacterial nucleophilic enzymes, they are of interest as inhibitors of viral nucleophilic serine and cysteine proteases. We describe the synthesis of penicillin derivatives which are potent Mpro inhibitors and investigate their mechanism of inhibition using mass spectrometric and crystallographic analyses. The results suggest that β-lactams have considerable potential as Mpro inhibitors via a mechanism involving reaction with the nucleophilic cysteine to form a stable acyl–enzyme complex as shown by crystallographic analysis. The results highlight the potential for inhibition of viral proteases employing nucleophilic catalysis by β-lactams and related acylating agents.
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May 2022
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Open Access
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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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Open Access
Abstract: 2-Oxoglutarate (2OG) oxygenases are validated agrochemical and human drug targets. The potential for modulating their activity with 2OG derivatives has not been explored, possibly due to concerns regarding selectivity. We report proof-of-principle studies demonstrating selective enhancement or inhibition of 2OG oxygenase activity by 2-oxo acids. The human 2OG oxygenases studied, factor inhibiting hypoxia-inducible transcription factor HIF-α (FIH) and aspartate/asparagine-β-hydroxylase (AspH), catalyze C3 hydroxylations of Asp/Asn-residues. Of 35 tested 2OG derivatives, 10 enhance and 17 inhibit FIH activity. Comparison with results for AspH reveals that 2OG derivatives selectively enhance or inhibit FIH or AspH. Comparison of FIH structures complexed with 2OG derivatives to those for AspH provides insight into the basis of the observed selectivity. 2-Oxo acid derivatives have potential as drugs, for use in biomimetic catalysis, and in functional studies. The results suggest that the in vivo activity of 2OG oxygenases may be regulated by natural 2-oxo acids other than 2OG.
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Nov 2021
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