I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269, 31440]
Open Access
Abstract: β-Lactamases, which hydrolyse β-lactam antibiotics, are key determinants of antibiotic resistance. Predicting the sites and effects of distal mutations in enzymes is challenging. For β-lactamases, the ability to make such predictions would contribute to understanding activity against, and development of, antibiotics and inhibitors to combat resistance. Here, using dynamical non-equilibrium molecular dynamics (D-NEMD) simulations combined with experiments, we demonstrate that intramolecular communication networks differ in three class A SulpHydryl Variant (SHV)-type β-lactamases. Differences in network architecture and correlated motions link to catalytic efficiency and β-lactam substrate spectrum. Further, the simulations identify a distal residue at position 89 in the clinically important Klebsiella pneumoniae carbapenemase 2 (KPC-2), as a participant in similar networks, suggesting that mutation at this position would modulate enzyme activity. Experimental kinetic, biophysical and structural characterisation of the naturally occurring, but previously biochemically uncharacterised, KPC-2G89D mutant with several antibiotics and inhibitors reveals significant changes in hydrolytic spectrum, specifically reducing activity towards carbapenems without effecting major structural or stability changes. These results show that D-NEMD simulations can predict distal sites where mutation affects enzyme activity. This approach could have broad application in understanding enzyme evolution, and in engineering of natural and de novo enzymes.
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Sep 2024
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I03-Macromolecular Crystallography
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Valentina
Villamil
,
Maria-Agustina
Rossi
,
Maria F.
Mojica
,
Philip
Hinchliffe
,
Verónica
Martínez
,
Valerie
Castillo
,
Cecilia
Saiz
,
Claudia
Banchio
,
Mario A.
Macías
,
James
Spencer
,
Robert A.
Bonomo
,
Alejandro
Vila
,
Diego M.
Moreno
,
Graciela
Mahler
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Antimicrobial resistance is a global public health threat. Metallo-β-lactamases (MBLs) inactivate β-lactam antibiotics, including carbapenems, are disseminating among Gram-negative bacteria, and lack clinically useful inhibitors. The evolving bisthiazolidine (BTZ) scaffold inhibits all three MBL subclasses (B1–B3). We report design, synthesis, and evaluation of BTZ analogues. Structure–activity relationships identified the BTZ thiol as essential, while carboxylate is replaceable, with its removal enhancing potency by facilitating hydrophobic interactions within the MBL active site. While the introduction of a flexible aromatic ring is neutral or detrimental for inhibition, a rigid (fused) ring generated nM benzobisheterocycle (BBH) inhibitors that potentiated carbapenems against MBL-producing strains. Crystallography of BBH:MBL complexes identified hydrophobic interactions as the basis of potency toward B1 MBLs. These data underscore BTZs as versatile, potent broad-spectrum MBL inhibitors (with activity extending to enzymes refractory to other inhibitors) and provide a rational approach to further improve the tricyclic BBH scaffold.
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Feb 2024
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212, 23269]
Open Access
Abstract: L1 is a dizinc subclass B3 metallo-β-lactamase (MBL) that hydrolyzes most β-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed β-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole) and carbapenem (tebipenem, doripenem and panipenem) classes. Despite differences in their structures, all the β-lactam-derived products hydrogen bond to Tyr33, Ser221 and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modelled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1β-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1β-hydrogen-containing carbapenems), or is displaced by the C-6 carboxylate (1β-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur non-enzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3’ leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (β) faces, and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and β-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.
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Mar 2023
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[172122, 23269]
Open Access
Abstract: KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-β-lactamase (SBL) responsible for extensive β-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate β-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent β-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25–1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165–170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different β-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.
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Mar 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Philip
Hinchliffe
,
Catherine L.
Tooke
,
Christopher R.
Bethel
,
Benlian
Wang
,
Christopher
Arthur
,
Kate J.
Heesom
,
Stuart
Shapiro
,
Daniela M.
Schlatzer
,
Krisztina M.
Papp-Wallace
,
Robert A.
Bonomo
,
James
Spencer
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: β-Lactamases hydrolyze β-lactam antibiotics and are major determinants of antibiotic resistance in Gram-negative pathogens. Enmetazobactam (formerly AAI101) and tazobactam are penicillanic acid sulfone (PAS) β-lactamase inhibitors that differ by an additional methyl group on the triazole ring of enmetazobactam, rendering it zwitterionic. In this study, ultrahigh-resolution X-ray crystal structures and mass spectrometry revealed the mechanism of PAS inhibition of CTX-M-15, an extended-spectrum β-lactamase (ESBL) globally disseminated among Enterobacterales. CTX-M-15 crystals grown in the presence of enmetazobactam or tazobactam revealed loss of the Ser70 hydroxyl group and formation of a lysinoalanine cross-link between Lys73 and Ser70, two residues critical for catalysis. Moreover, the residue at position 70 undergoes epimerization, resulting in formation of a D-amino acid. Cocrystallization of enmetazobactam or tazobactam with CTX-M-15 with a Glu166Gln mutant revealed the same cross-link, indicating that this modification is not dependent on Glu166-catalyzed deacylation of the PAS-acylenzyme. A cocrystal structure of enmetazobactam with CTX-M-15 with a Lys73Ala mutation indicates that epimerization can occur without cross-link formation and positions the Ser70 Cβ closer to Lys73, likely facilitating formation of the Ser70-Lys73 cross-link. A crystal structure of a tazobactam-derived imine intermediate covalently linked to Ser70, obtained after 30 min of exposure of CTX-M-15 crystals to tazobactam, supports formation of an initial acylenzyme by PAS inhibitors on reaction with CTX-M-15. These data rationalize earlier results showing CTX-M-15 deactivation by PAS inhibitors to involve loss of protein mass, and they identify a distinct mechanism of β-lactamase inhibition by these agents.
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May 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: Carbapenems are important antibacterials and are both substrates and inhibitors of some β-lactamases. We report studies on the reaction of the unusual carbapenem biapenem, with the subclass B1 metallo-β-lactamases VIM-1 and VIM-2 and the class A serine-β-lactamase KPC-2. X-ray diffraction studies with VIM-2 crystals treated with biapenem reveal the opening of the β-lactam ring to form a mixture of the (2S)-imine and enamine complexed at the active site. NMR studies on the reactions of biapenem with VIM-1, VIM-2, and KPC-2 reveal the formation of hydrolysed enamine and (2R)- and (2S)-imine products. The combined results support the proposal that SBL/MBL-mediated carbapenem hydrolysis results in a mixture of tautomerizing enamine and (2R)- and (2S)-imine products, with the thermodynamically favoured (2S)-imine being the major observed species over a relatively long-time scale. The results suggest that prolonging the lifetimes of β-lactamase carbapenem complexes by optimising tautomerisation of the nascently formed enamine to the (2R)-imine and likely more stable (2S)-imine tautomer is of interest in developing improved carbapenems.
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Mar 2022
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Jurgen
Brem
,
Tharindi
Panduwawala
,
Jon Ulf
Hansen
,
Joanne
Hewitt
,
Edgars
Liepins
,
Pawel
Donets
,
Laura
Espina
,
Alistair J. M.
Farley
,
Kirill
Shubin
,
Gonzalo Gomez
Campillos
,
Paula
Kiuru
,
Shifali
Shishodia
,
Daniel
Krahn
,
Robert K.
Leśniak
,
Juliane
Schmidt
,
Karina
Calvopina
,
María-Carmen
Turrientes
,
Madeline E.
Kavanagh
,
Dmitrijs
Lubriks
,
Philip
Hinchliffe
,
Gareth W.
Langley
,
Ali F.
Aboklaish
,
Anders
Eneroth
,
Maria
Backlund
,
Andrei G.
Baran
,
Elisabet I.
Nielsen
,
Michael
Speake
,
Janis
Kuka
,
John
Robinson
,
Solveiga
Grinberga
,
Lindsay
Robinson
,
Michael A.
Mcdonough
,
Anna M.
Rydzik
,
Thomas M.
Leissing
,
Juan Carlos
Jimenez-Castellanos
,
Matthew B.
Avison
,
Solange
Da Silva Pinto
,
Andrew D.
Pannifer
,
Marina
Martjuga
,
Emma
Widlake
,
Martins
Priede
,
Iva
Hopkins Navratilova
,
Marek
Gniadkowski
,
Anna Karin
Belfrage
,
Peter
Brandt
,
Jari
Yli-Kauhaluoma
,
Eric
Bacque
,
Malcolm G. P.
Page
,
Fredrik
Björkling
,
Jonathan M.
Tyrrell
,
James
Spencer
,
Pauline A.
Lang
,
Pawel
Baranczewski
,
Rafael
Cantón
,
Stuart P.
Mcelroy
,
Philip S.
Jones
,
Fernando
Baquero
,
Edgars
Suna
,
Angus
Morrison
,
Timothy R.
Walsh
,
Christopher J.
Schofield
Open Access
Abstract: Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-β-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential β-lactamase stable β-lactam mimics. Subsequent structure–activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL–carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.
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Dec 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212]
Abstract: Widespread bacterial resistance to carbapenem antibiotics is an increasing global health concern. Resistance has emerged due to carbapenem-hydrolyzing enzymes, including metallo-β-lactamases (MβLs), but despite their prevalence and clinical importance, MβL mechanisms are still not fully understood. Carbapenem hydrolysis by MβLs can yield alternative product tautomers with the potential to access different binding modes. Here, we show that a combined approach employing crystallography and quantum mechanics/molecular mechanics (QM/MM) simulations allow tautomer assignment in MβL:hydrolyzed antibiotic complexes. Molecular simulations also examine (meta)stable species of alternative protonation and tautomeric states, providing mechanistic insights into β-lactam hydrolysis. We report the crystal structure of the hydrolyzed carbapenem ertapenem bound to the L1 MβL from Stenotrophomonas maltophilia and model alternative tautomeric and protonation states of both hydrolyzed ertapenem and faropenem (a related penem antibiotic), which display different binding modes with L1. We show how the structures of both complexed β-lactams are best described as the (2S)-imine tautomer with the carboxylate formed after β-lactam ring cleavage deprotonated. Simulations show that enamine tautomer complexes are significantly less stable (e.g., showing partial loss of interactions with the L1 binuclear zinc center) and not consistent with experimental data. Strong interactions of Tyr32 and one zinc ion (Zn1) with ertapenem prevent a C6 group rotation, explaining the different binding modes of the two β-lactams. Our findings establish the relative stability of different hydrolyzed (carba)penem forms in the L1 active site and identify interactions important to stable complex formation, information that should assist inhibitor design for this important antibiotic resistance determinant.
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Oct 2021
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NONE-No attached Diamond beamline
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H. T. Henry
Chan
,
Marc A.
Moesser
,
Rebecca K.
Walters
,
Tika R.
Malla
,
Rebecca M.
Twidale
,
Tobias
John
,
Helen M.
Deeks
,
Tristan
Johnston-Wood
,
Victor
Mikhailov
,
Richard B.
Sessions
,
William
Dawson
,
Eidarus
Salah
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
C. David
Owen
,
Takahito
Nakajima
,
Katarzyna
Świderek
,
Alessio
Lodola
,
Vicent
Moliner
,
David R.
Glowacki
,
James
Spencer
,
Martin A.
Walsh
,
Christopher J.
Schofield
,
Luigi
Genovese
,
Deborah K.
Shoemark
,
Adrian J.
Mulholland
,
Fernanda
Duarte
,
Garrett M.
Morris
Open Access
Abstract: The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural Mpro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial Mpro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective ‘peptibitors’ inhibit Mpro competitively. Our combined results provide new insights and highlight opportunities for the development of Mpro inhibitors as anti-COVID-19 drugs.
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Oct 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Philip
Hinchliffe
,
Diego M.
Moreno
,
Maria-Agustina
Rossi
,
Maria F.
Mojica
,
Veronica
Martinez
,
Valentina
Villamil
,
Brad
Spellberg
,
George L.
Drusano
,
Claudia
Banchio
,
Graciela
Mahler
,
Robert A.
Bonomo
,
Alejandro J.
Vila
,
James
Spencer
Diamond Proposal Number(s):
[12342, 17212]
Abstract: Metallo-β-lactamase (MBL) production in Gram-negative bacteria is an important contributor to β-lactam antibiotic resistance. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcoming resistance, but MBL inhibitors are not available in the clinic. On the basis of zinc utilization and sequence, MBLs are divided into three subclasses, B1, B2, and B3, whose differing active-site architectures hinder development of BLIs capable of “cross-class” MBL inhibition. We previously described 2-mercaptomethyl thiazolidines (MMTZs) as B1 MBL inhibitors (e.g., NDM-1) and here show that inhibition extends to the clinically relevant B2 (Sfh-I) and B3 (L1) enzymes. MMTZs inhibit purified MBLs in vitro (e.g., Sfh-I, Ki 0.16 μM) and potentiate β-lactam activity against producer strains. X-ray crystallography reveals that inhibition involves direct interaction of the MMTZ thiol with the mono- or dizinc centers of Sfh-I/L1, respectively. This is further enhanced by sulfur-π interactions with a conserved active site tryptophan. Computational studies reveal that the stereochemistry at chiral centers is critical, showing less potent MMTZ stereoisomers (up to 800-fold) as unable to replicate sulfur-π interactions in Sfh-I, largely through steric constraints in a compact active site. Furthermore, in silico replacement of the thiazolidine sulfur with oxygen (forming an oxazolidine) resulted in less favorable aromatic interactions with B2 MBLs, though the effect is less than that previously observed for the subclass B1 enzyme NDM-1. In the B3 enzyme L1, these effects are offset by additional MMTZ interactions with the protein main chain. MMTZs can therefore inhibit all MBL classes by maintaining conserved binding modes through different routes.
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Aug 2021
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