I03-Macromolecular Crystallography
|
Michael
Beer
,
Philip
Hinchliffe
,
Marko
Hanževački
,
Christopher R.
Bethel
,
Catherine L.
Tooke
,
Marc W.
Van Der Kamp
,
Krisztina M.
Papp‐wallace
,
Robert A.
Bonomo
,
Stuart
Shapiro
,
Adrian J.
Mulholland
,
James
Spencer
Diamond Proposal Number(s):
[23269, 31440]
Open Access
Abstract: β-Lactamase-catalysed hydrolysis is the primary form of β-lactam antibiotic resistance in Gram-negative bacteria. The penicillanic acid sulfone (PAS) enmetazobactam is thought to inhibit extended-spectrum β-lactamases (ESBLs) by fragmentation of an initial acyl-enzyme to form an active-site lysinoalanine cross link. We investigate interactions of enmetazobactam and its congener tazobactam with GES-1, an ESBL with structural features of carbapenem-hydrolysing β-lactamases. Crystal structures show different breakdown products of the two inhibitors covalently bound to the catalytic Ser70, assigned using quantum mechanics/molecular mechanics (QM/MM) calculations. We find no evidence for lysinoalanine formation, with mass spectrometry indicating active enzyme regeneration, behaviour previously observed for carbapenem-hydrolysing enzymes, but not ESBLs. This work establishes that PAS inhibitors interact with diverse β-lactamases by differing mechanisms, which should inform development of future compounds.
|
Sep 2025
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Joseph F.
Hoff
,
Kirsty E.
Goudar
,
Karina
Calvopina
,
Michael
Beer
,
Philip
Hinchliffe
,
John M.
Shaw
,
Catherine L.
Tooke
,
Yuiko
Takebayashi
,
Andrew F.
Cadzow
,
Nicholas
Harmer
,
Adrian J.
Mulholland
,
Christopher J.
Schofield
,
James
Spencer
Diamond Proposal Number(s):
[23269, 31440]
Open Access
Abstract: Carbapenemases, β-lactamases hydrolysing carbapenem antibiotics, challenge treatment of multi-drug resistant bacteria. The OXA-48 carbapenemase is widely disseminated in Enterobacterales, necessitating new treatments for producer strains. Diazabicyclooctane (DBO) inhibitors, including avibactam and nacubactam, act on a wide range of enzymes to overcome β-lactamase-mediated resistance. Here we investigate avibactam and nacubactam activities towards OXA-48 and two variants, OXA-163 and OXA-405, with deletions in the β5 – β6 loop neighbouring the active site that modify activity towards different β-lactam classes. Nacubactam is c. 80-fold less potent than avibactam towards OXA-48, but this difference reduces in OXA-163 and OXA-405. Crystal structures and molecular dynamics simulations reveal electrostatic repulsion between Arg214 on the OXA-48 β5 – β6 active-site loop and nacubactam, but not avibactam, effects absent from simulations of OXA-163 and OXA-405, which lack Arg214. Crystallographic and mass spectrometry data demonstrate that all three enzymes support desulfation of bound DBOs. These data indicate that interactions with Arg214 affect DBO potency, suggesting that sequence variation in OXA-48-like β-lactamases affects reactivity towards inhibitors as well as β-lactam substrates.
|
Aug 2025
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[31440]
Open Access
Abstract: Antimicrobial resistance has emerged as a critical global public health threat, impacting human, animal and environmental health. An important mechanism of resistance is the production of β-lactamases, enzymes that hydrolyze the β-lactam ring, rendering β-lactam antibiotics ineffective. Metallo-β-lactamases (MBLs), which contain zinc ions in their active sites, are particularly challenging to counter as there are currently no inhibitors targeting these enzymes available on the market. Therefore, there is an urgent need for innovative drug discovery strategies to develop MBL-targeted therapies. New Delhi Metallo-β-Lactamase 1 (NDM-1) is the most widely disseminated MBL, with a global distribution in Enterobacterales. In this study, we used our library of fragment-sized chloroacetamides as a starting point to synthesize mercaptoacetamides as potential NDM-1 inhibitors. This resulted in a compound (14a) with an IC50 of 20 μM, which crystallography shows binds to NDM-1 in two different poses. Using this structure as a starting point for in silico design, we developed a series of larger thiol-based compounds designed to occupy more space in the active site and to utilize other novel zinc-binding groups. Although some showed minimal inhibition (which makes them valuable as decoys for metalloenzyme studies) one compound exhibited an IC50 of 14 μM, with crystallography indicating that an additional aromatic group, compared to 14a, interacts with hydrophobic residues on an NDM-1 active site loop. These data identify promising scaffolds for the further development of potent MBL inhibitors and show the utility of repurposing chemical libraries to target clinically important enzymes.
|
Jun 2025
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[31440]
Open Access
Abstract: Antimicrobial resistance is a significant global public health threat that limits treatment options for bacterial infections. This situation is aggravated by the environmental spread of β-lactamase genes. In particular, metallo-β-lactamases (MBLs) hydrolyze almost all available β-lactam antibiotics, including late-generation cephalosporins and carbapenems. Among MBLs, the New Delhi metallo-β-lactamase (NDM-1) of subclass B1 has shown the most ominous dissemination. NDM variants are the only MBLs of clinical importance that are membrane-anchored, a sub-cellular localization that endows them with high stability under conditions of metal limitation. However, antibiotic resistance predates modern antibiotic usage, and environmental bacteria serve as reservoirs for resistance genes. Here, we report the biochemical and structural characterization of two membrane-bound MBLs: CJO-1 and CIM-2, from Chryseobacterium joostei and Chryseobacterium indologenes, respectively. Both MBLs confer β-lactam resistance on producer bacterial strains and hydrolyze several antibiotics, although with impaired efficiency compared to NDM-1. Crystal structures reveal differences, compared to previously studied B1 MBLs, in the active site loops and their dynamic properties that impact activity. Specifically, a hindered access to the active site with the contribution of a Tyr residue in loop L10 and the presence of a positively charged Lys residue in loop L3 limit hydrolysis of cephalosporins with charged C3 substituents. Some of these novel features are preserved in other MBLs from Chryseobacterium spp. These findings suggest that Chryseobacterium spp. could act as reservoirs of MBL genes, while informing on the diversity of structure-function relationships and dynamic behaviors within the B1 subclass of these enzymes.
|
Jun 2025
|
|
I03-Macromolecular Crystallography
|
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.
|
Sep 2024
|
|
I03-Macromolecular Crystallography
|
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.
|
Feb 2024
|
|
I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
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.
|
Mar 2023
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
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.
|
Mar 2023
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
|
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.
|
May 2022
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
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.
|
Mar 2022
|
|
