Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable ...time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.
The β-lactams remain the most important antibacterials, but their use is increasingly compromised by resistance, importantly by β-lactamases. Although β-lactam and non-β-lactam inhibitors forming ...stable acyl–enzyme complexes with nucleophilic serine β-lactamases (SBLs) are widely used, these are increasingly susceptible to evolved SBLs and do not inhibit metallo-β-lactamases (MBLs). Boronic acids and boronate esters, especially cyclic ones, can potently inhibit both SBLs and MBLs. Vaborbactam, a monocyclic boronate, is approved for clinical use, but its β-lactamase coverage is limited. Bicyclic boronates rapidly react with SBLs and MBLs forming stable enzyme–inhibitor complexes that mimic the common anionic high-energy tetrahedral intermediates in SBL/MBL catalysis, as revealed by crystallography. The ability of boronic acids to ‘morph’ between sp2 and sp3 hybridisation states may help enable potent inhibition. There is limited structure–activity relationship information on the (bi)cyclic boronate inhibitors compared to β-lactams, hence scope for creativity towards new boron-based β-lactamase inhibitors/antibacterials.
β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as 'transition state analogue' inhibitors of nucleophilic serine ...enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.
Zinc ion-dependent β-lactamases (MBLs) catalyze the hydrolysis of almost all β-lactam antibiotics and resist the action of clinically available β-lactamase inhibitors. We report how application of in ...silico fragment-based molecular design employing thiol-mediated metal anchorage leads to potent MBL inhibitors. The new inhibitors manifest potent inhibition of clinically important B1 subfamily MBLs, including the widespread NDM-1, IMP-1, and VIM-2 enzymes; with lower potency, some of them also inhibit clinically relevant Class A and D serine-β-lactamases. The inhibitors show selectivity for bacterial MBL enzymes compared to that for human MBL fold nucleases. Cocrystallization of one inhibitor, which shows potentiation of Meropenem activity against MBL-expressing Enterobacteriaceae, with VIM-2 reveals an unexpected binding mode, involving interactions with residues from conserved active site bordering loops.
The effectiveness of β-lactam antibiotics is increasingly compromised by β-lactamases. Boron-containing inhibitors are potent serine-β-lactamase inhibitors, but the interactions of boron-based ...compounds with the penicillin-binding protein (PBP) β-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by β-lactamase-driven resistance.
β-Lactamase production is the major β-lactam resistance mechanism in Gram-negative bacteria. β-Lactamase inhibitors (BLIs) efficacious against serine β-lactamase (SBL) producers, especially strains ...carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant
We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum β-lactamases L2 (inhibition constant 3 μM) and CTX-M-15 (21 μM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 μM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration IC
). MIC assays indicate relebactam potentiates β-lactam (imipenem) activity against KPC-producing
, with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against
K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI-β-lactam combinations.
A new synthetic route for the preparation of the metallo-β-lactamase inhibitor pre-candidate EBL-3183 was developed and carried out on a kilogram scale. The described process starts from a ...commercially available indole-2-carboxylate and employs an Ellman auxiliary approach coupled with ruthenium-catalyzed stereoselective reduction for the introduction of chirality. The key spirocyclic cyclobutane motif was assembled utilizing an epoxide building block, which was conveniently obtained in diastereomerically pure form. The amount and quality of the prepared final target EBL-3183 were sufficient for the preclinical studies.
Modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) enables increased activity and selectivity toward phenylalanines and cinnamic acids ...monosubstituted with both electron-donating (−CH3, −OCH3) and electron-withdrawing (−CF3, −Br) groups at all positions (ortho, meta, para) of their aromatic ring. The results reveal specific residues involved in accommodating substituents at the ortho, meta, and para positions of the substrate’s phenyl ring. The predicted interactions were validated by crystallographic analysis of the binding mode of p-methoxy cinnamic acid complexed at the active site of PcPAL. The biocatalytic utility of the tailored PcPAL mutants was demonstrated by the efficient preparative-scale synthesis of (S)-m-bromo-phenylalanine (ee > 99%; yield 60%) and (R)-p-methyl-phenylalanine (ee 97%; yield 49%) using the corresponding ammonia addition and ammonia elimination reactions catalyzed by the L134A and I460V PcPAL variants, respectively. Overall, the results reveal the potential for structure-based protein engineering of PALs to provide enzymes with enhanced catalytic properties and which are specifically tailored for differently substituted phenylalanine analogues of high synthetic value.
β-Lactamases are the most important mechanisms of resistance to the β-lactam antibacterials. There are two mechanistic classes of β-lactamases: the serine β-lactamases (SBLs) and the zinc-dependent ...metallo-β-lactamases (MBLs). Avibactam, the first clinically useful non-β-lactam β-lactamase inhibitor, is a broad-spectrum SBL inhibitor, which is used in combination with a cephalosporin antibiotic (ceftazidime). There are multiple reports on the interaction of avibactam with SBLs but few such studies with MBLs. We report biochemical and biophysical studies on the binding and reactivity of avibactam with representatives from all 3 MBL subfamilies (B1, B2, and B3). Avibactam has only limited or no activity versus MBL-mediated resistance in pathogens. Avibactam does not inhibit MBLs and binds only weakly to most of the MBLs tested; in some cases, avibactam undergoes slow hydrolysis of one of its urea N-CO bonds followed by loss of CO2, in a process different from that observed with the SBLs studied. The results suggest that while the evolution of MBLs that more efficiently catalyze avibactam hydrolysis should be anticipated, pursuing the development of dual-action SBL and MBL inhibitors based on the diazabicyclooctane core of avibactam may be productive.
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