I04-Macromolecular Crystallography
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Clive S.
Mason
,
Tim
Avis
,
Chenlin
Hu
,
Nabeetha
Nagalingam
,
Manikhandan
Mudaliar
,
Chris
Coward
,
Khurshida
Begum
,
Kathleen
Gajewski
,
M. Jahangir
Alam
,
Eugenie
Bassères
,
Stephen
Moss
,
Stefanie
Reich
,
Esther
Duperchy
,
Keith R.
Fox
,
Kevin W.
Garey
,
David J.
Powell
Open Access
Abstract: Clostridioides difficile infection (CDI) causes substantial morbidity and mortality worldwide with limited antibiotic treatment options. Ridinilazole is a precision bisbenzimidazole antibiotic being developed to treat CDI and reduce unacceptably high rates of infection recurrence in patients. Although in late clinical development, the precise mechanism of action by which ridinilazole elicits its bactericidal activity has remained elusive. Here, we present conclusive biochemical and structural data to demonstrate that ridinilazole has a primary DNA binding mechanism, with a co-complex structure confirming binding to the DNA minor groove. Additional RNA-seq data indicated early pleiotropic changes to transcription, with broad effects on multiple C. difficile compartments and significant effects on energy generation pathways particularly. DNA binding and genomic localization was confirmed through confocal microscopy utilizing the intrinsic fluorescence of ridinilazole upon DNA binding. As such, ridinilazole has the potential to be the first antibiotic approved with a DNA minor groove binding mechanism of action.
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Apr 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[19800]
Open Access
Abstract: The β-lactamase of Mycobacterium tuberculosis, BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli. The variant that was found to confer most inhibitor resistance, K234R, as well as variant G132N that was found previously, were characterized using X-ray crystallography and NMR relaxation experiments to probe structural and dynamic properties. The G132N mutant exists in solution in two, almost equally populated conformations that exchange with a rate of ca. 88 s−1. The conformational change affects a broad region of the enzyme. The crystal structure reveals that the Asn132 side chain forces the peptide bond between Ser104 and Ile105 in a cis-conformation. The crystal structure suggests multiple conformations for several side chains (e.g. Ser104, Ser130) and a short loop (214-216). In the K234R mutant, the active site dynamics are significantly diminished with respect to the wild type enzyme. These results show that multiple evolutionary routes are available to increase inhibitor resistance in BlaC and that active site dynamics on the millisecond time scale are not required for catalytic function.
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May 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19069, 19458]
Open Access
Abstract: β-Lactam antibiotics are presently the most important treatments for infections by pathogenic Escherichia coli, but their use is increasingly compromised by β-lactamases, including the chromosomally encoded class C AmpC serine-β-lactamases (SBL). The diazabicyclooctane (DBO) avibactam is a potent AmpC inhibitor; the clinical success of avibactam combined with ceftazidime has stimulated efforts to optimise the DBO core. We report kinetic and structural studies, including four high resolution crystal structures, concerning inhibition of the AmpC serine-β-lactamase from E. coli (AmpCEC) by clinically relevant DBO-based inhibitors: avibactam, relebactam, nacubactam, and zidebactam. Kinetic analyses and mass spectrometry-based assays were used to study their mechanisms of AmpCEC inhibition. The results reveal that, under our assay conditions, zidebactam manifests increased potency (Kiapp 0.69 μM) against AmpCEC compared to the other DBOs (Kiapp 5.0-7.4 μM) due to an ∼ 10 fold accelerated carbamoylation-rate. However, zidebactam also has an accelerated off-rate and with sufficient preincubation time all the DBOs manifest similar potencies. Crystallographic analyses indicate a greater conformational freedom of the AmpCEC-zidebactam carbamoyl-complex compared to those for the other DBOs. The results suggest carbamoyl-complex lifetime should be a consideration in development of DBO-based SBL inhibitors for the clinically important class C SBLs.
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Nov 2020
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15868, 22906]
Abstract: Spectinomycin is a ribosome-binding antibiotic that blocks the translocation step of translation. A prevalent resistance mechanism is the modification of the drug by aminoglycoside nucleotidyl transferase (ANT) enzymes of the spectinomycin-specific ANT (9) family or by the dual-specificity ANT(3") (9) family that also acts on streptomycin. We previously reported the structural mechanism of streptomycin modification by the ANT(3") (9) AadA from Salmonella enterica. ANT (9) from Enterococcus faecalis adenylates the 9-hydroxyl of spectinomycin. We here present the first structures of spectinomycin bound to an ANT enzyme. Structures were solved for ANT (9) in apo form, in complex with ATP, spectinomycin and magnesium or in complex with only spectinomycin. ANT (9) shows similar overall structure as AadA with an N-terminal nucleotidyltransferase domain and a C-terminal α-helical domain. Spectinomycin binds close to the entrance of the interdomain cleft, while ATP is buried at the bottom. Upon drug binding, the C-terminal domain rotates by 14 degrees to close the cleft, allowing contacts of both domains with the drug. Comparison with AadA shows that spectinomycin specificity is explained by a straight α5 helix and a shorter α5-α6 loop that would clash with the larger streptomycin substrate. In the active site, we observe two magnesium ions, one of them in a previously un-observed position that may activate the 9-hydroxyl for deprotonation by the catalytic base Glu-86. The observed binding mode for spectinomycin suggests that also spectinamides and aminomethyl spectinomycins, recent spectinomycin analogues with expansions in position 4 of the C ring, will be subjected to modification by ANT (9) and ANT(3") (9) enzymes.
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Apr 2020
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I04-Macromolecular Crystallography
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Jodie C.
Hamrick
,
Jean-Denis
Docquier
,
Tsuyoshi
Uehara
,
Cullen L.
Myers
,
David A.
Six
,
Cassandra L.
Chatwin
,
Kaitlyn J.
John
,
Salvador F.
Vernacchio
,
Susan M.
Cusick
,
Robert E. L.
Trout
,
Cecilia
Pozzi
,
Filomena
De Luca
,
Manuela
Benvenuti
,
Stefano
Mangani
,
Bin
Liu
,
Randy W.
Jackson
,
Greg
Moeck
,
Luigi
Xerri
,
Christopher J.
Burns
,
Daniel C.
Pevear
,
Denis M.
Daigle
Diamond Proposal Number(s):
[8574]
Open Access
Abstract: As shifts in the epidemiology of β-lactamase-mediated resistance continue, carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are the most urgent threats. Although approved β-lactam (BL)–β-lactamase inhibitor (BLI) combinations address widespread serine β-lactamases (SBLs), such as CTX-M-15, none provide broad coverage against either clinically important serine-β-lactamases (KPC, OXA-48) or clinically important metallo-β-lactamases (MBLs; e.g., NDM-1). VNRX-5133 (taniborbactam) is a new cyclic boronate BLI that is in clinical development combined with cefepime for the treatment of infections caused by β-lactamase-producing CRE and CRPA. Taniborbactam is the first BLI with direct inhibitory activity against Ambler class A, B, C, and D enzymes. From biochemical and structural analyses, taniborbactam exploits substrate mimicry while employing distinct mechanisms to inhibit both SBLs and MBLs. It is a reversible covalent inhibitor of SBLs with slow dissociation and a prolonged active-site residence time (half-life, 30 to 105 min), while in MBLs, it behaves as a competitive inhibitor, with inhibitor constant (Ki) values ranging from 0.019 to 0.081 μM. Inhibition is achieved by mimicking the transition state structure and exploiting interactions with highly conserved active-site residues. In microbiological testing, taniborbactam restored cefepime activity in 33/34 engineered Escherichia coli strains overproducing individual enzymes covering Ambler classes A, B, C, and D, providing up to a 1,024-fold shift in the MIC. Addition of taniborbactam restored the antibacterial activity of cefepime against all 102 Enterobacterales clinical isolates tested and 38/41 P. aeruginosa clinical isolates tested with MIC90s of 1 and 4 μg/ml, respectively, representing ≥256- and ≥32-fold improvements, respectively, in antibacterial activity over that of cefepime alone. The data demonstrate the potent, broad-spectrum rescue of cefepime activity by taniborbactam against clinical isolates of CRE and CRPA.
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Feb 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212]
Open Access
Abstract: 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 treatment of infections by multi-drug resistant Enterobacteriaceae. 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 - 5 μM). Against purified class A SBLs, relebactam is an inferior inhibitor compared to the clinically approved DBO avibactam, (9 to 120-fold differences in IC50). Minimum inhibitory concentration assays indicate relebactam potentiates β-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia 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 compared to 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.
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Aug 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14980]
Abstract: The outer membrane of Gram-negative bacteria presents an efficient barrier to the permeation of antimicrobial molecules. One strategy pursued to circumvent this obstacle is to hijack transport systems for essential nutrients, such as iron. BAL30072 and MC-1 are two monobactams conjugated to a dihydroxypyridone siderophore that are active against Pseudomonas aeruginosa and Acinetobacter baumannii. Here, we investigated the mechanism of action of these molecules in A. baumannii. We identified two novel TonB-dependent receptors, termed Ab-PiuA and Ab-PirA, that are required for the antimicrobial activity of both agents. Deletion of either piuA or pirA in A. baumannii resulted in 4- to 8-fold-decreased susceptibility, while their overexpression in the heterologous host P. aeruginosa increased susceptibility to the two siderophore-drug conjugates by 4- to 32-fold. The crystal structures of PiuA and PirA from A. baumannii and their orthologues from P. aeruginosa were determined. The structures revealed similar architectures; however, structural differences between PirA and PiuA point to potential differences between their cognate siderophore ligands. Spontaneous mutants, selected upon exposure to BAL30072, harbored frameshift mutations in either the ExbD3 or the TonB3 protein of A. baumannii, forming the cytoplasmic-membrane complex providing the energy for the siderophore translocation process. The results of this study provide insight for the rational design of novel siderophore-drug conjugates against problematic Gram-negative pathogens.
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Mar 2017
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[8574]
Abstract: BEL-1 is an acquired class A extended-spectrum β-lactamase (ESBL) found in Pseudomonas aeruginosa clinical isolates from Belgium which is divergent from other ESBLs (maximum identity of 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem, and moxalactam. Crystals of BEL-1 were obtained at pH 5.6, and the structure of native BEL-1 was determined from orthorhombic and monoclinic crystal forms at 1.60-Å and 1.48-Å resolution, respectively. By soaking native BEL-1 crystals, complexes with imipenem (monoclinic form, 1.79-Å resolution) and moxalactam (orthorhombic form, 1.85-Å resolution) were also obtained. In the acyl-enzyme complexes, imipenem and moxalactam differ by the position of the α-substituent and of the carbonyl oxygen (in or out of the oxyanion hole). More surprisingly, the Ω-loop, which includes the catalytically relevant residue Glu166, was found in different conformations in the various subunits, resulting in the Glu166 side chain being rotated out of the active site or even in displacement of its Cα atom up to approximately 10 Å. A BEL-1 variant showing the single Leu162Phe substitution (BEL-2) confers a higher level of resistance to CAZ, CTX, and FEP and shows significantly lower Km values than BEL-1, especially with oxyiminocephalosporins. BEL-1 Leu162 is located at the beginning of the Ω-loop and is surrounded by Phe72, Leu139, and Leu148 (contact distances, 3.5 to 3.9 Å). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe162 found in BEL-2 without altering neighboring residues or the Ω-loop itself, thus likely causing an important alteration of the enzyme kinetic properties.
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Sep 2016
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[313]
Abstract: β-Lactamase production increasingly threatens the effectiveness of β-lactams, which remain a mainstay of antimicrobial chemotherapy. New activities emerge through both mutation of previously known β-lactamases and mobilization from environmental reservoirs. The spread of metallo-β-lactamases (MBLs) represents a particular challenge because of their typically broad-spectrum activities encompassing carbapenems, in addition to other β-lactam classes. Increasingly, genomic and metagenomic studies have revealed the distribution of putative MBLs in the environment, but in most cases their activity against clinically relevant β-lactams and, hence, the extent to which they can be considered a resistance reservoir remain uncharacterized. Here we characterize the product of one such gene, blaRm3, identified through functional metagenomic sampling of an environment with high levels of biocide exposure. blaRm3 encodes a subclass B3 MBL that, when expressed in a recombinant Escherichia coli strain, is exported to the bacterial periplasm and hydrolyzes clinically used penicillins, cephalosporins, and carbapenems with an efficiency limited by high Km values. An Rm3 crystal structure reveals the MBL superfamily αβ/βα fold, which more closely resembles that in mobilized B3 MBLs (AIM-1 and SMB-1) than other chromosomal enzymes (L1 or FEZ-1). A binuclear zinc site sits in a deep channel that is in part defined by a relatively extended N terminus. Structural comparisons suggest that the steric constraints imposed by the N terminus may limit its affinity for β-lactams. Sequence comparisons identify Rm3-like MBLs in numerous other environmental samples and species. Our data suggest that Rm3-like enzymes represent a distinct group of B3 MBLs with a wide distribution and can be considered an environmental reservoir of determinants of β-lactam resistance.
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Sep 2016
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I04-Macromolecular Crystallography
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Abstract: Metallo-β-lactamases (MBLs) are of increasing clinical significance; the development of clinically useful MBL inhibitors is challenged by the rapid evolution of variant MBLs. The Verona integron-borne metallo-β-lactamase (VIM) enzymes are among the most widely distributed MBLs, with >40 VIM variants having been reported. We report on the crystallographic analysis of VIM-5 and comparison of biochemical and biophysical properties of VIM-1, VIM-2, VIM-4, VIM-5, and VIM-38. Recombinant VIM variants were produced and purified, and their secondary structure and thermal stabilities were investigated by circular dichroism analyses. Steady-state kinetic analyses with a representative panel of β-lactam substrates were carried out to compare the catalytic efficiencies of the VIM variants. Furthermore, a set of metalloenzyme inhibitors were screened to compare their effects on the different VIM variants. The results reveal only small variations in the kinetic parameters of the VIM variants but substantial differences in their thermal stabilities and inhibition profiles. Overall, these results support the proposal that protein stability may be a factor in MBL evolution and highlight the importance of screening MBL variants during inhibitor development programs.
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Feb 2016
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