I03-Macromolecular Crystallography
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Gabriela
D'Amico González
,
María Margarita
Rodríguez
,
Pedro
Penzotti
,
Florencia
Brunetti
,
Barbara
Ghiglione
,
Luke A.
Moe
,
Daniela
Centrón
,
Gabriel
Gutkind
,
Lin
Gao
,
Shozeb
Haider
,
Rachel A.
Powers
,
Sebastián
Klinke
,
Pablo
Power
Diamond Proposal Number(s):
[29507]
Open Access
Abstract: Wild-type LRA-5, recovered from Alaskan soil samples, shares no more than 33% amino acid sequence identity with enzymes from pathogens like PER β-lactamases. Recombinant E. coli expressing wild-type LRA-5 and its engineered variants LRA-5Y69Q and LRA-5V166E showed MIC values equivalent to control strains. However, LRA-5Y69Q/V166E displayed MICs above the resistant breakpoint for some β-lactams. Kinetic parameters correlated with the MICs, showing that the catalytic efficiency of LRA-5Y69Q/V166E was comparable to those from class A β-lactamases, such as CTX-M-15, PER-2, and KPC-2. LRA-5Y69Q/V166E exhibited kcat/Km values up to 11,000-fold higher compared to wild-type LRA-5, which is associated with the presence of Glu166. The X-ray crystallographic structure of wild-type LRA-5 (1.80 Å; PDB 8EO5) shows that the lack of both Glu166 and a deacylation water molecule contributes to a biologically insignificant activity. Interactions observed between LRA-5 and ceftazidime (2.35 Å; PDB 8EO6) show structural conservation with other β-lactamases. In contrast, the crystallographic structure of LRA-5Y69Q/V166E (2.15 Å; PDB 8EO7) bears a deacylation water molecule that is associated with the increase in catalytic activity compared to the wild-type variant. Circular dichroism results confirm that amino acid substitutions in LRA-5 do not affect the overall content of the secondary/tertiary structures. Evidence suggests that alternative evolutionary paths could have occurred for β-lactamases like LRA-5, produced by environmental microorganisms: (i) proteins having similar structural features than active β-lactamases may accumulate a small number of mutations (e.g., Y69Q/V166E) to yield active enzymes and (ii) the β-lactamase fold may have lost key residues in the absence of antibiotics.
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Nov 2025
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Open Access
Abstract: β-lactams demonstrate promising in vitro activity against Mycobacterium species and are being explored for tuberculosis treatment; however, evidence of their in vivo efficacy versus Mycobacterium tuberculosis remains limited. To achieve broad clinically relevant potency, optimization of the classical β-lactam scaffolds or development of new or non-β-lactam inhibitors for mycobacterial transpeptidases is likely required. In mycobacteria, potential targets of β-lactams include l,d-transpeptidases (Ldts) and penicillin-binding proteins (PBPs). Reports suggest that dual inhibition of Ldts and PBPs may be necessary to achieve effective anti-mycobacterial activity, yet the specific contributions of Ldt and PBP inhibition to the β-lactam antibacterial mechanisms are poorly understood. We used fluorogenic substrate mimics to investigate the effects of β-lactams and reported LdtMt2 inhibitors on Mycobacterium smegmatis (Msm), assessing their impacts on Ldt and PBP transpeptidase activities in living cells. The results reveal a statistically significant correlation between both Ldt and PBP inhibition and Msm growth suppression; under the tested conditions, a stronger correlation between Ldt inhibition and Msm growth suppression was observed. Notably, apparent inhibition of both PBPs and Ldts was observed with all active inhibitors, though β-lactams manifest increased potency of PBP inhibition. The combination of the β-lactams meropenem and faropenem with selected LdtMt2 inhibitors manifested an additive inhibitory effect against Msm. Our results highlight the importance of further optimizing β-lactam efficacy versus mycobacterial PBPs and Ldt transpeptidases.
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Sep 2025
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I03-Macromolecular Crystallography
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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.
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Jun 2025
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I04-Macromolecular Crystallography
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Dario
Akaberi
,
Monireh
Pourghasemi Lati
,
Janina
Krambrich
,
Julia
Berger
,
Grace
Neilsen
,
Emilia
Strandback
,
S. Pauliina
Turunen
,
Johan
Wannberg
,
Hjalmar
Gullberg
,
Martin
Moche
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Praveen Kumar
Chinthakindi
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Tomas
Nyman
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Stefan G.
Sarafianos
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Anja
Sandström
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Josef D.
Järhult
,
Kristian
Sandberg
,
Åke
Lundkvist
,
Oscar
Verho
,
Johan
Lennerstrand
Diamond Proposal Number(s):
[21625]
Open Access
Abstract: In vitro screening of large compound libraries with automated high-throughput screening is expensive and time-consuming and requires dedicated infrastructures. Conversely, the selection of DNA-encoded chemical libraries (DECLs) can be rapidly performed with routine equipment available in most laboratories. In this study, we identified novel inhibitors of SARS-CoV-2 main protease (Mpro) through the affinity-based selection of the DELopen library (open access for academics), containing 4.2 billion compounds. The identified inhibitors were peptide-like compounds containing an N-terminal electrophilic group able to form a covalent bond with the nucleophilic Cys145 of Mpro, as confirmed by x-ray crystallography. This DECL selection campaign enabled the discovery of the unoptimized compound SLL11 (IC50 = 30 nM), proving that the rapid exploration of large chemical spaces enabled by DECL technology allows for the direct identification of potent inhibitors avoiding several rounds of iterative medicinal chemistry. As demonstrated further by x-ray crystallography, SLL11 was found to adopt a highly unique U-shaped binding conformation, which allows the N-terminal electrophilic group to loop back to the S1′ subsite while the C-terminal amino acid sits in the S1 subsite. MP1, a close analog of SLL11, showed antiviral activity against SARS-CoV-2 in the low micromolar range when tested in Caco-2 and Calu-3 (EC50 = 2.3 µM) cell lines. As peptide-like compounds can suffer from low cell permeability and metabolic stability, the cyclization of the compounds will be explored in the future to improve their antiviral activity.
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Aug 2024
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I04-Macromolecular Crystallography
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Damien
Bonnard
,
Erwann
Le Rouzic
,
Matthew
Singer
,
Zhe
Yu
,
Frédéric
Le Strat
,
Claire
Batisse
,
Julien
Batisse
,
Céline
Amadori
,
Sophie
Chasset
,
Valerie E.
Pye
,
Stéphane
Emiliani
,
Benoit
Ledoussal
,
Marc
Ruff
,
François
Moreau
,
Peter
Cherepanov
,
Richard
Benarous
Diamond Proposal Number(s):
[25587]
Open Access
Abstract: HIV-1 integrase-LEDGF allosteric inhibitors (INLAIs) share the binding site on the viral protein with the host factor LEDGF/p75. These small molecules act as molecular glues promoting hyper-multimerization of HIV-1 IN protein to severely perturb maturation of viral particles. Herein, we describe a new series of INLAIs based on a benzene scaffold that display antiviral activity in the single digit nanomolar range. Akin to other compounds of this class, the INLAIs predominantly inhibit the late stages of HIV-1 replication. A series of high-resolution crystal structures revealed how these small molecules engage the catalytic core and the C-terminal domains of HIV-1 IN. No antagonism was observed between our lead INLAI compound BDM-2 and a panel of 16 clinical antiretrovirals. Moreover, we show that compounds retained high antiviral activity against HIV-1 variants resistant to IN strand transfer inhibitors and other classes of antiretroviral drugs. The virologic profile of BDM-2 and the recently completed single ascending dose phase I trial (ClinicalTrials.gov identifier: NCT03634085) warrant further clinical investigation for use in combination with other antiretroviral drugs. Moreover, our results suggest routes for further improvement of this emerging drug class.
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Jun 2023
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I04-Macromolecular Crystallography
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Clive S.
Mason
,
Tim
Avis
,
Chenlin
Hu
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Nabeetha
Nagalingam
,
Manikhandan
Mudaliar
,
Chris
Coward
,
Khurshida
Begum
,
Kathleen
Gajewski
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M. Jahangir
Alam
,
Eugenie
Bassères
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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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