Epistasis arises from shifting the rate-limiting step during enzyme evolution of a ß-lactamase.

Epistasis, the non-additive effect of mutations, can provide combinatorial improvements to enzyme activity that substantially exceed the gains from individual mutations. Yet the molecular mechanisms of epistasis remain elusive, undermining our ability to predict pathogen evolution and engineer biocatalysts. Here we reveal how directed evolution of a ß-lactamase yielded highly epistatic activity enhancements. Evolution selected four mutations that increase antibiotic resistance 40-fold, despite their marginal individual effects (≤2-fold). Synergistic improvements coincided with the introduction of super-stochiometric burst kinetics, indicating that epistasis is rooted in the enzyme's conformational dynamics. Our analysis reveals that epistasis stemmed from distinct effects of each mutation on the catalytic cycle. The initial mutation increased protein flexibility and accelerated substrate binding, which is rate-limiting in the wild-type enzyme. Subsequent mutations predominantly boosted the chemical steps by fine-tuning substrate interactions. Our work identifies an overlooked cause for epistasis: changing the rate-limiting step can result in substantial synergy that boosts enzyme activity.

The Biofilm Lifestyle Shapes the Evolution of ß-Lactamases.

The evolutionary relationship between the biofilm lifestyle and antibiotic resistance enzymes remains a subject of limited understanding. Here, we investigate how ß-lactamases affect biofilm formation in Vibrio cholerae and how selection for a biofilm lifestyle impacts the evolution of these enzymes. Genetically diverse ß-lactamases expressed in V. cholerae displayed a strong inhibitory effect on biofilm production. To understand how natural evolution affects this antagonistic pleiotropy, we randomly mutagenized a ß-lactamase and selected for elevated biofilm formation. Our results revealed that biofilm evolution selects for ß-lactamase variants able to hydrolyze ß-lactams without inhibiting biofilms. Mutational analysis of evolved variants demonstrated that restoration of biofilm development was achieved either independently of enzymatic function or by actively leveraging enzymatic activity. Taken together, the biofilm lifestyle can impose a profound selective pressure on antimicrobial resistance enzymes. Shedding light on such evolutionary interplays is of importance to understand the factors driving antimicrobial resistance.

Fluorinated captopril analogues inhibit metallo-ß-lactamases and facilitate structure determination of NDM-1 binding pose.

Bacterial resistance to the majority of clinically used ß-lactam antibiotics is a global health threat and, consequently, the driving force for the development of metallo-ß-lactamase (MBL) inhibitors. The rapid evolution of new MBLs calls for new strategies and tools for inhibitor development. In this study, we designed and developed a series of trifluoromethylated captopril analogues as probes for structural studies of enzyme-inhibitor binding. The new compounds showed activity comparable to the non-fluorinated inhibitors against the New Delhi Metallo-ß-lactamase-1 (NDM-1). The most active compound, a derivative of D-captopril, exhibited an IC50 value of 0.3 µM. Several compounds demonstrated synergistic effects, restoring the effect of meropenem and reducing the minimum inhibitory concentration (MIC) values in NDM-1 (up to 64-fold), VIM-2 (up to 8-fold) and IMP-26 (up to 8-fold) harbouring Escherichia coli. NMR spectroscopy and molecular docking of one representative inhibitor determined the binding pose in NDM-1, demonstrating that fluorinated analogues of inhibitors are a valuable tool for structural studies of MBL-inhibitor complexes.

Kinetics, Thermodynamics, and Structural Effects of Quinoline-2-Carboxylates, Zinc-Binding Inhibitors of New Delhi Metallo-ß-lactamase-1 Re-sensitizing Multidrug-Resistant Bacteria for Carbapenems.

Carbapenem resistance mediated by metallo-ß-lactamases (MBL) such as New Delhi metallo-ß-lactamase-1 (NDM-1) has become a major factor threatening the efficacy of essential ß-lactam antibiotics. Starting from hit fragment dipicolinic acid (DPA), 8-hydroxy- and 8-sulfonamido-quinoline-2-carboxylic acids were developed as inhibitors of NDM-1 with highly improved inhibitory activity and binding affinity. The most active compounds formed reversibly inactive ternary protein-inhibitor complexes with two zinc ions as proven by native protein mass spectrometry and bio-layer interferometry. Modification of the NDM-1 structure with remarkable entropic gain was shown by isothermal titration calorimetry and NMR spectroscopy of isotopically labeled protein. The best compounds were potent inhibitors of NDM-1 and other representative MBL with no or little inhibition of human zinc-binding enzymes. These inhibitors significantly reduced the minimum inhibitory concentrations (MIC) of meropenem for multidrug-resistant bacteria recombinantly expressing blaNDM-1 as well as for several multidrug-resistant clinical strains at concentrations non-toxic to human cells.

Evolution of ß-lactamase-mediated cefiderocol resistance.

BACKGROUND: Cefiderocol is a novel siderophore ß-lactam with improved hydrolytic stability toward ß-lactamases, including carbapenemases, achieved by combining structural moieties of two clinically efficient cephalosporins, ceftazidime and cefepime. Consequently, cefiderocol represents a treatment alternative for infections caused by MDR Gram-negatives. OBJECTIVES: To study the role of cefiderocol on resistance development and on the evolution of ß-lactamases from all Ambler classes, including KPC-2, CTX-M-15, NDM-1, CMY-2 and OXA-48. METHODS: Directed evolution, using error-prone PCR followed by selective plating, was utilized to investigate how the production and the evolution of different ß-lactamases cause changes in cefiderocol susceptibility determined using microbroth dilution assays (MIC and IC50). RESULTS: We found that the expression of blaOXA-48 did not affect cefiderocol susceptibility. On the contrary, the expression of blaKPC-2, blaCMY-2, blaCTX-M-15 and blaNDM-1 substantially reduced cefiderocol susceptibility by 4-, 16-, 8- and 32-fold, respectively. Further, directed evolution on these enzymes showed that, with the acquisition of only 1-2 non-synonymous mutations, all ß-lactamases were evolvable to further cefiderocol resistance by 2- (NDM-1, CTX-M-15), 4- (CMY-2), 8- (OXA-48) and 16-fold (KPC-2). Cefiderocol resistance development was often associated with collateral susceptibility changes including increased resistance to ceftazidime and ceftazidime/avibactam as well as functional trade-offs against different ß-lactam drugs. CONCLUSIONS: The expression of contemporary ß-lactamase genes can potentially contribute to cefiderocol resistance development and the acquisition of mutations in these genes results in enzymes adapting to increasing cefiderocol concentrations. Resistance development caused clinically important cross-resistance, especially against ceftazidime and ceftazidime/avibactam.

Evolution of ß-lactamases and enzyme promiscuity.

ß-Lactamases represent one of the most prevalent resistance mechanisms against ß-lactam antibiotics. Beyond their clinical importance, they have also become key models in enzymology and evolutionary biochemistry. A global understanding of their evolution and sequence and functional diversity can therefore aid a wide set of different disciplines. Interestingly, ß-lactamases have evolved multiple times from distinct evolutionary origins, with ancestries that reach back billions of years. It is therefore no surprise that these enzymes exhibit diverse structural features and enzymatic mechanisms. In this review, we provide a bird's eye view on the evolution of ß-lactamases within the two enzyme superfamilies-i.e. the penicillin-binding protein-like and metallo-ß-lactamase superfamily-through phylogenetics. We further discuss potential evolutionary origins of each ß-lactamase class by highlighting signs of evolutionary connections in protein functions between ß-lactamases and other enzymes, especially cases of enzyme promiscuity.

Cryptic ß-Lactamase Evolution Is Driven by Low ß-Lactam Concentrations.

Our current understanding of how low antibiotic concentrations shape the evolution of contemporary ß-lactamases is limited. Using the widespread carbapenemase OXA-48, we tested the long-standing hypothesis that selective compartments with low antibiotic concentrations cause standing genetic diversity that could act as a gateway to developing clinical resistance. Here, we subjected Escherichia coli expressing blaOXA-48, on a clinical plasmid, to experimental evolution at sub-MICs of ceftazidime. We identified and characterized seven single variants of OXA-48. Susceptibility profiles and dose-response curves showed that they increased resistance only marginally. However, in competition experiments at sub-MICs of ceftazidime, they demonstrated strong selectable fitness benefits. Increased resistance was also reflected in elevated catalytic efficiencies toward ceftazidime. These changes are likely caused by enhanced flexibility of the Ω- and ß5-ß6 loops and fine-tuning of preexisting active site residues. In conclusion, low-level concentrations of ß-lactams can drive the evolution of ß-lactamases through cryptic phenotypes which may act as stepping-stones toward clinical resistance.IMPORTANCE Very low antibiotic concentrations have been shown to drive the evolution of antimicrobial resistance. While substantial progress has been made to understand the driving role of low concentrations during resistance development for different antimicrobial classes, the importance of ß-lactams, the most commonly used antibiotics, is still poorly studied. Here, we shed light on the evolutionary impact of low ß-lactam concentrations on the widespread ß-lactamase OXA-48. Our data indicate that the exposure to ß-lactams at very low concentrations enhances ß-lactamase diversity and drives the evolution of ß-lactamases by significantly influencing their substrate specificity. Thus, in contrast to high concentrations, low levels of these drugs may substantially contribute to the diversification and divergent evolution of these enzymes, providing a standing genetic diversity that can be selected and mobilized when antibiotic pressure increases.

Structural studies of triazole inhibitors with promising inhibitor effects against antibiotic resistance metallo-ß-lactamases.

Metallo-ß-lactamases (MBLs) are an emerging cause of bacterial antibiotic resistance by hydrolysing all classes of ß-lactams except monobactams, and the MBLs are not inhibited by clinically available serine-ß-lactamase inhibitors. Two of the most commonly encountered MBLs in clinical isolates worldwide - the New Delhi metallo-ß-lactamase (NDM-1) and the Verona integron-encoded metallo-ß-lactamase (VIM-2) - are included in this study. A series of several NH-1,2,3-triazoles was prepared by a three-step protocol utilizing Banert cascade reaction as the key step. The inhibitor properties were evaluated in biochemical assays against the MBLs VIM-2, NDM-1 and GIM-1, and VIM-2 showed IC50 values down to nanomolar range. High-resolution crystal structures of four inhibitors in complex with VIM-2 revealed hydrogen bonds from the triazole inhibitors to Arg228 and to the backbone of Ala231 or Asn233, along with hydrophobic interactions to Trp87, Phe61 and Tyr67. The inhibitors show reduced MIC in synergy assays with Pseudomonas aeruginosa and Escherichia coli strains harbouring VIM enzymes. The obtained results will be useful for further structural guided design of MBL inhibitors.

Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1.

BACKGROUND: MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to ß-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure-activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. OBJECTIVES: To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. METHODS: The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different ß-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). RESULTS: Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. CONCLUSIONS: These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

ZN148 Is a Modular Synthetic Metallo-ß-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo.

Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (ß-lactamases able to inactivate carbapenems) have been identified in both serine ß-lactamase (SBL) and metallo-ß-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 µM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

Synthesis and biological evaluation of zinc chelating compounds as metallo-ß-lactamase inhibitors.

The syntheses of metallo-ß-lactamase inhibitors comprising chelating moieties, with varying zinc affinities, and peptides partly inspired from bacterial peptide sequences, have been undertaken. The zinc chelator strength was varied using the following chelators, arranged in order of ascending binding affinity: dipicolylamine (DPA, tridentate), dipicolyl-1,2,3-triazolylmethylamine (DPTA, tetradentate) dipicolyl ethylenediamine (DPED, tetradentate) and trispicolyl ethylenediamine (TPED, pentadentate). The chosen peptides were mainly based on the known sequence of the C-terminus of the bacterial peptidoglycan precursors. Biological evaluation on clinical bacterial isolates, harbouring either the NDM-1 or VIM-2 metallo-ß-lactamase, showed a clear relationship between the zinc chelator strength and restoration of meropenem activity. However, evaluation of toxicity on different cancer cell lines demonstrated a similar trend, and thus inclusion of the bacterial peptides did possess rather high toxicity towards eukaryotic cells.

OXA-48-Mediated Ceftazidime-Avibactam Resistance Is Associated with Evolutionary Trade-Offs.

Infections due to carbapenemase-producing Gram-negative pathogens are associated with limited treatment options and consequently lead to increased mortality and morbidity. In response, combinations of existing ß-lactams and novel ß-lactamase inhibitors, such as ceftazidime-avibactam (CAZ-AVI), have been developed as alternative treatment options. To understand the development of resistance and evolutionary trajectories under CAZ-AVI exposure, we studied the effects of ceftazidime (CAZ) and CAZ-AVI on the carbapenemase OXA-48 and the epidemic OXA-48 plasmid in Escherichia coli Exposure of CAZ and CAZ-AVI resulted in single (P68A) and double (P68A,Y211S) amino acid substitutions in OXA-48, respectively. The antimicrobial susceptibility data and enzyme kinetics showed that the P68A substitution was responsible for an increased activity toward CAZ, whereas P68A,Y211S led to a decrease in the inhibitory activity of AVI. X-ray crystallography and molecular modeling of the mutants demonstrated increased flexibility within the active site, which could explain the elevated CAZ hydrolysis and reduced inhibitory activity of AVI. Interestingly, these substitutions resulted in collateral effects compromising the activity of OXA-48 toward carbapenems and penicillins. Moreover, exposure to CAZ-AVI selected for mutations within the OXA-48-encoding plasmid that severely reduced fitness in the absence of antimicrobial selection. These evolutionary trade-offs may contribute to limit the evolution of OXA-48-mediated CAZ and CAZ-AVI resistance, as well as potentially resensitize isolates toward other therapeutic alternatives.IMPORTANCE The recent introduction of novel ß-lactam/ß-lactamase inhibitor combinations like ceftazidime-avibactam has increased our ability to treat infections caused by multidrug-resistant Gram-negative bacteria, including carbapenemase-producing Enterobacterales However, the increasing number of cases of reported resistance to ceftazidime-avibactam is a concern. OXA-48 is a carbapenemase that has no significant effect on ceftazidime, but is inhibited by avibactam. Since isolates with OXA-48 frequently harbor extended-spectrum ß-lactamases that are inhibited by avibactam, it is likely that ceftazidime-avibactam will be used to treat infections caused by OXA-48-producing Enterobacterales. Our data show that exposure to ceftazidime-avibactam can lead to changes in OXA-48, resulting in increased ability to hydrolyze ceftazidime and withstand the inhibitory effect of avibactam. Thus, resistance toward ceftazidime-avibactam among OXA-48-producing Enterobacterales should be monitored. Interestingly, the compromising effect of the amino acid substitutions in OXA-48 on other ß-lactams and the effect of ceftazidime-avibactam exposure on the epidemic OXA-48 plasmid indicate that the evolution of ceftazidime-avibactam resistance comes with collateral effects.

Synthesis and Preclinical Evaluation of TPA-Based Zinc Chelators as Metallo-ß-lactamase Inhibitors.

The rise of antimicrobial resistance (AMR) worldwide and the increasing spread of multi-drug-resistant organisms expressing metallo-ß-lactamases (MBL) require the development of efficient and clinically available MBL inhibitors. At present, no such inhibitor is available, and research is urgently needed to advance this field. We report herein the development, synthesis, and biological evaluation of chemical compounds based on the selective zinc chelator tris-picolylamine (TPA) that can restore the bactericidal activity of Meropenem (MEM) against Pseudomonas aeruginosa and Klebsiella pneumoniae expressing carbapenemases Verona integron-encoded metallo-ß-lactamase (VIM-2) and New Delhi metallo-ß-lactamase 1 (NDM-1), respectively. These adjuvants were prepared via standard chemical methods and evaluated in biological assays for potentiation of MEM against bacteria and toxicity (IC50) against HepG2 human liver carcinoma cells. One of the best compounds, 15, lowered the minimum inhibitory concentration (MIC) of MEM by a factor of 32-256 at 50 µM within all tested MBL-expressing clinical isolates and showed no activity toward serine carbapenemase expressing isolates. Biochemical assays with purified VIM-2 and NDM-1 and 15 resulted in inhibition kinetics with kinact/ KI of 12.5 min-1 mM-1 and 0.500 min-1 mM-1, respectively. The resistance frequency of 15 at 50 µM was in the range of 10-7 to 10-9. 15 showed good tolerance in HepG2 cells with an IC50 well above 100 µM, and an in vivo study in mice showed no acute toxic effects even at a dose of 128 mg/kg.