ABSTRACT
Synonymous mutations result from the degeneracy of the genetic code. Most amino acids are encoded by two or more codons, and mutations that change a codon to another synonymous codon do not change the amino acid in the gene product. Historically, such mutations have been considered silent because they were assumed to have no to very little impact. However, research in the last few decades has produced several examples where synonymous mutations play important roles. These include optimizing expression by enhancing translation initiation and accelerating or decelerating translation elongation via codon usage and mRNA secondary structures, stabilizing mRNA molecules and preventing their breakdown before translation, and faulty protein folding or increased degradation due to enhanced ubiquitination and suboptimal secretion of proteins into the appropriate cell compartments. Some consequences of synonymous mutations, such as mRNA stability, can lead to different outcomes in prokaryotes and eukaryotes. Despite these examples, the significance of synonymous mutations in evolution and in causing disease in comparison to nonsynonymous mutations that do change amino acid residues in proteins remains controversial. Whether the molecular mechanisms described by which synonymous mutations affect organisms can be generalized remains poorly understood and warrants future research in this area.
Subject(s)
Protein Biosynthesis , Silent Mutation , Codon/genetics , Protein Biosynthesis/genetics , RNA, Messenger/genetics , Proteins/genetics , Amino Acids/genetics , Evolution, MolecularABSTRACT
Metallo-ß-lactamases (MBLs), also known as class B ß-lactamases (BBLs), are Zn(II)-containing enzymes able to inactivate a broad range of ß-lactams, the most commonly used antibiotics, including life-saving carbapenems. They have been known for about six decades, yet they have only gained much attention as a clinical problem for about three decades. The naming conventions of these enzymes have changed over time and followed various strategies, sometimes leading to confusion. We are summarizing the naming strategies of the currently known MBLs. These enzymes are quite diverse on the amino acid sequence level but structurally similar. Problems trying to describe conserved residues, such as Zn(II) ligands and other catalytically important residues, which have different numbers in different sequences, have led to the establishment of a standard numbering scheme for BBLs. While well intended, the standard numbering scheme is not trivial and has not been applied consistently. We revisit this standard numbering scheme and suggest some strategies for how its implementation could be made more accessible to researchers. Standard numbering facilitates the comparison of different enzymes as well as their interaction with novel antibiotics and BBL inhibitors.
ABSTRACT
Metallo-carbapenemases-mediated carbapenem-resistant Enterobacterales (CREs) has been acknowledged as "urgent threat" by the World Health Organization. The discovery of new strategies that block metallo-carbapenemases activity to reverse carbapenem resistance is an urgent need. In this study, a coumarin copper complex containing a PEG linker and glucose ligand, GluC-Cu, was used to reverse carbapenem resistance. Interestingly, it could effectively inhibit metallo-carbapenemases (NDM-1, IMP-1 and ImiS) with an IC50 value in the range of 0.23-1.21 µM, and simultaneously release the green fluorescence signal (GluC), therefore exhibiting self-reported inhibition performance. The inhibition mechanism of oxidizing Zn(II) thiolate site of NDM-1 from Cu2+ to Cu+ was verified by fluorescence assay, HR-MS, and XPS. Moreover, GluC-Cu in combination with meropenem showed excellent synergistic antibacterial effect to effectively combat E. coli expressing metallo-carbapenemases in vitro and in a mice infection model. This bifunctional metallo-carbapenemases inhibitor provides a novel chemical tool to overcome carbapenem resistance.
Subject(s)
Copper , Escherichia coli , Humans , Animals , Mice , Self Report , Copper/pharmacology , Anti-Bacterial Agents/pharmacology , beta-Lactamases , Carbapenems/pharmacology , Microbial Sensitivity TestsABSTRACT
The continuous emergence of multi-drug resistant pathogens co-expressing serine and metallo-carbapenemases seriously threatens the efficacy of carbapenem. Here, we report the first SeCN-derived dual inhibitor of serine and metallo-carbapenemases with IC50 values ranging from 0.0038 to 1.27 µg mL-1. The inhibitor was shown to form covalent bonds with Cys221 of NDM-1 and Ser70 of KPC-2, respectively, achieving selective labelling and cross-class inhibition for carbapenemases. Our results provide a potential strategy to develop clinically useful dual inhibitors targeting serine and metallo-carbapenemases to combat superbugs.
Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Anti-Bacterial Agents/chemistry , beta-Lactamases/chemistry , Carbapenems , Microbial Sensitivity TestsABSTRACT
The idea of personalized medicine came to fruition with sequencing the human genome; however, aside from a few cases, the genetic revolution has yet to materialize. Cardiovascular diseases are the leading cause of death globally, and hypertension is a common prelude to nearly all cardiovascular diseases. Thus, hypertension is an ideal candidate disease to apply tenants of personalized medicine to lessen cardiovascular disease. Herein is a survey that visually depicts the polymorphisms in the top eight antihypertensive targets. Although there are numerous genome-wide association studies regarding cardiovascular disease, few studies look at the effects of receptor polymorphisms on drug treatment. With 17,000+ polymorphisms in the combined target proteins examined, it is expected that some of the clinical variability in the treatment of hypertension is due to polymorphisms in the drug targets. Recent advances in techniques and technology, such as high throughput examination of single mutations, structure prediction, computational power for modeling, and CRISPR models of point mutations, allow for a relatively rapid and comprehensive examination of the effects of known and future polymorphisms on drug affinity and effects. As hypertension is easy to measure and has a plethora of clinically viable ligands, hypertension makes an excellent disease to study pharmacogenomics in the lab and the clinic. If the promises of personalized medicine are to materialize, a concerted effort to examine the effects polymorphisms have on drugs is required. A clinician with the knowledge of a patient's genotype can then prescribe drugs that are optimal for treating that specific patient.
Subject(s)
Cardiovascular Diseases , Hypertension , Antihypertensive Agents/pharmacology , Antihypertensive Agents/therapeutic use , Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/genetics , Genome-Wide Association Study , Humans , Hypertension/drug therapy , Hypertension/genetics , PharmacogeneticsABSTRACT
Serine/metallo-carbapenemase-coproducing pathogens, often referred to as "superbugs", are a significant clinical problem. They hydrolyze nearly all available ß-lactam antibiotics, especially carbapenems considered as last-resort antibiotics, seriously endangering efficacious antibacterial treatment. Despite the continuous global spread of carbapenem resistance, no dual-action inhibitors are available in therapy. This Perspective is the first systematic investigation of all chemotypes, modes of inhibition, and crystal structures of dual serine/metallo-carbapenemase inhibitors. An overview of the key strategy for designing dual serine/metallo-carbapenemase inhibitors and their mechanism of action is provided, as guiding rules for the development of clinically available dual inhibitors, coadministrated with carbapenems, to overcome the carbapenem resistance issue.
Subject(s)
Serine , beta-Lactamase Inhibitors , Anti-Bacterial Agents/pharmacology , Bacterial Proteins , Carbapenems/pharmacology , beta-Lactamase Inhibitors/chemistry , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/chemistryABSTRACT
ß-Lactams were the first class of antibiotics to be discovered and the second to be introduced into the clinic in the 1940s [...].
Subject(s)
Amino Acid Sequence , beta-Lactamase Inhibitors/chemistry , beta-Lactamases , Structure-Activity Relationship , beta-Lactamases/chemistry , beta-Lactamases/geneticsABSTRACT
Extremely drug-resistant (XDR) Acinetobacter baumannii is a notorious and frequently encountered pathogen demanding novel therapeutic interventions. An initial monoclonal antibody (MAb), C8, raised against A. baumannii capsule, proved a highly effective treatment against a minority of clinical isolates. To overcome this limitation, we broadened coverage by developing a second antibody for use in a combination regimen. We sought to develop an additional anti-A. baumannii MAb through hybridoma technology by immunizing mice with sublethal inocula of virulent, XDR clinical isolates not bound by MAb C8. We identified a new antibacterial MAb, 65, which bound to strains in a pattern distinct from and complementary to that of MAb C8. MAb 65 enhanced macrophage opsonophagocytosis of targeted strains and markedly improved survival in lethal bacteremic sepsis and aspiration pneumonia murine models of A. baumannii infection. MAb 65 was also synergistic with colistin, substantially enhancing protection compared to monotherapy. Treatment with MAb 65 significantly reduced blood bacterial density, ameliorated cytokine production (interleukin-1ß [IL-1ß], IL-6, IL-10, and tumor necrosis factor), and sepsis biomarkers. We describe a novel MAb targeting A. baumannii that broadens immunotherapeutic strain coverage, is highly potent and effective, and synergistically improves outcomes in combination with antibiotics.
Subject(s)
Acinetobacter Infections/immunology , Acinetobacter baumannii/immunology , Antibodies, Monoclonal/immunology , Acinetobacter Infections/blood , Acinetobacter Infections/microbiology , Animals , Anti-Bacterial Agents/immunology , Antibodies, Bacterial/immunology , Biomarkers/blood , Colistin/immunology , Cytokines/blood , Cytokines/immunology , Drug Resistance, Multiple, Bacterial/immunology , Mice , Microbial Sensitivity Tests/methods , Sepsis/blood , Sepsis/immunology , Sepsis/microbiologyABSTRACT
Nonsynonymous mutations are well documented in TEM ß-lactamases. The resulting amino acid changes often alter the conferred phenotype from broad spectrum (2b) conferred by TEM-1 to extended spectrum (2be), inhibitor resistant (2br), or both extended spectrum and inhibitor resistant (2ber). The encoding blaTEM genes also deviate in numerous synonymous mutations, which are not well understood. blaTEM-3 (2be), blaTEM-33 (2br), and blaTEM-109 (2ber) were studied in comparison to blaTEM-1blaTEM-33 was chosen for more detailed studies because it deviates from blaTEM-1 by a single nonsynonymous mutation and three additional synonymous mutations. Genes encoding the enzymes with only nonsynonymous or all (including synonymous) mutations plus all permutations between blaTEM-1 and blaTEM-33 were expressed in Escherichia coli cells. In disc diffusion assays, genes encoding TEM-3, TEM-33, and TEM-109 with all synonymous mutations resulted in higher resistance levels than genes without synonymous mutations. Disc diffusion assays with the 16 genes carrying all possible nucleotide change combinations between blaTEM-1 and blaTEM-33 indicated different susceptibilities for different variants. Nucleotide BLAST searches did not identify genes without synonymous mutations but did identify some without nonsynonymous mutations. Energies of possible secondary mRNA structures calculated with mfold are generally higher with synonymous mutations, suggesting that their role could be to destabilize the mRNA and facilitate its unfolding for efficient translation. In summary, our data indicate that transition from blaTEM-1 to other variant genes by simply acquiring the nonsynonymous mutations is not favored. Instead, synonymous mutations seem to support the transition to other variant genes with nonsynonymous mutations leading to different phenotypes.
Subject(s)
Escherichia coli Infections , beta-Lactamases , Escherichia coli/genetics , Humans , Microbial Sensitivity Tests , Silent Mutation/genetics , beta-Lactamases/geneticsABSTRACT
(1) Background: Metallo-ß-lactamases (MBLs) have raised concerns due to their ability to inactivate carbapenems and newer generation cephalosporins and the absence of clinically available MBL inhibitors. Their genes are often transferred horizontally, and the number of MBL variants has grown exponentially, with many newer variants showing enhanced enzyme activity or stability. In this study, we investigated a closely related group of variants from the IMP family that all contain the combination of mutations S115T and S119G relative to IMP-1. (2) Methods: The effects of each individual mutation and their combination in the IMP-1 sequence background in comparison to IMP-1 were investigated. Their ability to confer resistance and their in-cell expression levels were determined. All enzymes were purified, and their secondary structure and thermal stability were determined with circular dichroism. Their Zn(II) content and kinetic constants with a panel of ß-lactam antibiotics were determined. (3) Results: All four enzymes were viable and conferred resistance to all antibiotics tested except aztreonam. However, the single-mutant enzymes were slightly deficient, IMP-1S115T due to decreased enzyme activity and IMP-1-S119G due to decreased thermal stability and expression, while the double mutant did not show these defects. (4) Conclusions: These observations suggest that S119G was acquired due to its increased enzyme activity and S115T to suppress the thermal stability and expression defect introduced by S119G.
Subject(s)
Anti-Bacterial Agents/chemistry , Structure-Activity Relationship , beta-Lactamases/genetics , Amino Acid Substitution/genetics , Anti-Bacterial Agents/adverse effects , Gene Expression Regulation, Enzymologic , Humans , Inosine Monophosphate/chemistry , Kinetics , Microbial Sensitivity Tests , Mutation/genetics , Protein Structure, Secondary , beta-Lactam Resistance/genetics , beta-Lactamases/chemistryABSTRACT
The "superbug" infection caused by New Delhi metallo-ß-lactamase (NDM-1) has become an emerging threat. Monitoring NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. Here, we report an isothermal titration calorimetry (ITC) method that can monitor activity and inhibition of NDM-1 in live bacterial cells in real time. This method has been exemplified by monitoring of the activity and inhibition of the target enzyme and evaluating the breakdown of antibiotics by pathogenic bacteria expressing ß-lactamases. Cell-based studies demonstrate that the NDM-1 expressed in bacterial cells was inhibited by four known inhibitors ethylene diamine tetraacetic acid (EDTA), d-captopril, ebselen and azolylthioacetamide with fifty percent inhibitory concentration (IC50) values of 3.8, 48, 0.55, and 17.5 µM, respectively, which are in good agreement with the data from inhibition kinetics using UV-vis and NMR spectroscopy in vivo. This approach could be applied to screen and evaluate small molecule inhibitors of metallo-ß-lactamases (MßLs) in whole cells or to identify drug resistant bacteria.
Subject(s)
Anti-Bacterial Agents/metabolism , Calorimetry/methods , Escherichia coli/enzymology , beta-Lactamase Inhibitors/metabolism , beta-Lactamases/chemistry , beta-Lactamases/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cephalosporins/chemistry , Cephalosporins/metabolism , Cephalosporins/pharmacology , Drug Resistance, Bacterial , Escherichia coli/chemistry , Escherichia coli/drug effects , Escherichia coli/genetics , Kinetics , beta-Lactamase Inhibitors/chemistry , beta-Lactamase Inhibitors/pharmacologyABSTRACT
The global rise of metallo-ß-lactamases (MBLs) is problematic due to their ability to inactivate most ß-lactam antibiotics. MBL inhibitors that could be coadministered with and restore the efficacy of ß-lactams are highly sought after. In this study, we employ virtual screening of candidate MBL inhibitors without thiols or carboxylates to avoid off-target effects using the Avalanche software package, followed by experimental validation of the selected compounds. As target enzymes, we chose the clinically relevant B1 MBLs NDM-1, IMP-1, and VIM-2. Among 32 compounds selected from an approximately 1.5 million compound library, 6 exhibited IC50 values less than 40 µM against NDM-1 and/or IMP-1. The most potent inhibitors of NDM-1, IMP-1, and VIM-2 had IC50 values of 19 ± 2, 14 ± 1, and 50 ± 20 µM, respectively. While chemically diverse, the most potent inhibitors all contain combinations of hydroxyl, ketone, ester, amide, or sulfonyl groups. Docking studies suggest that these electron-dense moieties are involved in Zn(II) coordination and interaction with protein residues. These novel scaffolds could serve as the basis for further development of MBL inhibitors. A procedure for renaming NDM-1 residues to conform to the class B ß-lactamase (BBL) numbering scheme is also included.
Subject(s)
Drug Evaluation, Preclinical/methods , beta-Lactamase Inhibitors/chemistry , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/classification , beta-Lactamases/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Circular Dichroism , Computer Simulation , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Enzymologic , Mass Spectrometry , Models, Chemical , Molecular Structure , SoftwareABSTRACT
A series of rhodanines was constructed, their Z-configuration was confirmed by small molecule X-ray crystal structures, and their activity against metallo-ß-lactamases (MßLs) was measured. The obtained 26 molecules and a thioenolate specifically inhibited the MßL L1 with an IC50 range of 0.02-1.7 µM, and compounds 2h-m exhibited broad-spectrum inhibition of the MßLs NDM-1, VIM-2, ImiS, and L1 with IC50 values <16 µM. All inhibitors increased the antimicrobial effect of cefazolin against E. coli cells expressing L1, resulting in a 2-8-fold reduction in MIC. Docking studies suggested that the nitro (NDM-1, CphA, and L1) or carboxyl group (VIM-2) of 2l coordinates one or two Zn(II) ions, while the N-phenyl group of the inhibitor enhances its hydrophobic interaction with MßLs. These studies demonstrate that the diaryl-substituted rhodanines are good scaffolds for the design of future broad-spectrum inhibitors of MßLs.
ABSTRACT
INTRODUCTION: Objective measures for assessing teaching effectiveness and learning outcomes in the pharmacy curriculum are needed for improving quality of instruction and faculty development. The purpose of this article is to introduce a new teaching assessment method that focuses on self-assessed change in student comfort with the topics taught rather than evaluation of the instructor and to evaluate its performance in comparison to conventional student evaluations of teaching (SET). METHODS: Six successive cohorts of first-year pharmacy students were surveyed regarding their comfort level at the beginning and end of a 10-week pharmacology course. The change in self-assessed comfort level (ΔSACL) was interpreted as the amount of learning that occurred. This indicator was compared to ratings of a statement from SET designed to obtain the same information. RESULTS: An increasing ΔSACL suggests an increase in learning over time. Differences were observed between ΔSACL and corresponding results from SET, suggesting that there could be extrinsic factors influencing the results. DISCUSSION AND CONCLUSIONS: The use of ΔSACL could provide an alternative or complementary approach to assess teaching effectiveness that focuses less on the instructor and more on the actual student learning outcomes.
Subject(s)
Education, Pharmacy , Educational Measurement/methods , Self Efficacy , Students, Pharmacy , Teaching/standards , Female , Health Knowledge, Attitudes, Practice , Humans , Learning , Male , Pharmacology/education , Self-Assessment , Surveys and QuestionnairesABSTRACT
In an effort to develop new inhibitors of metallo-ß-lactamases (MßLs), twenty-eight azolylthioacetamides were synthesized and assayed against MßLs. The obtained benzimidazolyl and benzioxazolyl substituted 1-19 specifically inhibited the enzyme ImiS, and 10 was found to be the most potent inhibitor of ImiS with an IC50 value of 15â¯nM. The nitrobenzimidazolyl substituted 20-28 specifically inhibited NDM-1, with 27 being the most potent inhibitor with an IC50 value of 170â¯nM. Further studies with 10, 11, and 27 revealed a mixed inhibition mode with competitive and uncompetitive inhibition constants in a similar range as the IC50 values. These inhibitors resulted in a 2-4-fold decrease in imipenem MIC values using E. coli cells producing ImiS or NDM-1. While the source of uncompetitive (possibly allosteric) inhibition remains unclear, docking studies indicate that 10 and 11 may interact orthosterically with Zn2 in the active site of CphA, while 27 could bridge the two Zn(II) ions in the active site of NDM-1 via its nitro group.
Subject(s)
Anti-Bacterial Agents/pharmacology , Azoles/pharmacology , Escherichia coli/drug effects , Thioacetamide/analogs & derivatives , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/metabolism , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Azoles/chemical synthesis , Azoles/chemistry , Dose-Response Relationship, Drug , Escherichia coli/cytology , Escherichia coli/enzymology , Molecular Docking Simulation , Molecular Structure , Structure-Activity Relationship , Thioacetamide/chemical synthesis , Thioacetamide/chemistry , Thioacetamide/pharmacology , beta-Lactamase Inhibitors/chemical synthesis , beta-Lactamase Inhibitors/chemistryABSTRACT
Given the clinical importance of metallo-ß-lactamases (MßLs), a new scaffold, N-substituted carbamylmethyl mercaptoacetate thioether, was constructed. The obtained molecules 1-16 inhibited MßLs from all three subclasses, but preferentially L1 from subclass B3. Compound 9 with a p-carboxyphenyl substituent exhibited the broadest spectrum with at least 70% inhibition of enzymes from all subclasses at 100 µM, while compound 5 with a p-methylphenyl substituent was the most potent inhibitor of any individual enzyme, with 97% inhibition at 100 µM and an IC50 value of 0.41 µM against L1. Isothermal titration calorimetry assays corroborate findings from UV-vis spectrophotometric assays that the inhibition of L1 by 5 is dose-dependent. Docking studies suggest that the carboxyl group, the sulfide atom, and the carbonyl group of the carbamyl coordinate Zn2 in a chelating fashion. Using E. coli cells expressing L1, 6 and 8 were able to decrease cefazolin minimum inhibitory concentration 8-fold.
ABSTRACT
Although α-diazo-ß-ketoesters are synthetically versatile intermediates, methodology for introducing this functionality into complex molecules is still limited, most frequently involving a carboxylic acid precursor, which is then activated and transformed into a ß-ketoester, with the diazo group being subsequently added with a diazo transfer reagent. While introducing this highly functional moiety in a convergent one step process would be ideal, such an objective is limited by the relatively few studies which address functionalization of the α-diazo-ß-ketoester at the γ-position. In the present investigation, we evaluate strategies, both new and established, for functionalizing α-diazo-ß-ketoesters, particularly with regard to generating compounds prospectively useful in the synthesis of C1-substituted carbapenems. We report the first δ-aldehydo-α-diazo-ß-ketoester as well as a method for its oxidation to the corresponding methyl ester, and the formation of a new substituted pyrazole under basic conditions.
ABSTRACT
The emergence of antibiotic resistance caused by metallo-ß-lactamases (MßLs) is a global public health problem. Recently, we found amino acid thioesters to be a highly promising scaffold for inhibitors of the MßL L1. In order to optimize this series of inhibitors, nine new amino acid thioesters were developed by modifying the substituents on the N-terminus of the thioesters and the groups representing the amino acid side chain. Biological activity assays indicate that all of them are very potent inhibitors of L1 with an IC50 value range of 20-600nM, lower than those of most of the previously reported inhibitors of this scaffold. Analysis of structure-activity relationship reveals that big hydrophobic substituents on the N-terminus and a methionine amino acid side chain improves inhibitory activity of the thioesters. All these inhibitors are able to restore antibacterial activity of a ß-lactam antibiotic against Escherichia coli BL21(DE3) cells producing L1 to that against E. coli cells lacking a ß-lactamase. Docking studies reveal that a large N-terminal hydrophobic group results in a slightly different binding mode than smaller hydrophobic groups at the same position.
Subject(s)
Amino Acids/chemistry , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/drug effects , Esters , Structure-Activity RelationshipABSTRACT
The metallo-ß-lactamases (MßLs) cleave the ß-lactam ring of ß-lactam antibiotics, conferring resistance against these drugs to bacteria. Twenty-four triazolylthioacetamides were prepared and evaluated as inhibitors of representatives of the three subclasses of MßLs. All these compounds exhibited specific inhibitory activity against NDM-1 with an IC50 value range of 0.15-1.90 µM, but no activity against CcrA, ImiS, and L1 at inhibitor concentrations of up to 10 µM. Compounds 4d and 6c are partially mixed inhibitors with K i values of 0.49 and 0.63 µM using cefazolin as the substrate. Structure-activity relationship studies reveal that replacement of hydrogen on the aromatic ring by chlorine, heteroatoms, or alkyl groups can affect bioactivity, while leaving the aromatic ring of the triazolylthiols unmodified maintains the inhibitory potency. Docking studies reveal that the typical potent inhibitors of NDM-1, 4d and 6c, form stable interactions in the active site of NDM-1, with the triazole bridging Zn1 and Zn2, and the amide interacting with Lys 211 (Lys224).
ABSTRACT
Antibiotic resistance in bacteria is ever changing and adapting, as once-novel ß-lactam antibiotics are losing their efficacy, primarily due to the production of ß-lactamases. Metallo-ß-lactamases (MBLs) efficiently inactivate a broad range of ß-lactam antibiotics, including carbapenems, and are often coexpressed with other antibacterial resistance factors. The rapid dissemination of MBLs and lack of novel antibacterials pose an imminent threat to global health. In an effort to better counter these resistance-conferring ß-lactamases, an investigation of their natural evolution and resulting substrate specificity was employed. In this study, we elucidated the effects of different amino acid substitutions at position 67 in IMP-type MBLs on the ability to hydrolyze and confer resistance to a range of ß-lactam antibiotics. Wild-type ß-lactamases IMP-1 and IMP-10 and mutants IMP-1-V67A and IMP-1-V67I were characterized biophysically and biochemically, and MICs for Escherichia coli cells expressing these enzymes were determined. We found that all variants exhibited catalytic efficiencies (kcat/Km) equal to or higher than that of IMP-1 against all tested ß-lactams except penicillins, against which IMP-1 and IMP-1-V67I showed the highest kcat/Km values. The substrate-specific effects of the different amino acid substitutions at position 67 are discussed in light of their side chain structures and possible interactions with the substrates. Docking calculations were employed to investigate interactions between different side chains and an inhibitor used as a ß-lactam surrogate. The differences in binding affinities determined experimentally and computationally seem to be governed by hydrophobic interactions between residue 67 and the inhibitor and, by inference, the ß-lactam substrates.