Machine Learning Models Identify Inhibitors of New Delhi Metallo-ß-lactamase.

The worldwide spread of the metallo-ß-lactamases (MBL), especially New Delhi metallo-ß-lactamase-1 (NDM-1), is threatening the efficacy of ß-lactams, which are the most potent and prescribed class of antibiotics in the clinic. Currently, FDA-approved MBL inhibitors are lacking in the clinic even though many strategies have been used in inhibitor development, including quantitative high-throughput screening (qHTS), fragment-based drug discovery (FBDD), and molecular docking. Herein, a machine learning-based prediction tool is described, which was generated using results from HTS of a large chemical library and previously published inhibition data. The prediction tool was then used for virtual screening of the NIH Genesis library, which was subsequently screened using qHTS. A novel MBL inhibitor was identified and shown to lower minimum inhibitory concentrations (MICs) of Meropenem for a panel of E. coli and K. pneumoniae clinical isolates expressing NDM-1. The mechanism of inhibition of this novel scaffold was probed utilizing equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry, UV-vis spectrophotometry, and molecular docking. The uncovered inhibitor, compound 72922413, was shown to be 9-hydroxy-3-[(5-hydroxy-1-oxa-9-azaspiro[5.5]undec-9-yl)carbonyl]-4H-pyrido[1,2-a]pyrimidin-4-one.

Phage-layer interferometry: a companion diagnostic for phage therapy and a bacterial testing platform.

The continuing and rapid emergence of antibiotic resistance (AMR) calls for innovations in antimicrobial therapies. A promising, 're-emerging' approach is the application of bacteriophage viruses to selectively infect and kill pathogenic bacteria, referred to as phage therapy. In practice, phage therapy is personalized and requires companion diagnostics to identify efficacious phages, which are then formulated into a therapeutic cocktail. The predominant means for phage screening involves optical-based assays, but these methods cannot be carried out in complex media, such as colored solutions, inhomogeneous mixtures, or high-viscosity samples, which are often conditions encountered in vivo. Moreover, these assays cannot distinguish phage binding and lysis parameters, which are important for standardizing phage cocktail formulation. To address these challenges, we developed Phage-layer Interferometry (PLI) as a companion diagnostic. Herein, PLI is assessed as a quantitative phage screening method and prototyped as a bacterial detection platform. Importantly, PLI is amenable to automation and is functional in complex, opaque media, such as baby formula. Due to these newfound capabilities, we foresee immediate and broad impact of PLI for combating AMR and protecting against foodborne illnesses.

Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO.

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching. When purified or expressed in a human cell line, TC-hGAPDH had properties like native GAPDH and heme binding quenched its fluorescence by 45-65%, allowing it to report on GAPDH binding of mitochondrially-generated heme in live cells in real time. In cells with active mitochondrial heme synthesis, low-level NO exposure increased heme allocation to IDO1 while keeping the TC-hGAPDH heme level constant due to replenishment by mitochondria. When mitochondrial heme synthesis was blocked, low NO caused a near complete transfer of the existing heme in TC-hGAPDH to IDO1 in a process that required IDO1 be able to bind the heme and have an active hsp90 present. Higher NO exposure had the opposite effect and caused IDO1 heme to transfer back to TC-hGAPDH. This demonstrated: (i) flow of mitochondrial heme through GAPDH is tightly coupled to target delivery, (ii) NO up- or down-regulates IDO1 activity by promoting a conserved heme exchange with GAPDH that goes in either direction according to the NO exposure level. The ability to drive a concentration-dependent, reversible protein heme exchange is unprecedented and reveals a new role for NO in biology.

Visualizing Mitochondrial Heme Flow through GAPDH to Targets in Living Cells and its Regulation by NO.

Iron protoporphyrin IX (heme) is an essential cofactor that is chaperoned in mammalian cells by GAPDH in a process regulated by NO. To gain further understanding we generated a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after being expressed and labeled with fluorescent FlAsH reagent could indicate heme binding by fluorescence quenching. When purified or expressed in HEK293T mammalian cells, FlAsH-labeled TC-hGAPDH displayed physical, catalytic, and heme binding properties like native GAPDH and its heme binding (2 mol per tetramer) quenched its fluorescence by 45-65%. In live HEK293T cells we could visualize TC-hGAPDH binding mitochondrially-generated heme and releasing it to the hemeprotein target IDO1 by monitoring cell fluorescence in real time. In cells with active mitochondrial heme synthesis, a low-level NO exposure increased heme allocation into IDO1 while keeping steady the level of heme-bound TC-hGAPDH. When mitochondrial heme synthesis was blocked at the time of NO exposure, low NO caused cells to reallocate existing heme from TC-hGAPDH to IDO1 by a mechanism requiring IDO1 be present and able to bind heme. Higher NO exposure had an opposite effect and caused cells to reallocate existing heme from IDO1 to TC-hGAPDH. Thus, with TC-hGAPDH we could follow mitochondrial heme as it travelled onto and through GAPDH to a downstream target (IDO1) in living cells, and to learn that NO acted at or downstream from the GAPDH heme complex to promote a heme reallocation in either direction depending on the level of NO exposure.

Ubiquitination detection techniques.

Ubiquitination is an intricately regulated post-translational modification that involves the covalent attachment of ubiquitin to a substrate protein. The complex dynamic nature of the ubiquitination process regulates diverse cellular functions including targeting proteins for degradation, cell cycle, deoxyribonucleic acid (DNA) damage repair, and numerous cell signaling pathways. Ubiquitination also serves as a crucial mechanism in protein quality control. Dysregulation in ubiquitination could result in lethal disease conditions such as cancers and neurodegenerative diseases. Therefore, the ubiquitination cascade has become an attractive target for therapeutic interventions. Enormous efforts have been made to detect ubiquitination involving different detection techniques to better grasp the underlying molecular mechanisms of ubiquitination. This review discusses a wide range of techniques stretching from the simplest assays to real-time assays. This includes western blotting/immunoblotting, fluorescence assays, chemiluminescence assays, spectrophotometric assays, and nanopore sensing assays. This review compares these applications, and the inherent advantages and limitations.

The directed evolution of NDM-1.

ß-Lactam antibiotics are among the most frequently prescribed therapeutic agents. A common mechanism of resistance toward ß-lactam antibiotics is the production of ß-lactamases. These enzymes are capable of hydrolyzing the ß-lactam bond, rendering the drug inactive. Among the four described classes, the metallo- ß-lactamases (MBLs, class B) employ one or two zinc ions in the active site for catalysis. One of the three most clinically relevant MBLs is New Delhi Metallo- ß-Lactamase (NDM-1). The current study sought to investigate the in vitro protein evolution of NDM-1 ß-lactamase using error-prone polymerase chain reaction. Evaluation revealed that variants were not found to confer higher levels of resistance toward meropenem based on amino acid substitutions. Thus, we postulate that increases in transcription or changes in zinc transport may be clinically more relevant to meropenem resistance than amino acid substitutions.

Real-time bio-layer interferometry ubiquitination assays as alternatives to western blotting.

Ubiquitination is a crucial cellular pathway enabling normal cellular functions. Abnormalities in the ubiquitination process can lead to cellular dysfunction and cause a range of diseases. Efforts to screen and develop small molecule inhibitors targeting portions of the ubiquitination cascade require rapid and robust methods for detecting ubiquitination. Enormous efforts have been made in the field to detect ubiquitination using various techniques including fluorescence, spectrophotometry, chemiluminescence, NMR, and radioactive tracers. The most common method to detect ubiquitination is western blotting. However, western blotting is time-consuming and difficult to use when seeking fine-grained time course experiments. Here we present the use of bio-layer interferometry to rapidly assay ubiquitination in real-time. An E3 ligase auto-ubiquitination system and a substrate ubiquitination assay have been applied as tests for the newly developed assay. The developed BLI ubiquitination assay provides one-second time resolution and detects the formation of polyubiquitin chains directly on a biosensor-bound target. Results are returned instantaneously, and reagent concentrations are identical to those used by traditional western blot-based ubiquitination assays. The developed BLI ubiquitination assay is a viable alternative to traditional western blot assays to detect ubiquitination in a rapid real-time manner.

Transient receptor potential canonical type 6 (TRPC6) O-GlcNAcylation at Threonine-221 plays potent role in channel regulation.

Transient receptor potential canonical type 6 (TRPC6) is a non-voltage-gated channel that principally conducts calcium. Elevated channel activation contributes to fibrosis, hypertrophy, and proteinuria, often coupled to stimulation of nuclear factor of activated T-cells (NFAT). TRPC6 is post-translationally regulated, but a role for O-linked ß-N-acetyl glucosamine (O-GlcNAcylation) as elevated by diabetes, is unknown. Here we show TRPC6 is constitutively O-GlcNAcylated at Ser14, Thr70, and Thr221 in the N-terminus ankryn-4 (AR4) and linker (LH1) domains. Mutagenesis to alanine reveals T221 as a critical controller of resting TRPC6 conductance, and associated NFAT activity and pro-hypertrophic signaling. T→A mutations at sites homologous in closely related TRPC3 and TRPC7 also increases their activity. Molecular modeling predicts interactions between Thr221-O-GlcNAc and Ser199, Glu200, and Glu246, and combined alanine substitutions of the latter similarly elevates resting NFAT activity. Thus, O-GlcNAcylated T221 and interactions with coordinating residues is required for normal TRPC6 channel conductance and NFAT activation.

Polymer modification of SARS-CoV-2 spike protein impacts its ability to bind key receptor.

The global spread of SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has caused the loss of many human lives and severe economic losses. SARS-CoV-2 mediates its infection in humans via the spike glycoprotein. The receptor binding domain of the SARS-CoV-2 spike protein binds to its cognate receptor, angiotensin converting enzyme-2 (ACE2) to initiate viral entry. In this study, we examine how polymer modification of the spike protein receptor binding domain impacts binding to ACE2. The horseradish peroxidase conjugated receptor binding domain was modified with a range of polymers including hydrophilic N,N-dimethylacrylamide, hydrophobic N-isopropylacrylamide, cationic 3-(N,N-dimethylamino)propylacrylamide, and anionic 2-acrylamido-2-methylpropane sulfonic acid polymers. The effect of polymer chain length was observed using N,N-dimethylacrylamide polymers with degrees of polymerization of 5, 10 and 25. Polymer conjugation of the receptor binding domain significantly reduced the interaction with ACE2 protein, as determined by an enzyme-linked immunosorbent assay. Stability analysis showed that these conjugates remained highly stable even after seven days incubation at physiological temperature. Hence, this study provides a detailed view of the effect specific type of modification using a library of polymers with different functionalities in interrupting RBD-ACE2 interaction.

Fluorescence dequenching assay for the activity of TEV protease.

Tobacco etch virus (TEV) protease is a widely used protease for fusion tag cleavage. Despite its widespread usage, an assay to quickly and easily quantify its activity in laboratory settings is still lacking. Thus, researchers may encounter inefficient cleavage of the desired fusion proteins due to poor activity of a given TEV protease preparation. Here, we describe the development and implementation of a fluorescence dequenching-based assay to quantify TEV protease activity and assess kinetic parameters. The peptide substrate used in this assay consists of a C-terminal TAMRA fluorophore, an N-terminal fluorescein fluorophore, and the canonical TEV protease recognition sequence. The assay is based on a reduction of fluorescence quenching of fluorescein upon cleavage by TEV protease. The substrate peptide was studied spectroscopically to assess feasibility and to propose a plausible mechanism of the assay. The assay was optimized and applied to obtain rapid assessments of TEV protease activity in purified samples and crude lysate extracts. The kinetic data obtained from improved TEV protease variants were compared with a traditional SDS-PAGE assay. Finally, the assay was applied to determine the optimum pH for TEV protease. Further, the study found that the assay is a rapid and simple approach to quantify TEV protease activity. The findings of the assay on crude lysate extracts, activity assay of TEV protease variants, and assessment of optimum pH for TEV protease reactions demonstrate the robust utility of the assay.

Investigating the Impact of Polymer Length, Attachment Site, and Charge on Enzymatic Activity and Stability of Cellulase.

The thermophilic cellulase Cel5a from Fervidobacterium nodosum (FnCel5a) was conjugated with neutral, cationic, and anionic polymers of increasing molecular weights. The enzymatic activity toward an anionic soluble cellulose derivative, thermal stability, and functional chemical stability of these bioconjugates were investigated. The results suggest that increasing polymer chain length for polymers compatible with the substrate enhances the positive impact of polymer conjugation on enzymatic activity. Activity enhancements of nearly 100% were observed for bioconjugates with N,N-dimethyl acrylamide (DMAm) and N,N-dimethyl acrylamide-2-(N,N-dimethylamino)ethyl methacrylate (DMAm/DMAEMA) due to proposed polymer-substrate compatibility enabled by potential noncovalent interactions. Double conjugation of two functionally distinct polymers to wild-type and mutated FnCel5a using two conjugation methods was achieved. These doubly conjugated bioconjugates exhibited similar thermal stability to the unmodified wild-type enzyme, although enzymatic activity initially gained from conjugation was lost, suggesting that chain length may be a better tool for bioconjugate activity modulation than double conjugation.

Different Conformations Revealed by NMR Underlie Resistance to Ceftazidime/Avibactam and Susceptibility to Meropenem and Imipenem among D179Y Variants of KPC ß-Lactamase.

ß-Lactamase-mediated resistance to ceftazidime-avibactam (CZA) is a serious limitation in the treatment of Gram-negative bacteria harboring Klebsiella pneumoniae carbapenemase (KPC). Herein, the basis of susceptibility to carbapenems and resistance to ceftazidime (CAZ) and CZA of the D179Y variant of KPC-2 and -3 was explored. First, we determined that resistance to CZA in a laboratory strain of Escherichia coli DH10B was not due to increased expression levels of the variant enzymes, as demonstrated by reverse transcription PCR (RT-PCR). Using timed mass spectrometry, the D179Y variant formed prolonged acyl-enzyme complexes with imipenem (IMI) and meropenem (MEM) in KPC-2 and KPC-3, which could be detected up to 24 h, suggesting that IMI and MEM act as covalent ß-lactamase inhibitors more than as substrates for D179Y KPC-2 and -3. This prolonged acyl-enzyme complex of IMI and MEM by D179Y variants was not observed with wild-type (WT) KPCs. CAZ was studied and the D179Y variants also formed acyl-enzyme complexes (1 to 2 h). Thermal denaturation and differential scanning fluorimetry showed that the tyrosine substitution at position 179 destabilized the KPC ß-lactamases (KPC-2/3 melting temperature [Tm] of 54 to 55°C versus D179Y Tm of 47.5 to 51°C), and the D179Y protein was 3% disordered compared to KPC-2 at 318 K. Heteronuclear 1H/15N-heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy also revealed that the D179Y variant, compared to KPC-2, is partially disordered. Based upon these observations, we discuss the impact of disordering of the Ω loop as a consequence of the D179Y substitution. These conformational changes and disorder in the overall structure as a result of D179Y contribute to this unanticipated phenotype.

A single-dose, open-label, randomized, scintigraphic study to investigate the gastrointestinal behavior of 2 triple-combination cold products (acetaminophen, phenylephrine, and dextromethorphan) in healthy male volunteers.

BACKGROUND: Common cold symptoms may be mitigated by products in caplet, nasal spray, and oral solution formulations, although variations exist in the bioavailability of the active ingredients contained within these products. Rapid gastric emptying (GE) of these active ingredients is important for reducing the delay between drug absorption and onset of cold symptom relief. Hot drink cold remedies are associated with greater comfort and may enhance the bioavailability of active ingredients. The objective of this study was to characterize the gastrointestinal transit of powder (reconstituted in hot water) and caplet formulations of commercially available multisymptom cold medications. METHODS: This was an open-label, single-dose, parallel-group study. Healthy male adults under fasted conditions were randomized 1:1 to receive a single dose of radiolabeled Theraflu Daytime Severe Cold and Cough powder for oral solution or radiolabeled Theraflu ExpressMax Daytime Severe Cold and Cough caplet. External gamma scintigraphy was utilized to monitor GE and intestinal transit of two radiolabeled drug formulations. RESULTS: A total of 28 participants completed the study. The mean ± SE GE onset times were 1.1 ± 0.3 min and 8.5 ± 1.8 min for powder and caplet formulations, respectively. The mean ± SE GE completion times were 121 ± 13 min and 65 ± 13 min, respectively. Despite the similar mean times to GE25%, the powder had later mean GE50% (23 ± 3.0 vs 16 ± 3.2 min, respectively) and GE90% (85 ± 12 vs 36 ± 9 min, respectively) than caplets. Caplets had a shorter overall GE half-life, lower total gastric exposure, and faster transit time through the small intestine versus the powder formulation. No serious safety events were observed. CONCLUSION: The results of this study in healthy male adults suggest that the Theraflu powder formulation had a more rapid GE onset but longer time to GE completion than the caplet formulation. TRIAL REGISTRATION: ClinicalTrials.gov NCT03415243.

Hydrolytically Stable Maleimide-End-Functionalized Polymers for Site-Specific Protein Conjugation.

Site-specific conjugation to cysteines of proteins often uses ester groups to link maleimide or alkene groups to polymers. However, the ester group is susceptible to hydrolysis, potentially losing the benefits gained through bioconjugation. Here, we present a simple conjugation strategy that utilizes the amide bond stability of traditional 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide coupling while introducing site specificity. Hydrolytically stable maleimide-end-functionalized polymers for site-specific conjugation to free cysteines of proteins were synthesized using reversible addition-fragmentation chain-transfer (RAFT) polymerization. The alpha terminus of the polymers was amidated with a furan-protected aminoethyl maleimide using carbodiimide-based chemistry. Finally, the maleimide was exposed by a retro Diels-Alder reaction to yield the maleimide group, allowing for thiol-maleimide click chemistry for bioconjugation. A thermophilic cellulase from Fervidobacterium nodosum (FnCel5a) was conjugated using various strategies, including random 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling, site-specific hydroxyethyl maleimide (HEMI) end-functionalized coupling, hydroxyethyl acrylate (HEA) end-functionalized coupling, and amidoethyl maleimide (AEMI) end-functionalized coupling. Only the polymers conjugated by EDC and AEMI remained conjugated a week after attachment. This indicates that hydrolytically stable amide-based maleimides are an important bioconjugation strategy for conjugates that require long-term stability, while esters are better suited for systems that require debonding of polymers over time.

Structural Comparisons of Cefotaximase (CTX-M-ase) Sub Family 1.

The cefotaximase or CTX-M, family of serine-ß-lactamases represents a significant clinical concern due to the ability for these enzymes to confer resistance to a broad array of ß-lactam antibiotics an inhibitors. This behavior lends CTX-M-ases to be classified as extended spectrum ß-lactamases (ESBL). Across the family of CTX-M-ases most closely related to CTX-M-1, the structures of CTX-M-15 with a library of different ligands have been solved and serve as the basis of comparison within this review. Herein we focus on the structural changes apparent in structures of CTX-M-15 in complex with diazabicyclooctane (DABCO) and boronic acid transition state analog inhibitors. Interactions between a positive surface patch near the active site and complementary functional groups of the bound inhibitor play key roles in the dictating the conformations of active site residues. The insights provided by analyzing structures of CTX-M-15 in complex with DABCO and boronic acid transition state analog inhibitors and analyzing existing structures of CTX-M-64 offer opportunities to move closer to making predictions as to how CTX-M-ases may interact with potential drug candidates, setting the stage for the further development of new antibiotics and ß-lactamase inhibitors.

Two-distinct polymer ubiquitin conjugates by photochemical grafting-from.

Protein-polymer bioconjugates present a way to make enzymes more efficient and robust for industrial and medicinal applications. While much work has focused on mono-functional conjugates, i.e. conjugates with one type of polymer attached such as poly(ethylene glycol) or poly(N-isopropylacrylamide), there is a practical interest in gaining additional functionality by synthesizing well-defined bifunctional conjugates in a hetero-arm star copolymer architecture with protein as the core. Using ubiquitin as a model protein, a synthetic scheme was developed to attach two different polymers (OEOMA and DMAm) directly to the protein surface, using orthogonal conjugation chemistries and grafting-from by photochemical living radical polymerization techniques. The additional complexity arising from attempts to selectively modify multiple sites led to decreased polymerization performance and indicates that ICAR-ATRP and RAFT are not well-suited to bifunctional bioconjugates applications. Nonetheless, the polymerization conditions preserve the native fold of the ubiquitin and enable production of a hetero-arm star protein-polymer bioconjugate.

Spectroscopic and biochemical characterization of metallo-ß-lactamase IMP-1 with dicarboxylic, sulfonyl, and thiol inhibitors.

In an effort to probe the biophysical mechanisms of inhibition for ten previously-reported inhibitors of metallo-ß-lactamases (MBL) with MBL IMP-1, equilibrium dialysis, metal analyses coupled with atomic absorption spectroscopy (AAS), native state mass spectrometry (native MS), and ultraviolet-visible spectrophotometry (UV-VIS) were used. 6-(1H-tetrazol-5-yl) picolinic acid (1T5PA), ANT431, D/l-captopril, thiorphan, and tiopronin were shown to form IMP-1/Zn(II)/inhibitor ternary complexes, while dipicolinic acid (DPA) and 4-(3-aminophenyl)pyridine-2,6-dicarboxylic acid (3AP-DPA) stripped some metal from the active site of IMP but also formed ternary complexes. DPA and 3AP-DPA stripped less metal from IMP-1 than from VIM-2 but stripped more metal from IMP-1 than from NDM-1. In contrast to a previous report, pterostilbene does not appear to bind to IMP-1 under our conditions. These results, along with previous studies, demonstrate similar mechanisms of inhibition toward different MBLs for different MBL inhibitors.

Cytosolic protein quality control machinery: Interactions of Hsp70 with a network of co-chaperones and substrates.

The chaperone heat shock protein 70 (Hsp70) and its network of co-chaperones serve as a central hub of cellular protein quality control mechanisms. Domain organization in Hsp70 dictates ATPase activity, ATP dependent allosteric regulation, client/substrate binding and release, and interactions with co-chaperones. The protein quality control activities of Hsp70 are classified as foldase, holdase, and disaggregase activities. Co-chaperones directly assisting protein refolding included J domain proteins and nucleotide exchange factors. However, co-chaperones can also be grouped and explored based on which domain of Hsp70 they interact. Here we discuss how the network of cytosolic co-chaperones for Hsp70 contributes to the functions of Hsp70 while closely looking at their structural features. Comparison of domain organization and the structures of co-chaperones enables greater understanding of the interactions, mechanisms of action, and roles played in protein quality control.

Polymer Modification of Lipases, Substrate Interactions, and Potential Inhibition.

An industrially important enzyme, Candida antarctica lipase B (CalB), was modified with a range of functional polymers including hydrophilic, hydrophobic, anionic, and cationic character using a "grafting to" approach. We determined the impact of polymer chain length on CalB activity by synthesizing biohybrids of CalB with each polymer at three different chain lengths, using reversible addition-fragmentation chain transfer (RAFT) polymerization. The activity of CalB in both aqueous and aqueous-organic media mixtures was significantly enhanced for acrylamide (Am) and N,N-dimethyl acrylamide (DMAm) conjugates, with activity remaining approximately constant in 25 and 50% ethanol solvent systems. Interestingly, the activity of N,N-dimethylaminopropyl-acrylamide (DMAPA) conjugates increased gradually with increasing organic solvent content in the system. Contrary to other literature reports, our study showed significantly diminished activity for hydrophobic polymer-protein conjugates. Functional thermal stability assays also displayed a considerable enhancement of retained activity of Am, DMAm, and DMAPA conjugates compared to the native CalB enzyme. Thus, this study provides an insight into possible advances in lipase production, which can lead to new improved lipase bioconjugates with increased activity and stability.

Carbapenem Use Is Driving the Evolution of Imipenemase 1 Variants.

Metallo-ß-lactamases (MBLs) are a growing clinical threat because they inactivate nearly all ß-lactam-containing antibiotics, and there are no clinically available inhibitors. A significant number of variants have already emerged for each MBL subfamily. To understand the evolution of imipenemase (IMP) genes (blaIMP) and their clinical impact, 20 clinically derived IMP-1 like variants were obtained using site-directed mutagenesis and expressed in a uniform genetic background in Escherichia coli strain DH10B. Strains of IMP-1-like variants harboring S262G or V67F substitutions exhibited increased resistance toward carbapenems and decreased resistance toward ampicillin. Strains expressing IMP-78 (S262G/V67F) exhibited the largest changes in MIC values compared to IMP-1. In order to understand the molecular mechanisms of increased resistance, biochemical, biophysical, and molecular modeling studies were conducted to compare IMP-1, IMP-6 (S262G), IMP-10 (V67F), and IMP-78 (S262G/V67F). Finally, unlike most New Delhi metallo-ß-lactamase (NDM) and Verona integron-encoded metallo-ß-lactamase (VIM) variants, the IMP-1-like variants do not confer any additional survival advantage if zinc availability is limited. Therefore, the evolution of MBL subfamilies (i.e., IMP-6, -10, and -78) appears to be driven by different selective pressures.