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.

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.

Quercetin ameliorates memory impairment by inhibiting abnormal microglial activation in a mouse model of paradoxical sleep deprivation.

Paradoxical sleep deprivation (PSD) is prevalent in modern society, and impaired memory is one of its serious consequences. The pathogenic mechanism is still unclear, and the therapeutic strategies for PSD are limited. Here, we found that quercetin treatment ameliorated memory impairments caused by PSD in a dose-dependent manner in an animal model. Quercetin could restore the dynamic changes of the gamma band while the animals performed novel object recognition (NOR) tasks as determined by electroencephalogram analysis. Morphological analysis showed that quercetin, by targeting the hippocampal CA1 region, strikingly ameliorated the overactivation of microglia induced by PSD. Mechanistically, quercetin inhibited the toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa-b (NF-κB) cascade, which is critical for abnormal microglial activation following PSD stress. Our results provided experimental evidence for the therapeutic effects of quercetin on PSD-related memory impairments by suppressing TLR4/MyD88/NF-κB signaling that mediated abnormal microglia activation in the hippocampus.

Visualizing the Dynamic Metalation State of New Delhi Metallo-ß-lactamase-1 in Bacteria Using a Reversible Fluorescent Probe.

New Delhi metallo-ß-lactamase (NDM) grants resistance to a broad spectrum of ß-lactam antibiotics, including last-resort carbapenems, and is emerging as a global antibiotic resistance threat. Limited zinc availability adversely impacts the ability of NDM-1 to provide resistance, but a number of clinical variants have emerged that are more resistant to zinc scarcity (e.g., NDM-15). To provide a novel tool to better study metal ion sequestration in host-pathogen interactions, we describe the development of a fluorescent probe that reports on the dynamic metalation state of NDM within Escherichia coli. The thiol-containing probe selectively coordinates the dizinc metal cluster of NDM and results in a 17-fold increase in fluorescence intensity. Reversible binding enables competition and time-dependent studies that reveal fluorescence changes used to detect enzyme localization, substrate and inhibitor engagement, and changes to metalation state through the imaging of live E. coli using confocal microscopy. NDM-1 is shown to be susceptible to demetalation by intracellular and extracellular metal chelators in a live-cell model of zinc dyshomeostasis, whereas the NDM-15 metalation state is shown to be more resistant to zinc flux. The development of this reversible turn-on fluorescent probe for the metalation state of NDM provides a new tool for monitoring the impact of metal ion sequestration by host defense mechanisms and for detecting inhibitor-target engagement during the development of therapeutics to counter this resistance determinant.

Long non-coding RNA NORAD/miR-224-3p/MTDH axis contributes to CDDP resistance of esophageal squamous cell carcinoma by promoting nuclear accumulation of ß-catenin.

BACKGROUND: Cis-diamminedichloro-platinum (CDDP)-based chemotherapy regimens are the most predominant treatment strategies for patients with esophageal squamous cell carcinoma (ESCC). Dysregulated long non-coding RNAs (lncRNAs) contribute to CDDP resistance, which results in treatment failure in ESCC patients. However, the majority of lncRNAs involved in CDDP resistance in ESCC remain to be elucidated. METHODS: The public Gene Expression Omnibus (GEO) dataset GSE45670 was analysed to reveal potential lncRNAs involved in CDDP resistance of ESCC. Candidate upregulated lncRNAs were detected in ESCC specimens by qRT-PCR to identify crucial lncRNAs. Non-coding RNA activated by DNA damage (NORAD) was selected for further study. Kaplan-Meier analysis and a COX proportional regression model were performed to analyse the potential of NORAD for predicting prognosis of ESCC patients. The role of NORAD in CDDP resistance were determined by conducting gain and loss-of-function experiments in vitro. Fluorescence in situ hybridization (FISH) was performed to determine the subcellular location of NORAD in ESCC cells. A public GEO dataset and bioinformatic algorithms were used to predict the microRNAs (miRNAs) that might be latently sponged by NORAD. qRT-PCR was conducted to verify the expression of candidate miRNAs. Luciferase reporter and Argonaute-2 (Ago2)-RNA immunoprecipitation (RIP) assays were conducted to evaluate the interaction between NORAD and candidate miRNAs. A miRNA rescue experiment was performed to authenticate the NORAD regulatory axis and its effects on CDDP resistance in ESCC cells. Western blotting was conducted to confirm the precise downstream signalling pathway of NORAD. A xenograft mouse model was established to reveal the effect of NORAD on CDDP resistance in vivo. RESULTS: The expression of NORAD was higher in CDDP-resistant ESCC tissues and cells than in CDDP-sensitive tissues and cells. NORAD expression was negatively correlated with the postoperative prognosis of ESCC patients who underwent CDDP-based chemotherapy. NORAD knockdown partially arrested CDDP resistance of ESCC cells. FISH showed that NORAD was located in the cytoplasm in ESCC cells. Furthermore, overlapping results from bioinformatic algorithms analyses and qRT-PCR showed that NORAD could sponge miR-224-3p in ESCC cells. Ago2-RIP demonstrated that NORAD and miR-224-3p occupied the same Ago2 to form an RNA-induced silencing complex (RISC) and subsequently regulated the expression of metadherin (MTDH) in ESCC cells. The NORAD/miR-224-3p/MTDH axis promoted CDDP resistance and progression in ESCC cells by promoting nuclear accumulation of ß-catenin in vitro and in vivo. CONCLUSIONS: NORAD upregulates MTDH to promote CDDP resistance and progression in ESCC by sponging miR-224-3p. Our results highlight the potential of NORAD as a therapeutic target in ESCC patients receiving CDDP-based chemotherapy.

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.

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.

An integrated biophysical approach to discovering mechanisms of NDM-1 inhibition for several thiol-containing drugs.

Due to the rapid proliferation of antibiotic-resistant pathogenic bacteria, known as carbapenem-resistant enterobacteriaceae, the efficacy of ß-lactam antibiotics is threatened. ß-lactam antibiotics constitute over 50% of the available antibiotic arsenal. Recent efforts have been focused on developing inhibitors to these enzymes. In an effort to understand the mechanism of inhibition(s) of four FDA-approved thiol-containing drugs that were previously reported to be inhibitors of New Delhi metallo-ß-lactamase (NDM-1), various biochemical and spectroscopic techniques were used. Isothermal titration calorimetry demonstrated the binding affinity to NDM-1 corresponds to the reported IC50 values of the inhibitors. Equilibrium dialyses and metal analyses demonstrated that all of these inhibitors formed ternary complexes with ZnZn-NDM-1. Spectroscopic studies on CoCo-NDM-1 revealed two distinct binding modes for the thiol-containing compounds. These findings validate the need to further investigate the mechanism of inhibition of MBL inhibitors. Further research to identify inhibition capabilities beyond reported IC50 values is necessary for understanding the binding modes of these identified compounds and to provide the necessary foundation for developing clinically relevant MBL inhibitors.

Evolution of New Delhi metallo-ß-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity.

Infections by carbapenem-resistant Enterobacteriaceae are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically used ß-lactamase inhibitors to counter the activity of metallo-ß-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-ß-lactamase (NDM), which requires a di-nuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing melting temperature and refolding efficiency and also by spectroscopy (UV-visible, 1H NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining the minimum inhibitory concentrations of various antibiotics, pre-steady-state and steady-state kinetics, inhibitor binding, and zinc dependence of resistance and activity. We observed only minor differences among the fully loaded di-zinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants exhibited improved thermostability (up to ∼10 °C increased Tm ) and improved zinc affinity (up to ∼10-fold decreased Kd, Zn2). We also provide first evidence that some NDM variants have evolved the ability to function as mono-zinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/Km = 5 × 106 m-1 s-1). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of ß-lactam antibiotics and zinc deprivation.

A Noncanonical Metal Center Drives the Activity of the Sediminispirochaeta smaragdinae Metallo-ß-lactamase SPS-1.

In an effort to evaluate whether a recently reported putative metallo-ß-lactamase (MßL) contains a novel MßL active site, SPS-1 from Sediminispirochaeta smaragdinae was overexpressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equiv of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not ß-lactam substrates with bulky substituents at the 6/7 position. Spectroscopic studies of Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with a reduced level of spin coupling between the metal ions and a novel Zn1 metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn2 site that is similar to that in NDM-1 and other subclass B1 MßLs; however, the Zn1 metal ion is coordinated by two histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn1 metal is displaced nearly 1 Å from the position reported in other MßLs. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents at the 6/7 position of ß-lactam antibiotics. This study reveals a novel metal binding site in MßLs and suggests that the targeting of metal binding sites in MßLs with inhibitors is now more challenging with the identification of this new MßL.

Binding of Nitric Oxide in CDGSH-type [2Fe-2S] Clusters of the Human Mitochondrial Protein Miner2.

Iron-sulfur proteins are among the primary targets of nitric oxide in cells. Previous studies have shown that iron-sulfur clusters hosted by cysteine residues in proteins are readily disrupted by nitric oxide forming a protein-bound dinitrosyl iron complex, thiolate-bridged di-iron tetranitrosyl complex, or octanitrosyl cluster. Here we report that human mitochondrial protein Miner2 [2Fe-2S] clusters can bind nitric oxide without disruption of the clusters. Miner2 is a member of a new CDGSH iron-sulfur protein family that also includes two mitochondrial proteins: the type II diabetes-related mitoNEET and the Wolfram syndrome 2-linked Miner1. Miner2 contains two CDGSH motifs, and each CDGSH motif hosts a [2Fe-2S] cluster via three cysteine and one histidine residues. Binding of nitric oxide in the reduced Miner2 [2Fe-2S] clusters produces a major absorption peak at 422 nm without releasing iron or sulfide from the clusters. The EPR measurements and mass spectrometry analyses further reveal that nitric oxide binds to the reduced [2Fe-2S] clusters in Miner2, with each cluster binding one nitric oxide. Although the [2Fe-2S] cluster in purified human mitoNEET and Miner1 fails to bind nitric oxide, a single mutation of Asp-96 to Val in mitoNEET or Asp-123 to Val in Miner1 facilitates nitric oxide binding in the [2Fe-2S] cluster, indicating that a subtle change of protein structure may switch mitoNEET and Miner1 to bind nitric oxide. The results suggest that binding of nitric oxide in the CDGSH-type [2Fe-2S] clusters in mitochondrial protein Miner2 may represent a new nitric oxide signaling mode in cells.

Copper binding in IscA inhibits iron-sulphur cluster assembly in Escherichia coli.

Among the iron-sulphur cluster assembly proteins encoded by gene cluster iscSUA-hscBA-fdx in Escherichia coli, IscA has a unique and strong iron binding activity and can provide iron for iron-sulphur cluster assembly in proteins in vitro. Deletion of IscA and its paralogue SufA results in an E. coli mutant that fails to assemble [4Fe-4S] clusters in proteins under aerobic conditions, suggesting that IscA has a crucial role for iron-sulphur cluster biogenesis. Here we report that among the iron-sulphur cluster assembly proteins, IscA also has a strong and specific binding activity for Cu(I) in vivo and in vitro. The Cu(I) centre in IscA is stable and resistant to oxidation under aerobic conditions. Mutation of the conserved cysteine residues that are essential for the iron binding in IscA abolishes the copper binding activity, indicating that copper and iron may share the same binding site in the protein. Additional studies reveal that copper can compete with iron for the metal binding site in IscA and effectively inhibits the IscA-mediated [4Fe-4S] cluster assembly in E. coli cells. The results suggest that copper may not only attack the [4Fe-4S] clusters in dehydratases, but also block the [4Fe-4S] cluster assembly in proteins by targeting IscA in cells.

Iron and zinc binding activity of Escherichia coli topoisomerase I homolog YrdD.

YrdD, a homolog of the C-terminal zinc-binding region of Escherichia coli topoisomerase I, is highly conserved among proteobacteria and enterobacteria. However, the function of YrdD remains elusive. Here we report that YrdD purified from E. coli cells grown in LB media contains both zinc and iron. Supplement of exogenous zinc in the medium abolishes the iron binding of YrdD in E. coli cells, indicating that iron and zinc may compete for the same metal binding sites in the protein. While the zinc-bound YrdD is able to bind single-stranded (ss) DNA and protect ssDNA from the DNase I digestion in vitro, the iron-bound YrdD has very little or no binding activity for ssDNA, suggesting that the zinc-bound YrdD may have an important role in DNA repair by interacting with ssDNA in cells.

Targeting Mitochondrial Sirtuins in Age-Related Neurodegenerative Diseases and Fibrosis.

Aging is a natural and complex biological process that is associated with widespread functional declines in numerous physiological processes, terminally affecting multiple organs and tissues. Fibrosis and neurodegenerative diseases (NDs) often occur with aging, imposing large burdens on public health worldwide, and there are currently no effective treatment strategies for these diseases. Mitochondrial sirtuins (SIRT3-5), which are members of the sirtuin family of NAD+-dependent deacylases and ADP-ribosyltransferases, are capable of regulating mitochondrial function by modifying mitochondrial proteins that participate in the regulation of cell survival under various physiological and pathological conditions. A growing body of evidence has revealed that SIRT3-5 exert protective effects against fibrosis in multiple organs and tissues, including the heart, liver, and kidney. SIRT3-5 are also involved in multiple age-related NDs, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. Furthermore, SIRT3-5 have been noted as promising targets for antifibrotic therapies and the treatment of NDs. This review systematically highlights recent advances in knowledge regarding the role of SIRT3-5 in fibrosis and NDs and discusses SIRT3-5 as therapeutic targets for NDs and fibrosis.

Reversal of hyperactive higher-order thalamus attenuates defensiveness in a mouse model of PTSD.

Posttraumatic stress disorder (PTSD) is a highly prevalent and debilitating psychiatric disease often accompanied by severe defensive behaviors, preventing individuals from integrating into society. However, the neural mechanisms of defensiveness in PTSD remain largely unknown. Here, we identified that the higher-order thalamus, the posteromedial complex of the thalamus (PoM), was overactivated in a mouse model of PTSD, and suppressing PoM activity alleviated excessive defensive behaviors. Moreover, we found that diminished thalamic inhibition derived from the thalamic reticular nucleus was the major cause of thalamic hyperactivity in PTSD mice. Overloaded thalamic innervation to the downstream cortical area, frontal association cortex, drove abnormal defensiveness. Overall, our study revealed that the malfunction of the higher-order thalamus mediates defensive behaviors and highlighted the thalamocortical circuit as a potential target for treating PTSD-related overreactivity symptoms.

Nitric oxide reversibly binds the reduced [2Fe-2S] cluster in mitochondrial outer membrane protein mitoNEET and inhibits its electron transfer activity.

MitoNEET is a mitochondrial outer membrane protein that regulates energy metabolism, iron homeostasis, and production of reactive oxygen species in cells. Aberrant expression of mitoNEET in tissues has been linked to type II diabetes, neurodegenerative diseases, and several types of cancer. Structurally, the N-terminal domain of mitoNEET has a single transmembrane alpha helix that anchors the protein to mitochondrial outer membrane. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via an unusual ligand arrangement of three cysteine and one histidine residues. Here we report that the reduced [2Fe-2S] cluster in the C-terminal cytosolic domain of mitoNEET (mitoNEET45-108) is able to bind nitric oxide (NO) without disruption of the cluster. Importantly, binding of NO at the reduced [2Fe-2S] cluster effectively inhibits the redox transition of the cluster in mitoNEET45-108. While the NO-bound [2Fe-2S] cluster in mitoNEET45-108 is stable, light excitation releases NO from the NO-bound [2Fe-2S] cluster and restores the redox transition activity of the cluster in mitoNEET45-108. The results suggest that NO may regulate the electron transfer activity of mitoNEET in mitochondrial outer membrane via reversible binding to its reduced [2Fe-2S] cluster.

Differential Advising in Multiagent Reinforcement Learning.

Agent advising is one of the main approaches to improve agent learning performance by enabling agents to share advice. Existing advising methods have a common limitation that an adviser agent can offer advice to an advisee agent only if the advice is created in the same state as the advisee's state. However, in complex environments, it is a very strong requirement that two states are the same, because a state may consist of multiple dimensions and two states being the same means that all these dimensions in the two states are correspondingly identical. Therefore, this requirement may limit the applicability of existing advising methods to complex environments. In this article, inspired by the differential privacy scheme, we propose a differential advising method that relaxes this requirement by enabling agents to use advice in a state even if the advice is created in a slightly different state. Compared with the existing methods, agents using the proposed method have more opportunity to take advice from others. This article is the first to adopt the concept of differential privacy on advising to improve agent learning performance instead of addressing security issues. The experimental results demonstrate that the proposed method is more efficient in complex environments than the existing methods.

Long noncoding RNA DGCR5 involves in tumorigenesis of esophageal squamous cell carcinoma via SRSF1-mediated alternative splicing of Mcl-1.

Long noncoding RNAs (lncRNAs) emerge as essential roles in the regulation of alternative splicing (AS) in various malignancies. Serine- and arginine-rich splicing factor 1 (SRSF1)-mediated AS events are the most important molecular hallmarks in cancer. Nevertheless, the biological mechanism underlying tumorigenesis of lncRNAs correlated with SRSF1 in esophageal squamous cell carcinoma (ESCC) remains elusive. In this study, we found that lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) was upregulated in ESCC clinical samples, which associated with poor prognosis. Through RNA interference and overexpression approaches, we confirmed that DGCR5 contributed to promote ESCC cell proliferation, migration, and invasion while inhibited apoptosis in vitro. Mechanistically, DGCR5 could directly bind with SRSF1 to increase its stability and thus stimulate alternative splicing events. Furthermore, we clarified that SRSF1 regulated the aberrant splicing of myeloid cell leukemia-1 (Mcl-1) and initiated a significant Mcl-1L (antiapoptotic) isoform switch, which contributed to the expression of the full length of Mcl-1. Moreover, the cell-derived xenograft (CDX) model was validated that DGCR5 could facilitate the tumorigenesis of ESCC in vivo. Collectively, our findings identified that the key biological role of lncRNA DGCR5 in alternative splicing regulation and emphasized DGCR5 as a potential biomarker and therapeutic target for ESCC.

Iminodiacetic Acid as a Novel Metal-Binding Pharmacophore for New Delhi Metallo-ß-lactamase Inhibitor Development.

The fungal natural product aspergillomarasmine A (AMA) has been identified as a noncompetitive inhibitor of New Delhi metallo-ß-lactamase-1 (NDM-1) that inhibits by removing ZnII from the active-site. The nonselective metal-chelating properties and difficult synthesis and derivatization of AMA have hindered the development of this scaffold into a potent and selective inhibitor of NDM-1. Iminodiacetic acid (IDA) has been identified as the metal-binding pharmacophore (MBP) core of AMA that can be leveraged for inhibitor development. Herein, we report the use of IDA for fragment-based drug discovery (FBDD) of NDM-1 inhibitors. IDA (IC50 =120 µM) was developed into inhibitor 23 f (IC50 =8.6 µM, Ki =2.6 µM), which formed a ternary complex with NDM-1, as evidenced by protein thermal-shift and native-state electrospray ionization mass spectrometry (ESI-MS) experiments. Combining mechanistic analysis with inhibitor derivatization, the use of IDA as an alternative AMA scaffold for NDM-1 inhibitor development is detailed.

Elusive structural changes of New Delhi metallo-ß-lactamase revealed by ultraviolet photodissociation mass spectrometry.

We use mass spectrometry (MS), under denaturing and non-denaturing solution conditions, along with ultraviolet photodissociation (UVPD) to characterize structural variations in New Delhi metallo-ß-lactamase (NDM) upon perturbation by ligands or mutation. Mapping changes in the abundances and distributions of fragment ions enables sensitive detection of structural alterations throughout the protein. Binding of three covalent inhibitors was characterized: a pentafluorphenyl ester, an O-aryloxycarbonyl hydroxamate, and ebselen. The first two inhibitors modify Lys211 and maintain dizinc binding, although the pentafluorophenyl ester is not selective (Lys214 and Lys216 are also modified). Ebselen reacts with the sole Cys (Cys208) and ejects Zn2 from the active site. For each inhibitor, native UVPD-MS enabled simultaneous detection of the closing of a substrate-binding beta-hairpin loop, identification of covalently-modified residue(s), reporting of the metalation state of the enzyme, and in the case of ebselen, observation of the induction of partial disorder in the C-terminus of the protein. Owing to the ability of native UVPD-MS to track structural changes and metalation state with high sensitivity, we further used this method to evaluate the impact of mutations found in NDM clinical variants. Changes introduced by NDM-4 (M154L) and NDM-6 (A233V) are revealed to propagate through separate networks of interactions to direct zinc ligands, and the combination of these two mutations in NDM-15 (M154L, A233V) results in additive as well as additional structural changes. Insight from UVPD-MS helps to elucidate how distant mutations impact zinc affinity in the evolution of this antibiotic resistance determinant. UVPD-MS is a powerful tool capable of simultaneous reporting of ligand binding, conformational changes and metalation state of NDM, revealing structural aspects of ligand recognition and clinical variants that have proven difficult to probe.