Active Site Zn Research Articles

Efficacy of aspergillomarasmine A/meropenem combinations with and without avibactam against bacterial strains producing multiple β-lactamases.

The effectiveness of β-lactam antibiotics is increasingly threatened by resistant bacteria that harbor hydrolytic β-lactamase enzymes. Depending on the class of β-lactamase present, β-lactam hydrolysis can occur through one of two general molecular mechanisms. Metallo-β-lactamases (MBLs) require active site Zn2+ ions, whereas serine-β-lactamases (SBLs) deploy a catalytic serine residue. The result in both cases is drug inactivation via the opening of the β-lactam warhead of the antibiotic. MBLs confer resistance to most β-lactams and are non-susceptible to SBL inhibitors, including recently approved diazabicyclooctanes, such as avibactam; consequently, these enzymes represent a growing threat to public health. Aspergillomarasmine A (AMA), a fungal natural product, can rescue the activity of the β-lactam antibiotic meropenem against MBL-expressing bacterial strains. However, the effectiveness of this β-lactam/β-lactamase inhibitor combination against bacteria producing multiple β-lactamases remains unknown. We systematically investigated the efficacy of AMA/meropenem combination therapy with and without avibactam against 10 Escherichia coli and 10 Klebsiella pneumoniae laboratory strains tandemly expressing single MBL and SBL enzymes. Cell-based assays demonstrated that laboratory strains producing NDM-1 and KPC-2 carbapenemases were resistant to the AMA/meropenem combination but became drug-susceptible upon adding avibactam. We also probed these combinations against 30 clinical isolates expressing multiple β-lactamases. E. coli, Enterobacter cloacae, and K. pneumoniae clinical isolates were more susceptible to AMA, avibactam, and meropenem than Pseudomonas aeruginosa and Acinetobacter baumannii isolates. Overall, the results demonstrate that a triple combination of AMA/avibactam/meropenem has potential for empirical treatment of infections caused by multiple β-lactamase-producing bacteria, especially Enterobacterales.

Tail-Approach-Based Design and Synthesis of Coumarin-Monoterpenes as Carbonic Anhydrase Inhibitors and Anticancer Agents.

In this study, sixty novel coumarin-monoterpene compounds were synthesized in two series [thirty-two compounds (12-43) bearing a triazole ring in the first series, and twenty-eight compounds (44-71) bearing an alkyl chain in the second one]. Their inhibitory effects on the human carbonic anhydrase (hCA) isoforms I, II, IX, and XII and anticancer potentials were determined. All synthesized molecules selectively inhibited CA IX and XII. 23 and 42 were found to be the strongest inhibitors, with K i values of 1.9 nM against hCA IX. Also, 70 showed the highest inhibitory activity with a K i value of 4.9 nM against hCA XII. Moreover, their cytotoxic effects on colon adenocarcinoma (HT-29), prostate adenocarcinoma (PC-3), and breast adenocarcinoma (MCF-7) cell lines were evaluated. According to the cytotoxicity results, 14 (IC50 = 2.48 μM) and 63 (IC50 = 3.91 μM) exhibited the highest cytotoxicity on the MCF-7 cells, while 23 showed the strongest cytotoxic effect on both PC-3 (IC50 = 9.40 μM) and HT-29 (IC50 = 12.10 μM) cell lines. 14, 23, and 66 decreased CA IX and CA XII protein expression in HT-29 cells, while 23 and 66 showed the strongest reduction of both CA IX and CA XII in MCF-7 cells. All of the selected compounds increased total apoptosis in a concentration-dependent manner in HT-29 and MCF-7 cells. 14 has the strongest apoptotic effect in MCF-7 cells. 23 increased early apoptosis primarily, while 14 and 66 increased total apoptosis in HT-29. In addition, PI/Hoechst staining proves that apoptotic cells are increased in HT-29 with an effect of 14, 23, and 66. As a result of the modeling studies, it has been shown that only the open coumarin form of the compounds can interact directly with the active-site Zn2+ ion. It has been shown that coumarin-monoterpene structures with different alkyl and monoterpene groups both specifically inhibit CA IX and XII and exhibit specific cytotoxicity in different cell lines.

Structure of the catalytically active APOBEC3G bound to a DNA oligonucleotide inhibitor reveals tetrahedral geometry of the transition state

APOBEC3 proteins (A3s) are enzymes that catalyze the deamination of cytidine to uridine in single-stranded DNA (ssDNA) substrates, thus playing a key role in innate antiviral immunity. However, the APOBEC3 family has also been linked to many mutational signatures in cancer cells, which has led to an intense interest to develop inhibitors of A3’s catalytic activity as therapeutics as well as tools to study A3’s biochemistry, structure, and cellular function. Recent studies have shown that ssDNA containing 2′-deoxy-zebularine (dZ-ssDNA) is an inhibitor of A3s such as A3A, A3B, and A3G, although the atomic determinants of this activity have remained unknown. To fill this knowledge gap, we determined a 1.5 Å resolution structure of a dZ-ssDNA inhibitor bound to active A3G. The crystal structure revealed that the activated dZ-H2O mimics the transition state by coordinating the active site Zn2+ and engaging in additional stabilizing interactions, such as the one with the catalytic residue E259. Therefore, this structure allowed us to capture a snapshot of the A3’s transition state and suggests that developing transition-state mimicking inhibitors may provide a new opportunity to design more targeted molecules for A3s in the future.

A unique class of Zn2+-binding serine-based PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile

Treatment with β-lactam antibiotics, particularly cephalosporins, is a major risk factor for Clostridioides difficile infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), which are serine-based enzymes that assemble the bacterial cell wall. However, C. difficile has four different PBPs (PBP1-3 and SpoVD) with various roles in growth and spore formation, and their specific links to β-lactam resistance in this pathogen are underexplored. Here, we show that PBP2 (known to be essential for vegetative growth) is the primary bactericidal target for β-lactams in C. difficile. PBP2 is insensitive to cephalosporin inhibition, and this appears to be the main basis for cephalosporin resistance in this organism. We determine crystal structures of C. difficile PBP2, alone and in complex with β-lactams, revealing unique features including ligand-induced conformational changes and an active site Zn2+-binding motif that influences β-lactam binding and protein stability. The Zn2+-binding motif is also present in C. difficile PBP3 and SpoVD (which are known to be essential for sporulation), as well as in other bacterial taxa including species living in extreme environments and the human gut. We speculate that this thiol-containing motif and its cognate Zn2+ might function as a redox sensor to regulate cell wall synthesis for survival in adverse or anaerobic environments.

A combination strategy of structure-based virtual screening, MM-GBSA, cross docking, molecular dynamics and metadynamics simulations used to investigate natural compounds as potent and specific inhibitors of tumor linked human carbonic anhydrase IX

Cancer remains a serious health concern representing one of the leading causes of deaths worldwide. The enzyme human carbonic anhydrase IX (hCA IX) is found to be over-expressed in many cancer types and its selective inhibition over its cytosolic off-target isoform, human carbonic anhydrase II (hCA II), represents a potential area of research in the development of novel anticancer compounds. This work is concerned with the use of various in silico tools for the identification of natural product based molecules that can selectively inhibit hCA IX over hCA II. MM-GBSA assisted structure-based virtual screening against hCA IX was performed for nearly 225,000 natural products imported from the ZINC15 database. The obtained hits were checked for their potency by considering acetazolamide, the bound inhibitor of hCA IX, as the reference molecule, and 121 molecules were identified as potent hCA IX inhibitors. After ensuring their potency, cross-docking, followed by MM-GBSA calculations of the hits with hCA II, was performed, and their selectivity was assessed by considering the hCA IX selective compound SLC-0111 as the reference molecule, and 50 natural products were identified as potent as well as selective hCA IX inhibitors. Molecules with the quinoline scaffold showed the highest selectivity, and their selectivity was attributed to the strong electrostatic interactions of the zinc binding group (ZBG) with the active site Zn(II) ion. Furthermore, the stability of the binding modes of the top hCA IX selective hits was ensured by performing molecular dynamics (MD) simulations, which clearly proved that one of the short-listed molecules is truly selective, as it does not interact with the active site Zn(II) ion of hCA II, but interacts strongly with this ion in hCA IX. Bonding pose metadynamics studies revealed that the ligand moves to a more stable binding site from the one predicted by the docking studies and shows stronger interaction with the protein and Zn(II) at this binding site. The ligand is not likely to have issues with bioavailability. As a result, this ligand can be taken for bioassay testing and subsequently used as a feasible therapeutic treatment for a variety of cancer types. Communicated by Ramaswamy H. Sarma

First-in-Class Star-Shaped Triazine Dendrimers Endowed with MMP-9 Inhibition and VEGF Suppression Capacity: Design, Synthesis, and Anticancer Evaluation.

Off-target side effects are major challenges hindering the clinical success of matrix metalloproteinase (MMP) inhibitors. Various targeting strategies revitalized MMP research to eliminate this drawback. Herein, we developed s-triazine-based dendrimeric architecture not only amenable to tumor targeting but also decorated with pharmacophoric entities to endow MMP-9 inhibition for halting cancer progression. The design rationale utilized hydrazide branching chains as well as carboxylic and hydroxamic acid termini as Zn-binding groups to confer substantial MMP inhibitory potential. The carboxylic acids are tetherable to tumor targeting ligands and other cargo payloads as synergistic drugs via biodegradable linkages. The synthesized series were screened for cytotoxicity against normal fibroblasts (Wi-38) and two selected cancers (MDA-MB 231 and Caco-2) via MTT assay. The most active hexacarboxylic acid dendrimer 8a was more potent and safer than Dox against MDA-MB 231 and Caco-2 cells. It intrinsically inhibited MMP-9 with selectivity over MMP-2. Docking simulations demonstrated that the extended carboxylic acid termini of 8a could possibly chelate the active site Zn of MMP-9 and form hydrogen-bonding interactions with the ligand essential backbone Tyr423. In addition, it suppressed the correlated oncogenic mediators VEGF and cyclin D, upregulated p21 expression, induced apoptosis (>75%), and inhibited the tumor cell migration (∼84%) in the treated cancer cells. Thus, up to our knowledge, it is the first triazine-based MMP-9 inhibitor dendrimer endowed with VEGF suppression potential that can be employed as a bioactive carrier.

Bioorthogonal PEGylation Prolongs the Elimination Half-Life of N-TIMP2 While Retaining MMP Inhibition.

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of the matrix metalloproteinase (MMP) family of proteins, whose members are key regulators of the proteolysis of extracellular matrix components and hence of multiple biological processes. In particular, imbalanced activity of matrix metalloproteinase-14 (MMP-14) may lead to the development of cancer and cardiovascular and other diseases. This study aimed to engineer TIMP2, one of the four homologous TIMPs, as a potential therapeutic by virtue of its ability to bind to the active-site Zn2+ of MMP-14. However, the susceptibility to degradation of TIMP2 and its small size, which results in a short circulation half-life, limit its use as a therapeutic. PEGylation was thus used to improve the pharmacokinetic profile of TIMP2. PEGylation of the MMP-targeting N-terminal domain of TIMP2 (N-TIMP2), via either cysteine or lysine residues, resulted in a significant decrease in N-TIMP2 affinity toward MMP-14 or multisite conjugation and conjugate heterogeneity, respectively. Our strategy designed to address this problem was based on incorporating a noncanonical amino acid (NCAA) into N-TIMP2 to enable site-specific mono-PEGylation. The first step was to incorporate the NCAA propargyl lysine (PrK) at position S31 in N-TIMP2, which does not interfere with the N-TIMP2-MMP-14 binding interface. Thereafter, site-specific PEGylation was achieved via a click chemistry reaction between N-TIMP2-S31PrK and PEG-azide-20K. Inhibition studies showed that PEGylated N-TIMP2-S31PrK did indeed retain its inhibitory activity toward MMP-14. The modified protein also showed improved serum stability vs non-PEGylated N-TIMP2. In vivo pharmacokinetic studies in mice revealed a significant 8-fold increase in the elimination half-life of PEGylated N-TIMP2 vs the non-PEGylated protein. This study shows that site-specific bioorthogonal mono-PEGylation extends the half-life of N-TIMP2 without impairing its biological activity, thereby highlighting the advantage of this strategy for generating potent PEGylated proteins.

Celecoxib, Glipizide, Lapatinib, and Sitagliptin as potential suspects of aggravating SARS-CoV-2 (COVID-19) infection: a computational approach

COVID-19 caused by SARS-CoV-2 has emerged as a potential threat to human life, especially to people suffering from chronic diseases. In this study, we investigated the ability of selected FDA-approved drugs to inhibit TACE (tumor necrosis factor α converting enzyme), which is responsible for the shedding of membrane-bound ACE2 (angiotensin-converting enzyme2) receptors into soluble ACE2. The inhibition of TACE would lead to an increased population of membrane-bound ACE2, which would facilitate ACE2-Spike protein interaction and viral entry. A total of 50 drugs prescribed in treating various chronic diseases in Saudi Arabia were screened by performing molecular docking using AutoDock4.2. Based on docking energy (≤ −9.00 kcal mol−1), four drugs (Celecoxib, Glipizide, Lapatinib, and Sitagliptin) were identified as potential inhibitors of TACE, with binding affinities up to 106–107 M −1. Analysis of the molecular docking suggests that these drugs were bound to TACE's catalytic domain and interact with the key residues such as His405, Glu406, and His415, which are involved in active site Zn2+ ion chelation. Molecular dynamics simulation was performed to confirm the stability of TACE-drugs complexes. RMSD (root mean square deviation), RMSF (root mean square fluctuation), Rg (radius of gyration), and SASA (solvent accessible surface area) were within the acceptable limits. Free energy calculations using Prime-MM/GBSA suggest that Celecoxib formed the most stable complex with TACE, followed by Glipizide, Sitagliptin, and Lapatinib. The finding of this study suggests a mechanism for drugs to aggravate SARS-CoV-2 infection and hence high mortality in patients suffering from chronic diseases. Communicated by Ramaswamy H. Sarma

Comprehensive Understanding of the Relationship between Bioactive Compounds of Black Tea and its Angiotensin Converting Enzyme (ACE) Inhibition and Antioxidant Activity

Long term regular intake of black tea (BT) can lower blood pressure, which is probably due to its antioxidant activity and angiotensin converting enzymes (ACE) inhibitory activity. This study achieves a comprehensive understanding of the relationship between bioactive compounds of BT and its ACE inhibitory activity and antioxidant activity. Phenolic compounds are closely related to antioxidant activity and ACE inhibitory activity. Catechin (C) exhibits stronger inhibitory activity on ACE enzyme than that of other compounds. Molecular docking demonstrates that C could directly bind to ACE active site pockets and Zn(II). Other bioactive compounds are involved in antioxidant and ACE inhibitory activity in varying degrees but no obvious trend is established. Our study proposes a conjecture that some bioactive compounds of BT regulate antioxidant defenses through mechanisms that involve ACE. The mixed mode of in vitro inhibition of ACE and oxidant of BT bioactive compounds needs to be further investigated.

Hydroxypyridinethione Inhibitors of Human Insulin-Degrading Enzyme.

Insulin-degrading enzyme (IDE) is a human mononuclear Zn2+ -dependent metalloenzyme that is widely regarded as the primary peptidase responsible for insulin degradation. Despite its name, IDE is also critically involved in the hydrolysis of several other disparate peptide hormones, including glucagon, amylin, and the amyloid β-protein. As such, the study of IDE inhibition is highly relevant to deciphering the role of IDE in conditions such as type-2 diabetes mellitus and Alzheimer disease. There have been few reported IDE inhibitors, and of these, inhibitors that directly target the active-site Zn2+ ion have yet to be fully explored. In an effort to discover new, zinc-targeting inhibitors of IDE, a library of ∼350 metal-binding pharmacophores was screened against IDE, resulting in the identification of 1-hydroxypyridine-2-thione (1,2-HOPTO) as an effective Zn2+ -binding scaffold. Screening a focused library of HOPTO compounds identified 3-sulfonamide derivatives of 1,2-HOPTO as inhibitors of IDE (Ki values of ∼50 μM). Further structure-activity relationship studies yielded several thiophene-sulfonamide HOPTO derivatives with good, broad-spectrum activity against IDE that have the potential to be useful pharmacological tools for future studies of IDE.

Structure of a reaction intermediate mimic in t6A biosynthesis bound in the active site of the TsaBD heterodimer from Escherichia coli.

The tRNA modification N6-threonylcarbamoyladenosine (t6A) is universally conserved in all organisms. In bacteria, the biosynthesis of t6A requires four proteins (TsaBCDE) that catalyze the formation of t6A via the unstable intermediate l-threonylcarbamoyl-adenylate (TC-AMP). While the formation and stability of this intermediate has been studied in detail, the mechanism of its transfer to A37 in tRNA is poorly understood. To investigate this step, the structure of the TsaBD heterodimer from Escherichia coli has been solved bound to a stable phosphonate isosteric mimic of TC-AMP. The phosphonate inhibits t6A synthesis in vitro with an IC50 value of 1.3 μM in the presence of millimolar ATP and L-threonine. The inhibitor binds to TsaBD by coordination to the active site Zn atom via an oxygen atom from both the phosphonate and the carboxylate moieties. The bound conformation of the inhibitor suggests that the catalysis exploits a putative oxyanion hole created by a conserved active site loop of TsaD and that the metal essentially serves as a binding scaffold for the intermediate. The phosphonate bound crystal structure should be useful for the rational design of potent, drug-like small molecule inhibitors as mechanistic probes or potentially novel antibiotics.

Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer Disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded β-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.

Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer’s disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded β-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.

Structure–activity relationship of ipglycermide binding to phosphoglycerate mutases

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex.

1250. Novel Boronic Acid Transition State Analogs (BATSI) with in vitro inhibitory activity against class A, B and C β-lactamases

BackgroundCatalytic mechanisms of serine β-lactamases (SBL; classes A, C and D) and metallo-β-lactamases (MBLs) have directed divergent strategies towards inhibitor design. SBL inhibitors act as high affinity substrates that -as in BATSIs- form a reversible, dative covalent bond with the conserved active site Ser. MBL inhibitors bind the active-site Zn2+ ions and displace the nucleophilic OH-. Herein, we explore the efficacy of a series of BATSI compounds with a free-thiol group at inhibiting both SBL and MBL.MethodsExploratory compounds were synthesized using stereoselective homologation of (+) pinandiol boronates to introduce the amino group on the boron-bearing carbon atom, which was subsequently acylated with mercaptopropanoic acid. Representative SBL (KPC-2, ADC-7, PDC-3 and OXA-23) and MBL (IMP-1, NDM-1 and VIM-2) were purified and used for the kinetic characterization of the BATSIs. In vitro activity was evaluated by a modified time-kill curve assay, using SBL and MBL-producing strains.ResultsKinetic assays revealed that IC50 values ranged from 1.3 µM to >100 µM for this series. The best compound, s08033, demonstrated inhibitory activity against KPC-2, VIM-2, ADC-7 and PDC-3, with IC50 in the low μM range. Reduction of at least 1.5 log10-fold of viable cell counts upon exposure to sub-lethal concentrations of antibiotics (AB) + s08033, compared to the cells exposed to AB alone, demonstrated the microbiological activity of this novel compound against SBL- and MBL-producing E. coli (Table 1).Table 1 ConclusionAddition of a free-thiol group to the BATSI scaffold increases the range of these compounds resulting in a broad-spectrum inhibitor toward clinically important carbapenemases and cephalosporinases.DisclosuresRobert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Discovery and Design of Peptides as MMP9 Inhibitors through Structure-Based Molecular Docking for Targeted Mantle Cell Lymphoma Therapy.

Enhanced expression and activation of metalloproteinase-9 (MMP9) are associated with Mantle Cell Lymphoma (MCL) progression, invasion and metastasis. To find a potential peptide inhibitor against MMP9, which, in turn, could inhibit MCL progression. We performed CCK8 assay, western blot, and transwell assays for RNAi activity. Molecular Operating Environment (MOE) software was applied for structural optimization as MMP9 and peptides were docked. We used gelatin zymography and confocal microscopy to confirm that the peptides can inhibit MMP9 activity. We applied CCK8 and transwell assay to evaluate cell proliferation and metastasis, and flow cytometry to evaluate cell cycle progression and apoptosis. High MMP9 expression was observed in 49 of 88 samples (55.7%). Patients with high MMP9 expression were more likely to present with high stage (Stage 3-4, P=0.01), bone marrow invasion (P=0.033), and high-level LDH (P=0.000). High MMP9 expression was associated with significantly shorter overall survival (OS, HR=2.378, P=0.012) and progression-free survival (PFS, HR=2.068, P=0.03). Multivariate analysis identified high MMP9 expression (P= 0.027), high-risk mantle cell lymphoma international prognostic index (MIPI, HR=2.327, P=0.023), and no radiation therapy (P=0.035) as adverse prognostic factors. The silencing of MMP9 in Jeko-1 cells by RNAi suppressed cells migration and invasion in vitro (P<0.05). According to the docking results, peptide M3 bound deeply in the binding pocket of MMP9 and had interaction with the active-site Zn2+ ion in the catalytic domain. M3 was not only compatible with MMP9, but also inhibited its activity. M3 inhibited Jeko-1 cells proliferation, metastasis, and cell cycle progression, and promoted cell apoptosis rate (P<0.05). We designed M3 through structure-based molecular docking, which can specifically bind to MMP9 and inhibit the activity of MMP9. M3 could be a potential antagonist in the treatment of MCL with MMP9 overexpression.

Structural basis for the hydrolytic dehalogenation of the fungicide chlorothalonil

Cleavage of aromatic carbon-chlorine bonds is critical for the degradation of toxic industrial compounds. Here, we solved the X-ray crystal structure of chlorothalonil dehalogenase (Chd) from Pseudomonas sp. CTN-3, with 15 of its N-terminal residues truncated (ChdT), using single-wavelength anomalous dispersion refined to 1.96 Å resolution. Chd has low sequence identity (<15%) compared with all other proteins whose structures are currently available, and to the best of our knowledge, we present the first structure of a Zn(II)-dependent aromatic dehalogenase that does not require a coenzyme. ChdT forms a "head-to-tail" homodimer, formed between two α-helices from each monomer, with three Zn(II)-binding sites, two of which occupy the active sites, whereas the third anchors a structural site at the homodimer interface. The catalytic Zn(II) ions are solvent-accessible via a large hydrophobic (8.5 × 17.8 Å) opening to bulk solvent and two hydrophilic branched channels. Each active-site Zn(II) ion resides in a distorted trigonal bipyramid geometry with His117, His257, Asp116, Asn216, and a water/hydroxide as ligands. A conserved His residue, His114, is hydrogen-bonded to the Zn(II)-bound water/hydroxide and likely functions as the general acid-base. We examined substrate binding by docking chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile, TPN) into the hydrophobic channel and observed that the most energetically favorable pose includes a TPN orientation that coordinates to the active-site Zn(II) ions via a CN and that maximizes a π-π interaction with Trp227 On the basis of these results, along with previously reported kinetics data, we propose a refined catalytic mechanism for Chd-mediated TPN dehalogenation.

Structural Basis of 7‐Deazaguanine Modification of DNA in Bacteria and Phage

Two important modifications of tRNA, the 7‐deazaguanine nucleosides queuosine (Q) and archaeosine (G+), are biosynthesized from GTP in bacteria and archaea, respectively, in a well characterized multi‐enzyme pathway leading to the shared advanced intermediate, 7‐cyano‐7‐deazaguanine (preQ0). In bacteria, preQ0 is converted to an aminomethyl derivative that is then inserted in tRNA by the bacterial tRNA‐guanine transglycosylase (TGT) enzyme. In archaea, preQ0 is inserted directly in tRNA by the archaeal TGT before conversion to G+. Recently, in 230 bacterial and phage genomes, a genomic island that contains a paralog of TGT was identified, which led to the discovery of Q, G+ and preQ0 deoxy derivatives in the DNA of some of these organisms. This gene cluster was renamed DpdA‐K, for “7‐deazapurine in DNA.” The G+‐modified DNA of E. coli bacteriophage 9g has been shown to resist restriction by &gt;140 Type II restriction endonucleases (REases), consistent with a role of the modification as a defense mechanism. Recent experiments have shown that DpdA is a DNA‐guanine transglycosylase that catalyzes the insertion of preQ0 at a specific palindromic sequence in DNA. Here we report overexpression, purification, and crystallographic analysis of S. Montevideo DpdA (SmDpdA, ~47 kDa). The crystal structure, determined at 2.25‐Å resolution by multi‐wavelength anomalous diffraction methods, reveals a TIM‐barrel enzyme similar to the bacterial TGT in overall fold, active site and structural Zn2+ site. However, a large DpdA‐specific insertion in the TIM barrel provides an extended positively charged 26‐Å wide surface groove, consistent with a DNA binding surface. Free docking of a DNA duplex using the HADDOCK server places DNA to this surface in a fashion reminiscent of DNA binding to the ubiquitous Zn2+‐dependent DNA repair enzyme apurinic/apyrimidinic endonuclease IV. The structure and docking model suggest that DpdA bends its substrate DNA by 60° and flips the modification‐site guanine base out of the helix for transglycosylation. The results also inform the design of a substrate DNA duplex for future crystallization of the nucleoprotein complex.Support or Funding InformationNIGMS grant GM110588 and GM132254 to M.A.S., and The California Metabolic Research Foundation (SDSU).

Understanding structure-based dynamic interactions of antihypertensive peptides extracted from food sources

Functional foods are emerging as essential healthy nutritional component due to their abundant wellbeing benefits. Especially the food-derived peptides are considered as key components for playing their biologically active roles. One such robust therapeutics that already exploited with food peptides that help treating high blood pressure via targeting Angiotensin-Converting Enzyme (ACE). This in silico study demonstrated the inhibitory potential of antihypertensive peptides derived from food sources. This study involves an intensive structure-based analysis of enzyme-peptide interactions using Molecular Dynamics (MD) simulations. Interestingly, this study will help us to get deeper understanding on how food peptides achieve successful inhibition of ACE. In this study, the peptide-enzyme complexes revealed two binding pockets, A and B, on either side of the active site Zn atom. Pocket B has a smaller binding site volume than pocket A, comprised of β-sheets and the active site opening cleft. The interface of the binding sites showed that the enzyme structure was negative to neutral charge, and the peptide structure was positive to neutral charge. The dynamics of complex structures of seven highly potential peptides were performed for 20 ns each at 300 K. Comparative analysis of RMSD, RMSF and binding energies show the enzyme-peptide complexes and the overall stability of apo-enzyme. Importantly, two peptides AFKAWAVAR and IWHHTF showed the highest variation in their RMSD as compared to the apo-enzyme. This study will further be useful for the assessment of the characteristics to predict novel inhibitory peptides that can be generated from food proteins.Communicated by Ramaswamy H. Sarma

New thiopyrimidine-benzenesulfonamide conjugates as selective carbonic anhydrase II inhibitors: synthesis, in vitro biological evaluation, and molecular docking studies

In the present work, a new series of thiopyrimidine-benzenesulfonamide conjugates was designed, synthesized and tested as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. Our design strategy was based on the molecular hybridization of the benzenesulfonamide moiety as a zinc binding group (ZBG), an alkylated thiopyrimidine moiety as a spacer and (un)substituted phenyl moieties with various electronic and hydrophobic environments as a tail. The designed and synthesized compounds were evaluated against four human (h) CA isoforms hCA I, hCA II, hCA IX and hCA XII. Series 6 showed promising activity and selectivity toward the cytosolic isoforms hCA I and hCA II versus the membrane bound isoforms hCA IX and hCA XII. Compounds 6e and 6f showed Ki of 0.04 µM against hCA II with a selectivity of 15.8- to 980-fold towards hCA II over hCA I, hCA IX, hCA XII isoforms. Molecular docking in the hCA II active site attributed the promising inhibitory activity of series 6 to the interaction of their sulfonamide moiety with the active site Zn2+ ion as well as its hydrogen bonding with the key amino acids Thr199 and Thr200. Through hydrophobic interaction, the benzenesulfonamide and the thiopyrimidine moieties interact with the hydrophobic side chains of the amino acids Val121/Leu198 and Ile91/Phe131, respectively. These results indicated that the designed and synthesized series is an interesting scaffold that can be further optimized for the development of selective antiglaucoma drugs.