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Bacterial cell-wall hydrolases must be tightly regulated during bacterial cell division to prevent aberrant cell lysis and to allow final separation of viable daughter cells. In a multidisciplinary work, we disclose the molecular dialogue between the cell-wall hydrolase LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP in Streptococcus pneumoniae. After characterizing the peptidoglycan recognition mode by the catalytic domain of LytB, we further demonstrate that LytB possesses a modular organization allowing the specific binding to wall teichoic acids and to the protein kinase StkP. Structural and cellular studies notably reveal that the temporal and spatial localization of LytB is governed by the interaction between specific modules of LytB and the final PASTA domain of StkP. Our data collectively provide a comprehensive understanding of how LytB performs final separation of daughter cells and highlights the regulatory role of eukaryotic-like kinases on lytic machineries in the last step of cell division in streptococci., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The penicillin-binding proteins are the enzyme catalysts of the critical transpeptidation crosslinking polymerization reaction of bacterial peptidoglycan synthesis and the molecular targets of the penicillin antibiotics. Here, we report a combined crystallographic, small-angle X-ray scattering (SAXS) in-solution structure, computational and biophysical analysis of PBP1 of  Staphylococcus aureus (sa PBP1), providing mechanistic clues about its function and regulation during cell division. The structure reveals the pedestal domain, the transpeptidase domain, and most of the linker connecting to the "penicillin-binding protein and serine/threonine kinase associated" (PASTA) domains, but not its two PASTA domains, despite their presence in the construct. To address this absence, the structure of the PASTA domains was determined at 1.5 Å resolution. Extensive molecular-dynamics simulations interpret the PASTA domains of  sa PBP1 as conformationally mobile and separated from the transpeptidase domain. This conclusion was confirmed by SAXS experiments on the full-length protein in solution. A series of crystallographic complexes with β-lactam antibiotics (as inhibitors) and penta-Gly (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. The observed mobility of the PASTA domain in  sa PBP1 could play a crucial role for  in vivo  interaction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the coronavirus disease of 2019 (COVID-19). Its genome encodes two open reading frames for two large proteins, PP1a and PP1ab. Within the two polypeptide stretches, there are two proteases that process the large proteins into 15 discrete proteins essential for the assembly of the virion during its replication. We describe herein the cloning of the genes for these discrete proteins optimized for expression in Escherichia coli , production of the proteins, and their purification to homogeneity. These included all but six: NSP6, which possesses eight transmembrane regions, and five that are small proteins/peptides (E, ORF3b, ORF6, ORF7b, and ORF10). These proteins are intended for experimental validation of small-molecule binders as molecular template hits. The proof of concept was established with the ADP-ribosylhydrolase (ARH) domain of NSP3 in discovery of small-molecule templates that could serve as the basis for further optimization. The hit molecules include one submicromolar and a few low-micromolar binders to the ARH domain. Availability of these proteins in soluble forms opens up the opportunity for discoveries of novel templates with the potential for anti-COVID-19 pharmaceuticals., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The need for new classes of antibacterials is genuine in light of the dearth of clinical options for the treatment of bacterial infections. The prodigious discoveries of antibiotics during the 1940s to 1970s, a period wistfully referred to as the Golden Age of Antibiotics, have not kept up in the face of emergence of resistant bacteria in the past few decades. There has been a renewed interest in old drugs, the repurposing of the existing antibiotics and pairing of synergistic antibiotics or of an antibiotic with an adjuvant. Notwithstanding, discoveries of novel classes of these life-saving drugs have become increasingly difficult, calling for new paradigms. We describe, herein, three strategies from our laboratories toward discoveries of new antibacterials and adjuvants using computational and multidisciplinary experimental methods. One approach targets penicillin-binding proteins (PBPs), biosynthetic enzymes of cell-wall peptidoglycan, for discoveries of non-β-lactam inhibitors. Oxadiazoles and quinazolinones emerged as two structural classes out of these efforts. Several hundred analogs of these two classes of antibiotics have been synthesized and fully characterized in our laboratories. A second approach ventures into inhibition of allosteric regulation of cell-wall biosynthesis. The mechanistic details of allosteric regulation of PBP2a of Staphylococcus aureus , discovered in our laboratories, is outlined. The allosteric site in this protein is at 60 Å distance to the active site, whereby ligand binding at the former makes access to the latter by the substrate possible. We have documented that both quinazolinones and ceftaroline, a fifth-generation cephalosporin, bind to the allosteric site in manifestation of the antibacterial activity. Attempts at inhibition of the regulatory phosphorylation events identified three classes of antibacterial adjuvants and one class of antibacterials, the picolinamides. The chemical structures for these hits went through diversification by synthesis of hundreds of analogs. These analogs were characterized in various assays for identification of leads with adjuvant and antibacterial activities. Furthermore, we revisited the mechanism of bulgecins, a class of adjuvants discovered and abandoned in the 1980s. These compounds potentiate the activities of β-lactam antibiotics by the formation of bulges at the sites of septum formation during bacterial replication, which are points of structural weakness in the envelope. These bulges experience rupture, which leads to bacterial death. Bulgecin A inhibits the lytic transglycosylase Slt of Pseudomonas aeruginosa as a likely transition-state mimetic for its turnover of the cell-wall peptidoglycan. Once damage to cell wall is inflicted by a β-lactam antibiotic, the function of Slt is to repair the damage. When Slt is inhibited by bulgecin A, the organism cannot cope with it and would undergo rapid lysis. Bulgecin A is an effective adjuvant of β-lactam antibiotics. These discoveries of small-molecule classes of antibacterials or of adjuvants to antibacterials hold promise in strategies for treatment of bacterial infections.

We report herein the syntheses of 79 derivatives of the 4(3 H )-quinazolinones and their structure-activity relationship (SAR) against methicillin-resistant Staphylococcus aureus (MRSA). Twenty one analogs were further evaluated in in vitro assays. Subsequent investigation of the pharmacokinetic properties singled out compound 73 (( E )-3-(5-carboxy-2-fluorophenyl)-2-(4-cyanostyryl)quinazolin-4(3 H )-one) for further study. The compound synergized with piperacillin-tazobactam (TZP) both in vitro and in vivo in a clinically relevant mouse model of MRSA infection. The TZP combination lacks activity against MRSA, yet it synergized with compound 73 to kill MRSA in a bactericidal manner. The synergy is rationalized by the ability of the quinazolinones to bind to the allosteric site of penicillin-binding protein (PBP)2a, resulting in opening of the active site, whereby the β-lactam antibiotic now is enabled to bind to the active site in its mechanism of action. The combination effectively treats MRSA infection, for which many antibiotics (including TZP) have faced clinical obsolescence.

BglX is a heretofore uncharacterized periplasmic glycoside hydrolase (GH) of the human pathogen Pseudomonas aeruginosa . X-ray analysis identifies it as a protein homodimer. The two active sites of the homodimer comprise catalytic residues provided by each monomer. This arrangement is seen in <2% of the hydrolases of known structure. In vitro substrate profiling shows BglX is a catalyst for β-(1→2) and β-(1→3) saccharide hydrolysis. Saccharides with β-(1→4) or β-(1→6) bonds, and the β-(1→4) muropeptides from the cell-wall peptidoglycan, are not substrates. Additional structural insights from X-ray analysis (including structures of a mutant enzyme-derived Michaelis complex, two transition-state mimetics, and two enzyme-product complexes) enabled the comprehensive description of BglX catalysis. The half-chair ( 4 H 3 ) conformation of the transition-state oxocarbenium species, the approach of the hydrolytic water molecule to the oxocarbenium species, and the stepwise release of the two reaction products were also visualized. The substrate pattern for BglX aligns with the [β-(1→2)-Glc] x and [β-(1→3)-Glc] x periplasmic osmoregulated periplasmic glucans, and possibly with the Psl exopolysaccharides, of P. aeruginosa . Both polysaccharides are implicated in biofilm formation. Accordingly, we show that inactivation of the bglX gene of P. aeruginosa PAO1 attenuates biofilm formation.

SPOR domains are widely present in bacterial proteins that recognize cell-wall peptidoglycan strands stripped of the peptide stems. This type of peptidoglycan is enriched in the septal ring as a product of catalysis by cell-wall amidases that participate in the separation of daughter cells during cell division. Here, we document binding of synthetic denuded glycan ligands to the SPOR domain of the lytic transglycosylase RlpA from Pseudomonas aeruginosa (SPOR-RlpA) by mass spectrometry and structural analyses, and demonstrate that indeed the presence of peptide stems in the peptidoglycan abrogates binding. The crystal structures of the SPOR domain, in the apo state and in complex with different synthetic glycan ligands, provide insights into the molecular basis for recognition and delineate a conserved pattern in other SPOR domains. The biological and structural observations presented here are followed up by molecular-dynamics simulations and by exploration of the effect on binding of distinct peptidoglycan modifications.

The quinazolinones are a new class of antibacterials with in vivo efficacy against methicillin-resistant Staphylococcus aureus (MRSA). The quinazolinones target cell wall biosynthesis and have a unique mechanism of action by binding to the allosteric site of penicillin-binding protein 2a (PBP 2a). We investigated the potential for synergism of a lead quinazolinone with several antibiotics of different classes using checkerboard and time-kill assays. The quinazolinone synergized with β-lactam antibiotics. The combination of the quinazolinone with commercial piperacillin-tazobactam showed bactericidal synergy at sub-MICs of all three drugs. We demonstrated the efficacy of the triple-drug combination in a mouse MRSA neutropenic thigh infection model. The proposed mechanism for the synergistic activity in MRSA involves inhibition of the β-lactamase by tazobactam, which protects piperacillin from hydrolysis, which can then inhibit its target, PBP 2. Furthermore, the quinazolinone binds to the allosteric site of PBP 2a, triggering the allosteric response. This leads to the opening of the active site, which, in turn, binds another molecule of piperacillin. In other words, PBP 2a, which is not normally inhibited by piperacillin, becomes vulnerable to inhibition in the presence of the quinazolinone. The collective effect is the impairment of cell wall biosynthesis, with bactericidal consequence. Two crystal structures for complexes of the antibiotics with PBP 2a provide support for the proposed mechanism of action., (Copyright © 2019 American Society for Microbiology.)

Lytic transglycosylases (LTs) are bacterial enzymes that catalyze the cleavage of the glycan strands of the bacterial cell wall. The mechanism of this cleavage is a remarkable intramolecular transacetalization reaction, accomplished by an ensemble of active-site residues. Because the LT reaction occurs in parallel with the cell wall bond-forming reactions catalyzed by the penicillin-binding proteins, simultaneous inhibition of both enzymes can be particularly bactericidal to Gram-negative bacteria. The MltE lytic transglycosylase is the smallest of the eight LTs encoded by the Escherichia coli genome. Prior crystallographic and computational studies identified four active-site residues-E64, S73, S75, and Y192-as playing roles in catalysis. Each of these four residues was individually altered by mutation to give four variant enzymes (E64Q, S73A, S75A, and Y192F). All four variants showed reduced catalytic activity [soluble wild type (100%) > soluble Y192F and S75A (both 40%) > S73A (4%) > E64Q (≤1%)]. The crystal structure of each variant protein was determined at the resolution of 2.12 Å for E64Q, 2.33 Å for Y192F, 1.38 Å for S73A, and 1.35 Å for S75A. These variants show alteration of the hydrogen-bond interactions of the active site. Within the framework of a prior computational study of the LT mechanism, we suggest the mechanistic role of these four active-site residues in MltE catalysis.

Diabetic foot ulcers (DFUs) are a significant health problem. A single existing FDA-approved drug for this ailment, becaplermin, is not standard-of-care. We previously demonstrated that upregulation of active matrix metalloproteinase (MMP)-9 is the reason that the diabetic wound in mice is recalcitrant to healing and that MMP-8 participates in wound repair. In the present study, we validate the target MMP-9 by identifying and quantifying active MMP-8 and MMP-9 in human diabetic wounds using an affinity resin that binds exclusively to the active forms of MMPs coupled with proteomics. Furthermore, we synthesize and evaluate enantiomerically pure ( R)- and ( S)-ND-336, as inhibitors of the detrimental MMP-9, and show that the ( R)-enantiomer has superior efficacy in wound healing over becaplermin. Our results reveal that the mechanisms of pathology and repair are similar in diabetic mice and diabetic humans and that ( R)-ND-336 holds promise for the treatment of DFUs as a first-in-class therapeutic.

The metalloproteinase ADAM10 has been reported as an important target for drug discovery in several human diseases. In this vein, (6 S ,7 S )- N -hydroxy-5-methyl-6-(4-(5-(trifluoromethyl)pyridin-2-yl)piperazine-1-carbonyl)-5-azaspiro[2.5]octane-7-carboxamide (compound 1 ) has been reported as a selective ADAM10 inhibitor. We synthesized this compound and document that it lacks both potency and selectivity in inhibition of ADAM10. This finding necessitated a structure-based computational analysis to investigate potency and selectivity of ADAM10 inhibition. The model that emerged indeed excluded compound 1 as an inhibitor for ADAM10, while suggesting another reported compound, (1 R ,3 S ,4 S )-3-(hydroxycarbamoyl)-4-(4-phenylpiperidine-1-carbonyl)cyclohexyl pyrrolidine-1-carboxylate (compound 2 ), as an ADAM10 selective inhibitor. Compound 2 was synthesized and its potency, and selectivity in inhibition of ADAM10 were documented with a panel of several related enzymes. Pharmacokinetic studies of compound 2 in mice documented that the compound crosses the blood-brain barrier and may be useful as a pharmacological agent or mechanistic tool to delineate the role of ADAM10 in neurological diseases., Competing Interests: The authors declare no competing financial interest.

β-Lactam antibiotics inhibit cell-wall transpeptidases, preventing the peptidoglycan, the major constituent of the bacterial cell wall, from cross-linking. This causes accumulation of long non-cross-linked strands of peptidoglycan, which leads to bacterial death. Pseudomonas aeruginosa , a nefarious bacterial pathogen, attempts to repair this aberrantly formed peptidoglycan by the function of the lytic transglycosylase Slt. We document in this report that Slt turns over the peptidoglycan by both exolytic and endolytic reactions, which cause glycosidic bond scission from a terminus or in the middle of the peptidoglycan, respectively. These reactions were characterized with complex synthetic peptidoglycan fragments that ranged in size from tetrasaccharides to octasaccharides. The X-ray structure of the wild-type apo Slt revealed it to be a doughnut-shaped protein. In a series of six additional X-ray crystal structures, we provide insights with authentic substrates into how Slt is enabled for catalysis for both the endolytic and exolytic reactions. The substrate for the exolytic reaction binds Slt in a canonical arrangement and reveals how both the glycan chain and the peptide stems are recognized by the Slt. We document that the apo enzyme does not have a fully formed active site for the endolytic reaction. However, binding of the peptidoglycan at the existing subsites within the catalytic domain causes a conformational change in the protein that assembles the surface for binding of a more expansive peptidoglycan between the catalytic domain and an adjacent domain. The complexes of Slt with synthetic peptidoglycan substrates provide an unprecedented snapshot of the endolytic reaction., Competing Interests: The authors declare no conflict of interest.

Transpeptidases, members of the penicillin-binding protein (PBP) families, catalyze cross-linking of the bacterial cell wall. This transformation is critical for the survival of bacteria, and it is the target of inhibition by β-lactam antibiotics. We report herein our structural insights into catalysis by the essential PBP2x of Streptococcus pneumoniae by disclosing a total of four X-ray structures, two computational models based on the crystal structures, and molecular-dynamics simulations. The X-ray structures are for the apo PBP2x, the enzyme modified covalently in the active site by oxacillin (a penicillin antibiotic), the enzyme modified by oxacillin in the presence of a synthetic tetrasaccharide surrogate for the cell-wall peptidoglycan, and a noncovalent complex of cefepime (a cephalosporin antibiotic) bound to the active site. A prerequisite for catalysis by transpeptidases, including PBP2x, is the molecular recognition of nascent peptidoglycan strands, which harbor pentapeptide stems. We disclose that the recognition of nascent peptidoglycan by PBP2x takes place by complexation of one pentapeptide stem at an allosteric site located in the PASTA domains of this enzyme. This binding predisposes the third pentapeptide stem in the same nascent peptidoglycan strand to penetration into the active site for the turnover events. The complexation of the two pentapeptide stems in the same peptidoglycan strand is a recognition motif for the nascent peptidoglycan, critical for the cell-wall cross-linking reaction.

Lytic transglycosylases (LTs) catalyze the non-hydrolytic cleavage of the bacterial cell wall by an intramolecular transacetalization reaction. This reaction is critically and broadly important in modifications of the bacterial cell wall in the course of its biosynthesis, recycling, manifestation of virulence, insertion of structural entities such as the flagellum and the pili, among others. The first QM/MM analysis of the mechanism of reaction of an LT, that for the Escherichia coli MltE, is undertaken. The study reveals a conformational itinerary consistent with an oxocarbenium-like transition state, characterized by a pivotal role for the active-site glutamic acid in proton transfer. Notably, an oxazolinium intermediate, as a potential intermediate, is absent. Rather, substrate-assisted catalysis is observed through a favorable dipole provided by the N-acetyl carbonyl group of MurNAc saccharide. This interaction stabilizes the incipient positive charge development in the transition state. This mechanism coincides with near-synchronous acetal cleavage and acetal formation.

The N-acetylglucosaminidase NagZ of Pseudomonas aeruginosa catalyzes the first cytoplasmic step in recycling of muropeptides, cell-wall-derived natural products. This reaction regulates gene expression for the β-lactam resistance enzyme, β-lactamase. The enzyme catalyzes hydrolysis of N-acetyl-β-d-glucosamine-(1→4)-1,6-anhydro-N-acetyl-β-d-muramyl-peptide (1) to N-acetyl-β-d-glucosamine (2) and 1,6-anhydro-N-acetyl-β-d-muramyl-peptide (3). The structural and functional aspects of catalysis by NagZ were investigated by a total of seven X-ray structures, three computational models based on the X-ray structures, molecular-dynamics simulations and mutagenesis. The structural insights came from the unbound state and complexes of NagZ with the substrate, products and a mimetic of the transient oxocarbenium species, which were prepared by synthesis. The mechanism involves a histidine as acid/base catalyst, which is unique for glycosidases. The turnover process utilizes covalent modification of D244, requiring two transition-state species and is regulated by coordination with a zinc ion. The analysis provides a seamless continuum for the catalytic cycle, incorporating large motions by four loops that surround the active site.

Matrix metalloproteinases (MMPs) have numerous physiological functions and share a highly similar catalytic domain. Differential dynamical information on the closely related human MMP-8, -13, and -14 was integrated onto the benzoxazinone molecular template. An in silico library of 28,099 benzoxazinones was generated and evaluated in the context of the molecular-dynamics information. This led to experimental evaluation of 19 synthesized compounds and identification of selective inhibitors, which have potential utility in delineating the physiological functions of MMPs. Moreover, the approach serves as an example of how dynamics of closely related active sites may be exploited to achieve selective inhibition by small molecules and should find applications in other enzyme families with similar active sites.

The mechanism of the β-lactam antibacterials is the functionally irreversible acylation of the enzymes that catalyze the cross-linking steps in the biosynthesis of their peptidoglycan cell wall. The Gram-positive pathogen Staphylococcus aureus uses one primary resistance mechanism. An enzyme, called penicillin-binding protein 2a (PBP2a), is brought into this biosynthetic pathway to complete the cross-linking. PBP2a effectively discriminates against the β-lactam antibiotics as potential inhibitors, and in favor of the peptidoglycan substrate. The basis for this discrimination is an allosteric site, distal from the active site, that when properly occupied concomitantly opens the gatekeeper residues within the active site and realigns the conformation of key residues to permit catalysis. We address the molecular basis of this regulation using crystallographic studies augmented by computational analyses. The crystal structures of three β-lactams (oxacillin, cefepime, ceftazidime) complexes with PBP2a-each with the β-lactam in the allosteric site-defined (with preceding PBP2a structures) as the "open" or "partially open" PBP2a states. A particular loop motion adjacent to the active site is identified as the driving force for the active-site conformational change that accompanies active-site opening. Correlation of this loop motion to effector binding at the allosteric site, in order to identify the signaling pathway, was accomplished computationally in reference to the known "closed" apo-PBP2a X-ray crystal structure state. This correlation enabled the computational simulation of the structures coinciding with initial peptidoglycan substrate binding to PBP2a, acyl enzyme formation, and acyl transfer to a second peptidoglycan substrate to attain cross-linking. These studies offer important insights into the structural bases for allosteric site-to-active site communication and for β-lactam mimicry of the peptidoglycan substrates, as foundational to the mechanistic understanding of emerging PBP2a resistance mutations.

Active in the aqueous cellular environment where a massive excess of water is perpetually present, enzymes that catalyze the transfer of an electrophile to a non-water nucleophile (transferases) require specific strategies to inhibit mechanistically related hydrolysis reactions. To identify principles that confer transferase versus hydrolase reaction specificity, we exploited two enzymes that use highly similar catalytic apparatuses to catalyze the transglycosylation (a transferase reaction) or hydrolysis of α-1,3-glucan linkages in the cyclic tetrasaccharide cycloalternan (CA). We show that substrate binding to non-catalytic domains and a conformationally stable active site promote CA transglycosylation, whereas a distinct pattern of active site conformational change is associated with CA hydrolysis. These findings defy the classic view of induced-fit conformational change and illustrate a mechanism by which a stable hydrophobic binding site can favor transferase activity and disfavor hydrolysis. Application of these principles could facilitate the rational reengineering of transferases with desired catalytic properties., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinase (MMP)-2 knockout (KO) mice show impaired neurological recovery after spinal cord injury (SCI), suggesting that this proteinase is critical to recovery processes. However, this finding in the KO has been confounded by a compensatory increase in MMP-9. We synthesized the thiirane mechanism-based inhibitor ND-378 and document that it is a potent (nanomolar) and selective slow-binding inhibitor of MMP-2 that does not inhibit the closely related MMP-9 and MMP-14. ND-378 crosses the blood-spinal cord barrier, achieving therapeutic concentrations in the injured spinal cord. Spinal-cord injured mice treated with ND-378 showed no change in long-term neurological outcomes, suggesting that MMP-2 is not a key determinant of locomotor recovery.

Bacteria grow and divide without loss of cellular integrity. This accomplishment is notable, as a key component of their cell envelope is a surrounding glycopeptide polymer. In Gram-negative bacteria this polymer-the peptidoglycan-grows by the difference between concurrent synthesis and degradation. The regulation of the enzymatic ensemble for these activities is poorly understood. We report herein the structural basis for the control of one such enzyme, the lytic transglycosylase MltF of Pseudomonas aeruginosa. Its structure comprises two modules: an ABC-transporter-like regulatory module and a catalytic module. Occupancy of the regulatory module by peptidoglycan-derived muropeptides effects a dramatic and long-distance (40 Å) conformational change, occurring over the entire protein structure, to open its active site for catalysis. This discovery of the molecular basis for the allosteric control of MltF catalysis is foundational to further study of MltF within the complex enzymatic orchestration of the dynamic peptidoglycan., (Copyright © 2016. Published by Elsevier Ltd.)

A family of 11 lytic transglycosylases in Pseudomonas aeruginosa, an opportunistic human pathogen, turn over the polymeric bacterial cell wall in the course of its recycling, repair, and maturation. The functions of these enzymes are not fully understood. We disclose herein that SltB3 of P. aeruginosa is an exolytic lytic transglycosylase. We characterize its reaction and its products by the use of peptidoglycan-based molecules. The enzyme recognizes a minimum of four sugars in its substrate but can process a substrate comprised of a peptidoglycan of 20 sugars. The ultimate product of the reaction is N-acetylglucosamine-1,6-anhydro-N-acetylmuramic acid. The X-ray structure of this enzyme is reported for the first time. The enzyme is comprised of four domains, arranged within an annular conformation. The polymeric linear peptidoglycan substrate threads through the opening of the annulus, as it experiences turnover.

The oxadiazole antibacterials, a class of newly discovered compounds that are active against Gram-positive bacteria, target bacterial cell-wall biosynthesis by inhibition of a family of essential enzymes, the penicillin-binding proteins. Ligand-based 3D-QSAR analyses by comparative molecular field analysis (CoMFA), comparative molecular shape indices analysis (CoMSIA) and Field-Based 3D-QSAR evaluated a series of 102 members of this class. This series included inactive compounds as well as compounds that were moderately to strongly antibacterial against Staphylococcus aureus. Multiple models were constructed using different types of energy minimization and charge calculations. CoMFA derived contour maps successfully defined favored and disfavored regions of the molecules in terms of steric and electrostatic properties for substitution., (Copyright © 2016. Published by Elsevier Ltd.)

Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)-induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV(+) lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas., (© 2015 by The American Society of Hematology.)

The lytic transglycosylases are essential bacterial enzymes that catalyze the nonhydrolytic cleavage of the glycan strands of the bacterial cell wall. We describe here the structural and catalytic properties of MltC, one of the seven lytic transglycosylases found in the genome of the Gram-negative bacterium Escherichia coli. The 2.3 Å resolution X-ray structure of a soluble construct of MltC shows a unique, compared to known lytic transglycosylase structures, two-domain structure characterized by an expansive active site of 53 Å length extending through an interface between the domains. The structures of three complexes of MltC with cell wall analogues suggest the positioning of the peptidoglycan in the active site both as a substrate and as a product. One complex is suggested to correspond to an intermediate in the course of sequential and exolytic cleavage of the peptidoglycan. Moreover, MltC partitioned its reactive oxocarbenium-like intermediate between trapping by the C6-hydroxyl of the muramyl moiety (lytic transglycosylase activity, the major path) and by water (muramidase activity). Genomic analysis identifies the presence of an MltC homologue in no less than 791 bacterial genomes. While the role of MltC in cell wall assembly and maturation remains uncertain, we propose a functional role for this enzyme as befits the uniqueness of its two-domain structure.

Discoidin domain receptor 1 (DDR1) belongs to a unique family of receptor tyrosine kinases that signal in response to collagens. DDR1 undergoes autophosphorylation in response to collagen binding with a slow and sustained kinetics that is unique among members of the receptor tyrosine kinase family. DDR1 dimerization precedes receptor activation suggesting a structural inhibitory mechanism to prevent unwarranted phosphorylation. However, the mechanism(s) that maintains the autoinhibitory state of the DDR1 dimers is unknown. Here, we report that N-glycosylation at the Asn(211) residue plays a unique role in the control of DDR1 dimerization and autophosphorylation. Using site-directed mutagenesis, we found that mutations that disrupt the conserved (211)NDS N-glycosylation motif, but not other N-glycosylation sites (Asn(260), Asn(371), and Asn(394)), result in collagen I-independent constitutive phosphorylation. Mass spectrometry revealed that the N211Q mutant undergoes phosphorylation at Tyr(484), Tyr(520), Tyr(792), and Tyr(797). The N211Q traffics to the cell surface, and its ectodomain displays collagen I binding with an affinity similar to that of the wild-type DDR1 ectodomain. However, unlike the wild-type receptor, the N211Q mutant exhibits enhanced receptor dimerization and sustained activation upon ligand withdrawal. Taken together, these data suggest that N-glycosylation at the highly conserved (211)NDS motif evolved to act as a negative repressor of DDR1 phosphorylation in the absence of ligand. The presence of glycan moieties at that site may help to lock the collagen-binding domain in the inactive state and prevent unwarranted signaling by receptor dimers. These studies provide a novel insight into the structural mechanisms that regulate DDR activation.

Brain metastasis occurs in 20-40% of cancer patients. Treatment is mostly palliative, and the inability of most drugs to penetrate the brain presents one of the greatest challenges in the development of therapeutics for brain metastasis. Matrix metalloproteinase-2 (MMP-2) plays important roles in invasion and vascularization of the central nervous system and represents a potential target for treatment of brain metastasis. Carbonate, O-phenyl carbamate, urea, and N-phenyl carbamate derivatives of SB-3CT, a selective and potent gelatinase inhibitor, were synthesized and evaluated. The O-phenyl carbamate and urea variants were selective and potent inhibitors of MMP-2. Carbamate 5b was metabolized to the potent gelatinase inhibitor 2, which was present at therapeutic concentrations in the brain. In contrast, phenyl urea 6b crossed the blood-brain barrier, however, higher doses would result in therapeutic brain concentrations. Carbamate 5b and urea 6b show potential for intervention of MMP-2-dependent diseases such as brain metastasis.

The discoidin domain receptors (DDRs) are receptor tyrosine kinases that upon binding to collagens undergo receptor phosphorylation, which in turn activates signal transduction pathways that regulate cell-collagen interactions. We report here that collagen-dependent DDR1 activation is partly regulated by the proteolytic activity of the membrane-anchored collagenases, MT1-, MT2-, and MT3-matrix metalloproteinase (MMP). These collagenases cleave DDR1 and attenuate collagen I- and IV-induced receptor phosphorylation. This effect is not due to ligand degradation, as it proceeds even when the receptor is stimulated with collagenase-resistant collagen I (r/r) or with a triple-helical peptide harboring the DDR recognition motif in collagens. Moreover, the secreted collagenases MMP-1 and MMP-13 and the glycosylphosphatidylinositol-anchored membrane-type MMPs (MT4- and MT6-MMP) have no effect on DDR1 cleavage or activation. N-terminal sequencing of the MT1-MMP-mediated cleaved products and mutational analyses show that cleavage of DDR1 takes place within the extracellular juxtamembrane region, generating a membrane-anchored C-terminal fragment. Metalloproteinase inhibitor studies show that constitutive shedding of endogenous DDR1 in breast cancer HCC1806 cells is partly mediated by MT1-MMP, which also regulates collagen-induced receptor activation. Taken together, these data suggest a role for the collagenase of membrane-type MMPs in regulation of DDR1 cleavage and activation at the cell-matrix interface.

Semisynthetic 8,8-dialkyldihydroberberines (8,8-DDBs) were found to possess mid- to low-nanomolar potency against Plasmodium falciparum blood-stage parasites, Leishmania donovani intracellular amastigotes, and Trypanosoma brucei brucei bloodstream forms. For example, 8,8-diethyldihydroberberine chloride (5b) exhibited in vitro IC50 values of 77, 100, and 5.3 nM against these three parasites, respectively. In turn, two 8,8-dialkylcanadines, obtained by reduction of the corresponding 8,8-DDBs, were much less potent against these parasites in vitro. While the natural product berberine is a weak DNA binder, the 8,8-DDBs displayed no affinity for DNA, as assessed by changes in the melting temperature of poly(dA·dT) DNA. Selected 8,8-DDBs showed efficacy in mouse models of visceral leishmaniasis and African trypanosomiasis, with 8,8-dimethyldihydroberberine chloride (5a) reducing liver parasitemia by 46% in L. donovani-infected BALB/c mice when given at an intraperitoneal dose of 10 mg/kg/day for five days. The 8,8-DDBs may thus serve as leads for discovering new antimalarial, antileishmanial, and antitrypanosomal drug candidates.

The discoidin domain receptors (DDRs) are receptor tyrosine kinases that recognize collagens as their ligands. DDRs display unique structural features and distinctive activation kinetics, which set them apart from other members of the kinase superfamily. DDRs regulate cell-collagen interactions in normal and pathological conditions and thus are emerging as major sensors of collagen matrices and potential novel therapeutic targets. New structural and biological information has shed light on the molecular mechanisms that regulate DDR signaling, turnover, and function. This minireview provides an overview of these areas of DDR research with the goal of fostering further investigation of these intriguing and unique receptors.

Kinase targets have been demonstrated to undergo major conformational reorganization upon ligand binding. Such protein conformational plasticity remains a significant challenge in structure-based virtual screening methodology and may be approximated by screening against an ensemble of diverse protein conformations. Maternal embryonic leucine zipper kinase (MELK), a member of serine-threonine kinase family, has been recently found to be involved in the tumerogenic state of glioblastoma, breast, ovarian, and colon cancers. We therefore modeled several conformers of MELK utilizing the available chemogenomic and crystallographic data of homologous kinases. We carried out docking pose prediction and virtual screening enrichment studies with these conformers. The performances of the ensembles were evaluated by their ability to reproduce known inhibitor bioactive conformations and to efficiently recover known active compounds early in the virtual screen when seeded with decoy sets. A few of the individual MELK conformers performed satisfactorily in reproducing the native protein-ligand pharmacophoric interactions up to 50% of the cases. By selecting an ensemble of a few representative conformational states, most of the known inhibitor binding poses could be rationalized. For example, a four conformer ensemble is able to recover 95% of the studied actives, especially with imperfect scoring function(s). The virtual screening enrichment varied considerably among different MELK conformers. Enrichment appears to improve by selection of a proper protein conformation. For example, several holo and unliganded active conformations are better to accommodate diverse chemotypes than ATP-bound conformer. These results prove that using an ensemble of diverse conformations could give a better performance. Applying this approach, we were able to screen a commercially available library of half a million compounds against three conformers to discover three novel inhibitors of MELK, one from each template. Among the three compounds validated via experimental enzyme inhibition assays, one is relatively potent (15; K(d) = 0.37 μM), one moderately active (12; K(d) = 3.2 μM), and one weak but very selective (9; K(d) = 18 μM). These novel hits may be utilized to assist in the development of small molecule therapeutic agents useful in diseases caused by deregulated MELK, and perhaps more importantly, the approach demonstrates the advantages of choosing an appropriate ensemble of a few conformers in pursuing compound potency, selectivity, and novel chemotypes over using single target conformation for structure-based drug design in general.

The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a critical role in the viral life cycle. The C-terminal domain (CTD) of CA binds to human lysyl-tRNA synthetase (hLysRS), and this interaction facilitates packaging of host cell tRNA(Lys,3), which serves as the primer for reverse transcription. Here, we report the library synthesis, high-throughput screening, and identification of cyclic peptides (CPs) that bind HIV-1 CA. Scrambling or single-residue changes of the selected peptide sequences eliminated binding, suggesting a sequence-specific mode of interaction. Two peptides (CP2 and CP4) subjected to detailed analysis also inhibited hLysRS/CA interaction in vitro. Nuclear magnetic resonance spectroscopy and mutagenesis studies revealed that both CPs bind to a site proximal to helix 4 of the CA-CTD, which is the known site of hLysRS interaction. These results extend the current repertoire of CA-binding molecules to a new class of peptides targeting a novel site with potential for development into novel antiviral agents.

We performed a hierarchical structure-based virtual screening utilizing a comparative model of the human α4β2 neuronal nicotinic acetylcholine receptor (nAChR) extracellular domain. Compounds were selected for experimental testing based on structural diversity, binding pocket location, and standard error of the free energy scoring function used in the screening. Four of the eleven in silico hit compounds showed promising activity with low micromolar IC50 values in a calcium accumulation assay. Two of the antagonists were also proven to be selective for human α4β2 vs human α3β4 nAChRs. This is the first report of successful discovery of novel nAChR antagonists through the use of structure-based virtual screening with a human nAChR homology model. These compounds may serve as potential novel scaffolds for further development of selective nAChR antagonists.

The current chemotherapy for second stage human African trypanosomiasis is unsatisfactory. A synthetic optimization study based on the lead antitrypanosomal compound 1,2-dihydro-2,2,4-trimethylquinolin-6-yl 3,5-dimethoxybenzoate (TDR20364, 1a) was undertaken in an attempt to discover new trypanocides with potent in vivo activity. While 6-ether derivatives were less active than the lead compound, several N1-substituted derivatives displayed nanomolar IC(50) values against T. b. rhodesiense STIB900 in vitro, with selectivity indexes up to >18000. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate (10a) displayed an IC(50) value of 0.014 microM against these parasites and a selectivity index of 1700. Intraperitoneal administration of 10a at 50 (mg/kg)/day for 4 days caused a promising prolongation of lifespan in T. b. brucei STIB795-infected mice (>14 days vs 7.75 days for untreated controls). Reactive oxygen species were produced when T. b. brucei were exposed to 10a in vitro, implicating oxidative stress in the trypanocidal mode of action of these 1,2-dihydroquinoline derivatives.

Dinitroanilines are of interest as antiprotozoal lead compounds because of their selective activity against the tubulin of these organisms, but concern has been raised due to the potentially mutagenic nitro groups. Analogues of N(1)-phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (GB-II-150, compound 2b), a selective antimitotic agent against African trypanosomes and Leishmania, have been prepared where the nitro groups are replaced with amino, chloro, cyano, carboxylate, methyl ester, amide, and methyl ketone moieties. Dicyano compound 5 displays IC(50) values that are comparable to 2b against purified leishmanial tubulin assembly (6.6 vs 7.4 microM), Trypanosoma brucei brucei growth in vitro (0.26 vs 0.18 microM), Leishmania donovani axenic amastigote growth in vitro (4.4 vs 2.3 microM), and in vitro toxicity against Vero cells (16 vs 9.7 microM). Computational studies provide a rationale for the antiparasitic order of activity of these analogues and further insight into the role of the substituents at the 3 and 5 positions of the sulfanilamide ring.