Structure-Activity Relationship for the Picolinamide Antibacterials that Selectively Target Clostridioides difficile.

Clostridioides difficile is a leading health threat. This pathogen initiates intestinal infections during gut microbiota dysbiosis caused by oral administration of antibiotics. C. difficile is difficult to eradicate due to its ability to form spores, which are not susceptible to antibiotics. To address the urgent need for treating recurrent C. difficile infection, antibiotics that selectively target C. difficile over common gut microbiota are needed. We herein describe the class of picolinamide antibacterials which show potent and selective activity against C. difficile. The structure-activity relationship of 108 analogues of isonicotinamide 4, a compound that is equally active against methicillin-resistant Staphylococcus aureus and C. difficile, was investigated. Introduction of the picolinamide core as exemplified by analogue 87 resulted in exquisite potency and selectivity against C. difficile. The ability of the picolinamide class to selectively target C. difficile and to prevent gut dysbiosis holds promise for the treatment of recurrent C. difficile infection.

Turnover Chemistry and Structural Characterization of the Cj0843c Lytic Transglycosylase of Campylobacter jejuni.

The soluble lytic transglycosylase Cj0843c from Campylobacter jejuni breaks down cell-wall peptidoglycan (PG). Its nonhydrolytic activity sustains cell-wall remodeling and repair. We report herein our structure-function studies probing the substrate preferences and recognition by this enzyme. Our studies show that Cj0843c exhibits both exolytic and endolytic activities and forms the N-acetyl-1,6-anhydromuramyl (anhMurNAc) peptidoglycan termini, the typical transformation catalyzed by lytic transglycosylase. Cj0843c shows a trend toward a preference for substrates with anhMurNAc ends and those with peptide stems. Mutagenesis revealed that the catalytic E390 is critical for activity. In addition, mutagenesis showed that R388 and K505, located in the positively charged pocket near E390, also serve important roles. Mutation of R326, on the opposite side of this positively charged pocket, enhanced activity. Our data point to different roles for positively charged residues in this pocket for productive binding of the predominantly negatively charged PG. We also show by X-ray crystallography and by molecular dynamics simulations that the active site of Cj0843c is still capable of binding GlcNAc containing di- and trisaccharides without MurNAc moieties, without peptide stems, and without the anhMurNAc ends.

Discovery of a Potent Picolinamide Antibacterial Active against Clostridioides difficile.

A major challenge for chemotherapy of bacterial infections is perturbation of the intestinal microbiota. Clostridioides difficile is a Gram-positive bacterium of the gut that can thrive under this circumstance. Its production of dormant and antibiotic-impervious spores results in chronic disruption of normal gut flora and debilitating diarrhea and intestinal infection. C. difficile is responsible for 12,800 deaths per year in the United States. Here, we report the discovery of 2-(4-(3-(trifluoromethoxy)phenoxy)picolinamido)benzo[d]oxazole-5-carboxylate as an antibacterial with potent and selective activity against C. difficile. Its MIC50 and MIC90 (the concentration required to inhibit the growth of 50% and 90% of all the tested strains, respectively) values, documented across 101 strains of C. difficile, are 0.12 and 0.25 µg/mL, respectively. The compound targets cell wall biosynthesis, as assessed by macromolecular biosynthesis assays and by scanning electron microscopy. Animals infected with a lethal dose of C. difficile and treated with compound 1 had a similar survival compared to treatment with vancomycin, which is the frontline antibiotic used for C. difficile infection.

Exploration of the Structural Space in 4(3H)-Quinazolinone Antibacterials.

We report herein the syntheses of 79 derivatives of the 4(3H)-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(3H)-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 ß-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.

Structure-Activity Relationship for the Oxadiazole Class of Antibacterials.

A structure-activity relationship (SAR) for the oxadiazole class of antibacterials was evaluated by syntheses of 72 analogs and determination of the minimal-inhibitory concentrations (MICs) against the ESKAPE panel of bacteria. Selected compounds were further evaluated for in vitro toxicity, plasma protein binding, pharmacokinetics (PK), and a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) infection. Oxadiazole 72c shows potent in vitro antibacterial activity, exhibits low clearance, a high volume of distribution, and 41% oral bioavailability, and shows efficacy in mouse models of MRSA infection.

Structural basis of denuded glycan recognition by SPOR domains in bacterial cell division.

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.

Cinnamonitrile Adjuvants Restore Susceptibility to ß-Lactams against Methicillin-Resistant Staphylococcus aureus.

ß-Lactams are used routinely to treat Staphylococcus aureus infections. However, the emergence of methicillin-resistant S. aureus (MRSA) renders them clinically precarious. We describe a class of cinnamonitrile adjuvants that restore the activity of oxacillin (a penicillin member of the ß-lactams) against MRSA. The lead adjuvants were tested against six important strains of MRSA, one vancomycin-intermediate S. aureus (VISA) strain, and one linezolid-resistant S. aureus strain. Five compounds out of 84 total compounds showed broad potentiation. At 8 µM (E)-3-(5-(3,4-dichlorobenzyl)-2-(trifluoromethoxy)phenyl)-2-(methylsulfonyl)acrylonitrile (26) potentiated oxacillin with a >4000-fold reduction of its MIC (from 256 to 0.06 mg·L-1). This class of adjuvants holds promise for reversal of the resistance phenotype of MRSA.

Slt, MltD, and MltG of Pseudomonas aeruginosa as Targets of Bulgecin A in Potentiation of ß-Lactam Antibiotics.

The interplay between the activities of lytic transglycosylases (LTs) and penicillin-binding proteins (PBPs) is critical for the health of the bacterial cell wall. Bulgecin A (a natural-product inhibitor of LTs) potentiates the activity of ß-lactam antibiotics (inhibitors of PBPs), underscoring this intimate mechanistic interdependence. Bulgecin A in the presence of an appropriate ß-lactam causes bulge deformation due to the formation of aberrant peptidoglycan at the division site of the bacterium. As Pseudomonas aeruginosa, a nefarious human pathogen, has 11 LT paralogs, the answer as to which LT activity correlates with ß-lactam potentiation is important and is currently unknown. Growth of P. aeruginosa PAO1 strains harboring individual transposon-insertion mutants at each of the 11 genes for LTs, in the presence of the ß-lactam antibiotic ceftazidime or meropenem, implicated the gene products of slt, mltD, and mltG (of the 11), in bulge formation and potentiation. Hence, the respective enzymes would be the targets of inhibition by bulgecin A, which was indeed documented. We further demonstrated by imaging in real time and by SEM that cell lysis occurs by the structural failure of this bulge. Upon removal of the ß-lactam antibiotic prior to lysis, P. aeruginosa experiences delayed recovery from the elongation and bulge phenotype in the presence of bulgecin A. These observations argue for a collaborative role for the target LTs in the repair of the aberrant cell wall, the absence of activities of which in the presence of bulgecin A results in potentiation of the ß-lactam antibiotic.

Validation of Matrix Metalloproteinase-9 (MMP-9) as a Novel Target for Treatment of Diabetic Foot Ulcers in Humans and Discovery of a Potent and Selective Small-Molecule MMP-9 Inhibitor That Accelerates Healing.

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.

A Structural Dissection of the Active Site of the Lytic Transglycosylase MltE from Escherichia coli.

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.

Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa.

ß-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.

Deciphering the Nature of Enzymatic Modifications of Bacterial Cell Walls.

The major constituent of bacterial cell walls is peptidoglycan, which, in its crosslinked form, is a polymer of considerable complexity that encases the entire bacterium. A functional cell wall is indispensable for survival of the organism. There are several dozen enzymes that assemble and disassemble the peptidoglycan dynamically within each bacterial generation. Understanding of the nature of these transformations is critical knowledge for these events. Octasaccharide peptidoglycans were prepared and studied with seven recombinant cell-wall-active enzymes (SltB1, MltB, RlpA, mutanolysin, AmpDh2, AmpDh3, and PBP5). With the use of highly sensitive mass spectrometry methods, we described the breadth of reactions that these enzymes catalyzed with peptidoglycan and shed light on the nature of the cell wall alteration performed by these enzymes. The enzymes exhibit broadly distinct preferences for their substrate peptidoglycans in the reactions that they catalyze.

From Genome to Proteome to Elucidation of Reactions for All Eleven Known Lytic Transglycosylases from Pseudomonas aeruginosa.

An enzyme superfamily, the lytic transglycosylases (LTs), occupies the space between the two membranes of Gram-negative bacteria. LTs catalyze the non-hydrolytic cleavage of the bacterial peptidoglycan cell-wall polymer. This reaction is central to the growth of the cell wall, for excavating the cell wall for protein insertion, and for monitoring the cell wall so as to initiate resistance responses to cell-wall-acting antibiotics. The nefarious Gram-negative pathogen Pseudomonas aeruginosa encodes eleven LTs. With few exceptions, their substrates and functions are unknown. Each P. aeruginosa LT was expressed as a soluble protein and evaluated with a panel of substrates (both simple and complex mimetics of their natural substrates). Thirty-one distinct products distinguish these LTs with respect to substrate recognition, catalytic activity, and relative exolytic or endolytic ability. These properties are foundational to an understanding of the LTs as catalysts and as antibiotic targets.

Activation by Allostery in Cell-Wall Remodeling by a Modular Membrane-Bound Lytic Transglycosylase from Pseudomonas aeruginosa.

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.

Turnover of Bacterial Cell Wall by SltB3, a Multidomain Lytic Transglycosylase of Pseudomonas aeruginosa.

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 Natural Product Essramycin and Three of Its Isomers Are Devoid of Antibacterial Activity.

Four possible isomers of essramycin, a natural product from a marine Streptomyces species isolated from the Egyptian Mediterranean coast, were synthesized. The structures for the isomers were assigned unequivocally by (1)H NMR, (13)C NMR, high-resolution mass spectrometry, and X-ray crystal structure determinations. Notwithstanding the earlier report of broad-spectrum antibacterial activity for the natural product, none of the four isomers exhibited any such activity.

Structure-Activity Relationship for the 4(3H)-Quinazolinone Antibacterials.

We recently reported on the discovery of a novel antibacterial (2) with a 4(3H)-quinazolinone core. This discovery was made by in silico screening of 1.2 million compounds for binding to a penicillin-binding protein and the subsequent demonstration of antibacterial activity against Staphylococcus aureus. The first structure-activity relationship for this antibacterial scaffold is explored in this report with evaluation of 77 variants of the structural class. Eleven promising compounds were further evaluated for in vitro toxicity, pharmacokinetics, and efficacy in a mouse peritonitis model of infection, which led to the discovery of compound 27. This new quinazolinone has potent activity against methicillin-resistant (MRSA) strains, low clearance, oral bioavailability and shows efficacy in a mouse neutropenic thigh infection model.

Three-dimensional QSAR analysis and design of new 1,2,4-oxadiazole antibacterials.

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.

Water-Soluble MMP-9 Inhibitor Reduces Lesion Volume after Severe Traumatic Brain Injury.

SB-3CT is a potent and selective inhibitor of matrix metalloproteinase (MMP)-2 and -9, which has shown efficacy in an animal model of severe traumatic brain injury (TBI). However, SB-3CT is poorly water-soluble and is metabolized primarily to p-hydroxy SB-3CT (2), a more potent inhibitor than SB-3CT. We synthesized the O-phosphate prodrug (3) of compound 2 to enhance its water solubility by more than 2000-fold. The prodrug 3 was a poor MMP inhibitor, but readily hydrolyzed to the active 2 in human blood. Pharmacokinetics and brain distribution studies in mice showed that 2 crossed the blood-brain barrier (BBB) and achieved therapeutic concentrations in the brain. The prodrug 3/compound 2 was evaluated in a mouse model of severe TBI and found to significantly decrease the brain lesion volume and improve neurological outcomes. MMP-9 inhibition by a water-soluble thiirane inhibitor is a promising therapy for treatment of TBI.