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.

X-ray Structure of Catenated Lytic Transglycosylase SltB1.

Formation of catenanes by proteins is rare, with few known examples. We report herein the X-ray structure of a catenane dimer of lytic transglycosylase SltB1 of Pseudomonas aeruginosa. The enzyme is soluble and exists in the periplasmic space, where it modifies the bacterial cell wall. The catenane dimer exhibits the protein monomers in a noncovalent chain-link arrangement, whereby a stretch of 51 amino acids (to become a loop and three helices) from one monomer threads through the central opening of the structure of the partner monomer. The protein folds after threading in a manner that leaves two helices (α1 and α2) as stoppers to impart stability to the dimer structure. The symmetric embrace by the two SltB1 molecules occludes both active sites entirely, an arrangement that is sustained by six electrostatic interactions between the two monomers. In light of the observation of these structural motifs in all members of Family 3 lytic transglycosylases, catenanes might be present for those enzymes, as well. The dimeric catenane might represent a regulated form of SltB1.

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.

Exploration of the structure-activity relationship of 1,2,4-oxadiazole antibiotics.

We have recently disclosed the discovery of the class of 1,2,4-oxadiazole antibiotics, which emerged from in silico docking and scoring efforts. This class of antibacterials exhibits Gram-positive activity, particularly against Staphylococcus aureus. We define the structure-activity relationship (SAR) of this class of antibiotics with the synthesis and evaluation of a series of 59 derivatives with variations in the C ring or C and D rings. A total of 17 compounds showed activity against S. aureus. Four derivatives were evaluated against a panel of 16 Gram-positive strains, inclusive of several methicillin-resistant S. aureus strains. These compounds are broadly active against Gram-positive bacteria.

Structure-activity relationship for the oxadiazole class of antibiotics.

The structure-activity relationship (SAR) for the newly discovered oxadiazole class of antibiotics is described with evaluation of 120 derivatives of the lead structure. This class of antibiotics was discovered by in silico docking and scoring against the crystal structure of a penicillin-binding protein. They impair cell-wall biosynthesis and exhibit activities against the Gram-positive bacterium Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) and vancomycin-resistant and linezolid-resistant S. aureus. 5-(1H-Indol-5-yl)-3-(4-(4-(trifluoromethyl)phenoxy)phenyl)-1,2,4-oxadiazole (antibiotic 75b) was efficacious in a mouse model of MRSA infection, exhibiting a long half-life, a high volume of distribution, and low clearance. This antibiotic is bactericidal and is orally bioavailable in mice. This class of antibiotics holds great promise in recourse against infections by MRSA.

Discovery of a new class of non-ß-lactam inhibitors of penicillin-binding proteins with Gram-positive antibacterial activity.

Infections caused by hard-to-treat methicillin-resistant Staphylococcus aureus (MRSA) are a serious global public-health concern, as MRSA has become broadly resistant to many classes of antibiotics. We disclose herein the discovery of a new class of non-ß-lactam antibiotics, the oxadiazoles, which inhibit penicillin-binding protein 2a (PBP2a) of MRSA. The oxadiazoles show bactericidal activity against vancomycin- and linezolid-resistant MRSA and other Gram-positive bacterial strains, in vivo efficacy in a mouse model of infection, and have 100% oral bioavailability.

Cell-wall remodeling by the zinc-protease AmpDh3 from Pseudomonas aeruginosa.

Bacterial cell wall is a polymer of considerable complexity that is in constant equilibrium between synthesis and recycling. AmpDh3 is a periplasmic zinc protease of Pseudomonas aeruginosa , which is intimately involved in cell-wall remodeling. We document the hydrolytic reactions that this enzyme performs on the cell wall. The process removes the peptide stems from the peptidoglycan, the major constituent of the cell wall. We document that the majority of the reactions of this enzyme takes place on the polymeric insoluble portion of the cell wall, as opposed to the fraction that is released from it. We show that AmpDh3 is tetrameric both in crystals and in solution. Based on the X-ray structures of the enzyme in complex with two synthetic cell-wall-based ligands, we present for the first time a model for a multivalent anchoring of AmpDh3 onto the cell wall, which lends itself to its processive remodeling.

Reaction products and the X-ray structure of AmpDh2, a virulence determinant of Pseudomonas aeruginosa.

The zinc protease AmpDh2 is a virulence determinant of Pseudomonas aeruginosa, a problematic human pathogen. The mechanism of how the protease manifests virulence is not known, but it is known that it turns over the bacterial cell wall. The reaction of AmpDh2 with the cell wall was investigated, and nine distinct turnover products were characterized by LC/MS/MS. The enzyme turns over both the cross-linked and noncross-linked cell wall. Three high-resolution X-ray structures, the apo enzyme and two complexes with turnover products, were solved. The X-ray structures show how the dimeric protein interacts with the inner leaflet of the bacterial outer membrane and that the two monomers provide a more expansive surface for recognition of the cell wall. This binding surface can accommodate the 3D solution structure of the cross-linked cell wall.

pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor.

The stromelysin-1 catalytic domain(83-247) (SCD) is stable for at least 16 h at pHs 6.0-8.4. At pHs 5.0 and 9.0 there is exponential irreversible denaturation with half lives of 38 and 68 min respectively. At pHs 4.5 and 10.0 irreversible denaturation is biphasic. At 25°C, C-terminal truncation of stromelysin-1 decreases the stability of the stromelysin-1 catalytic domain at pH values >8.4 and <6.0. We describe the conversion of the carboxylate group of (ßR)-ß-[[[(1S)-1-[[[(1S)-2-Methoxy-1-phenylethyl]amino]carbonyl]-2,2-dimethylpropyl]amino]carbonyl]-2-methyl-[1,1'-biphenyl]-4-hexanoic acid (UK-370106-COOH) a potent inhibitor of the metalloprotease stromelysin-1 to a glyoxal group (UK-370106-CO(13)CHO). At pH 5.5-6.5 the glyoxal inhibitor is a potent inhibitor of stromelysin-1 (K(i)=~1µM). The aldehyde carbon of the glyoxal inhibitor was enriched with carbon-13 and using carbon-13 NMR we show that the glyoxal aldehyde carbon is fully hydrated when it is in aqueous solutions (90.4ppm) and also when it is bound to SCD (~92.0ppm). We conclude that the hemiacetal hydroxyl groups of the glyoxal inhibitor are not ionised when the glyoxal inhibitor is bound to SCD. The free enzyme pK(a) values associated with inhibitor binding were 5.9 and 6.2. The formation and breakdown of the signal at ~92ppm due to the bound UK-370106-CO(13)CHO inhibitor depends on pK(a) values of 5.8 and 7.8 respectively. No strong hydrogen bonds are present in free SCD or in SCD-inhibitor complexes. We conclude that the inhibitor glyoxal group is not directly coordinated to the catalytic zinc atom of SCD.

Determination of the structure of tetrahedral transition state analogues bound at the active site of chymotrypsin using 18O and 2H isotope shifts in the 13C NMR spectra of glyoxal inhibitors.

The peptide-derived glyoxal inhibitor Z-Ala-Pro-Phe-glyoxal, where Z is benzyloxycarbonyl, is an extremely potent inhibitor of chymotrypsin. When it is bound to chymotrypsin both the glyoxal (RCOCHO) keto and aldehyde carbons are sp3 hybridized with chemical shifts of 100.7 and 91.4 ppm, respectively. However it is has not been shown whether these carbons are bound as hydrates or whether the active-site serine has reacted with them to form the corresponding hemiketal or hemiacetal. In this study we use 18O isotope shifts to determine whether one or two exchangeable oxygen atoms are attached to the glyoxal keto or aldehyde carbons when it is free in water or bound to alpha-chymotrypsin. Both the 18O isotope shifts at the free and enzyme-bound aldehyde carbons were approximately 0.04 ppm showing that it is hydrated in both the free and bound forms. The 18O isotope shift for the free hydrated keto carbon at 96.6 ppm was 0.046-0.049 ppm, but this was reduced to 0.026 ppm when the glyoxal inhibitor was bound to alpha-chymotrypsin showing that the nonexchangeable serine hydroxyl group has formed a hemiketal with glyoxal keto carbon. Deuterium isotope shifts on the 13C NMR signals from the glyoxal inhibitor when it free and hydrated, when it is bound to chymotrypsin, as well as when it forms a model hemiketal confirm that the serine hydroxyl group has formed a hemiketal with the glyoxal keto carbon. The reasons for the different reaction specificities of glyoxal inhibitors for the active-site nucleophiles of serine and cysteine proteases are discussed.

13C and 1H NMR studies of ionizations and hydrogen bonding in chymotrypsin-glyoxal inhibitor complexes.

Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-Ala-Phe-glyoxal have both been shown to be inhibitors of alpha-chymotrypsin with minimal Ki values of 19 and 344 nM, respectively, at neutral pH. These Ki values increased at low and high pH with pKa values of approximately 4.0 and approximately 10.5, respectively. By using surface plasmon resonance, we show that the apparent association rate constant for Z-Ala-Pro-Phe-glyoxal is much lower than the value expected for a diffusion-controlled reaction. 13C NMR has been used to show that at low pH the glyoxal keto carbon is sp3-hybridized with a chemical shift of approximately 100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of approximately 91.6 ppm. The signal at approximately 100.7 ppm is assigned to the hemiketal formed between the hydroxy group of serine 195 and the keto carbon of the glyoxal. In a slow exchange process controlled by a pKa of approximately 4.5, the aldehyde carbon dehydrates to give a signal at approximately 205.5 ppm and the hemiketal forms an oxyanion at approximately 107.0 ppm. At higher pH, the re-hydration of the glyoxal aldehyde carbon leads to the signal at 107 ppm being replaced by a signal at 104 ppm (pKa approximately 9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-Ala-Ala-Phe-glyoxal to alpha-chymotrypsin at 4 and 25 degrees C, 1H NMR is used to show that the binding of these glyoxal inhibitors raises the pKa value of the imidazolium ion of histidine 57 to a value of >11 at both 4 and 25 degrees C. We discuss the mechanistic significance of these results, and we propose that it is ligand binding that raises the pKa value of the imidazolium ring of histidine 57 allowing it to enhance the nucleophilicity of the hydroxy group of the active site serine 195 and lower the pKa value of the oxyanion forming a zwitterionic tetrahedral intermediate during catalysis.