Matrix Metalloproteinase-14 as an Instigator of Fibrosis in Human Pterygium and Its Pharmacological Intervention.

There exists a paucity of information on the pathogenesis of pterygium, a benign ocular tumor that scars the cornea and can lead to vision loss. The main recourse for pterygium is surgery; however, recurrence is observed. Matrix metalloproteinases (MMPs) are involved in the pathology of pterygium. The determination of the specific MMP involved among the 24 human enzymes has not been established due to challenges in MMP profiling. We used an affinity resin that binds specifically to the active forms of MMPs in the complex mixture of the cellular proteome. The proteomics analysis identified active MMP-14 and three related metalloproteinases, ADAM9, ADAM10, and ADAM17, in human pterygia. Inhibition of MMP-14 with the small-molecule inhibitor (R)-ND-336 was assessed in cell migration and collagen contraction assays. (R)-ND-336 attenuated human conjunctiva fibroblast migration and mitigated collagen contraction, both activities required for the formation of pterygium. (R)-ND-336 holds the promise of a therapeutic recourse for pterygium as an orphan disease.

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.

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.

Dioxygen and nitric oxide scavenging by Treponema denticola flavodiiron protein: a mechanistic paradigm for catalysis.

Flavodiiron proteins (FDPs) contain a unique active site consisting of a non-heme diiron carboxylate site proximal to a flavin mononucleotide (FMN). FDPs serve as the terminal components for reductive scavenging of dioxygen (to water) or nitric oxide (to nitrous oxide), which combats oxidative or nitrosative stress in many bacteria. Characterizations of FDPs from spirochetes or from any oral microbes have not been previously reported. Here, we report characterization of an FDP from the anaerobic spirochete, Treponema (T.) denticola, which is associated with chronic periodontitis. The isolated T. denticola FDP exhibited efficient four-electron dioxygen reductase activity and lower but significant anaerobic nitric oxide reductase activity. A mutant T. denticola strain containing the inactivated FDP-encoding gene was significantly more air-sensitive than the wild-type strain. Single turnover reactions of the four-electron-reduced FDP (FMNH2-Fe(II)Fe(II)) (FDPred) with O2 monitored on the milliseconds to seconds time scale indicated initial rapid formation of a spectral feature consistent with a cis-µ-1,2-peroxo-diferric intermediate, which triggered two-electron oxidation of FMNH2. Reaction of FDPred with NO showed apparent cooperativity between binding of the first and second NO to the diferrous site. The resulting diferrous dinitrosyl complex triggered two-electron oxidation of the FMNH2. Our cumulative results on this and other FDPs indicate that smooth two-electron FMNH2 oxidation triggered by the FDPred/substrate complex and overall four-electron oxidation of FDPred to FDPox constitutes a mechanistic paradigm for both dioxygen and nitric oxide reductase activities of FDPs. Four-electron reductive O2 scavenging by FDPs could contribute to oxidative stress protection in many other oral bacteria.

Bioinspired catalytic nitrile hydration by dithiolato, sulfinato/thiolato, and sulfenato/sulfinato ruthenium complexes.

A series of Ru(II) catalysts inspired by the metalloenzyme nitrile hydratase catalyze the hydration of benzonitrile with up to 242 turnovers under neutral conditions with very low catalysts loading. Catalysts with an oxidized sulfur environment are less susceptible to product inhibition increasing the catalytic efficiency at low nitrile : water ratios.

Influence of sequential thiolate oxidation on a nitrile hydratase mimic probed by multiedge X-ray absorption spectroscopy.

Nitrile hydratases (NHases) are Fe(III)- and Co(III)-containing hydrolytic enzymes that convert nitriles into amides. The metal-center is contained within an N(2)S(3) coordination motif with two post-translationally modified cysteinates contained in a cis arrangement, which have been converted into a sulfinate (R-SO(2)(-)) and a sulfenate (R-SO(-)) group. Herein, we utilize Ru L-edge and ligand (N-, S-, and P-) K-edge X-ray absorption spectroscopies to probe the influence that these modifications have on the electronic structure of a series of sequentially oxidized thiolate-coordinated Ru(II) complexes ((bmmp-TASN)RuPPh(3), (bmmp-O(2)-TASN)RuPPh(3), and (bmmp-O(3)-TASN)RuPPh(3)). Included is the use of N K-edge spectroscopy, which was used for the first time to extract N-metal covalency parameters. We find that upon oxygenation of the bis-thiolate compound (bmmp-TASN)RuPPh(3) to the sulfenato species (bmmp-O(2)-TASN)RuPPh(3) and then to the mixed sulfenato/sulfinato speices (bmmp-O(3)-TASN)RuPPh(3) the complexes become progressively more ionic, and hence the Ru(II) center becomes a harder Lewis acid. These findings are reinforced by hybrid DFT calculations (B(38HF)P86) using a large quadruple-ζ basis set. The biological implications of these findings in relation to the NHase catalytic cycle are discussed in terms of the creation of a harder Lewis acid, which aids in nitrile hydrolysis.

Controlled sulfur oxygenation of the ruthenium dithiolate (4,7-bis-(2'-methyl-2'-mercaptopropyl)-1-thia-4,7-diazacyclononane)RuPPh(3) under limiting O(2) conditions yields thiolato/sulfinato, sulfenato/sulfinato, and bis-sulfinato derivatives.

The ruthenium(II) dithiolate complex (bmmp-TASN)RuPPh(3) (1) reacts with O(2) under limiting conditions to yield isolable sulfur oxygenated derivatives as a function of reaction time. With this approach, a family of sulfur-oxygenates has been prepared and isolated without the need for O-atom transfer agents or column chromatography. Addition of 5 equiv of O(2) to 1 yields the thiolato/sulfinato complex (bmmp-O(2)-TASN)RuPPh(3) (2) in 70% yield within 5 min. Increasing the reaction time to 12 h yields the sulfenato/sulfinato derivative (bmmp-O(3)-TASN)RuPPh(3) (3) in 82% yield. Longer reaction times and/or additional O(2) exposure yield the bis-sulfinato complex (bmmp-O(4)-TASN)RuPPh(3) (4). All products remain in the Ru(II) oxidation state under the conditions employed. Stoichiometric hydrolysis of acetonitrile to acetamide by 2 and 3 is observed in mixed acetonitrile, methanol, PIPES buffer (pH = 7.0) mixtures. The Ru(III)/(II) reduction potential of -0.85 V (versus ferrocenium/ferrocene) for 1 shifts to -0.39 and -0.26 V for 2 and 3, respectively, because of the decreased donor ability of sulfur upon oxygenation. X-ray diffraction studies reveal a decrease in Ru-S bond distances upon oxygenation by 0.045(1) and 0.158(1) Å for the sulfenato and sulfinato donors, respectively. Conversely, sulfur-oxygenation increases the Ru-P bond distance by 0.061(1) Å from 1 to 2 and an additional 0.027(1) Å from 2 to 3. Density functional theory investigations using the BP86 and B3LYP functionals with a LANL2DZ basis set for Ru and the 6-31G(d) basis set for all other atoms reveal a direct correlation between the oxygenation level and the Ru-P distance with an increase of 0.031 Å per O-atom.

Asymmetric oxygenation of a ruthenium dithiolate mimics the mixed sulfenato/sulfinato donor sets of nitrile hydratase and thiocyanate hydrolase.

The dithiolate complex (bmmp-TASN)RuPPh(3) reacts with O(2) under limiting conditions to yield the mixed sulfenato/sulfinato product (bmmp-O(3)-TASN)RuPPh(3) in 82% yield. Isotopic labeling studies confirm O(2) as the sole source of O atoms in the product complex. X-ray crystallographic studies reveal decreases in the Ru-S bond distances of 0.026(1) and 0.151(1) A for the sulfenato and sulfinato donors, respectively, and a 0.088(1) A increase in the Ru-PPh(3) bond distance upon oxygenation.