Systematic analysis and integrative discovery of active-site subpocket-specific dehydroquinate synthase inhibitors combating antibiotic-resistant Staphylococcus aureus infection

2018 ◽  
Vol 16 (06) ◽  
pp. 1850027
Author(s):  
Quanfeng Liu ◽  
Liping Li ◽  
Fei Xu
Keyword(s):  
Staphylococcus Aureus ◽  
Active Site ◽  
Shikimate Pathway ◽  
In Vitro Susceptibility ◽  
Systematic Analysis ◽  
Antibiotic Resistant ◽  
Enzyme Active Site ◽  
Specific Inhibitors ◽  
Dehydroquinate Synthase

Shikimate pathway plays an essential role in the biosynthesis of aromatic amino acids in various plants and bacteria, which consists of seven key enzymes and they are all attractive targets for antibacterial agent development due to their absence in humans. The Staphylococcus aureus dehydroquinate synthase (SaDHQS) is involved in the second step of shikimate pathway, which catalyzes the NAD[Formula: see text]-dependent conversion of 3-deoxy-D-arabino-heptulosonate-7-phosphate to dehydroquinate via multiple steps. The enzyme active site can be characterized by two spatially separated subpockets 1 and 2, which represent the reaction center of substrate adduct with NAD[Formula: see text] nicotinamide moiety and the assistant binding site of NAD[Formula: see text] adenine moiety, respectively. In silico virtual screening is performed against a biogenic compound library to discover SaDHQS subpocket-specific inhibitors, which were then tested against both antibiotic-sensitive and antibiotic-resistant S. aureus strains by using in vitro susceptibility test. The activity profile of hit compounds has no considerable difference between the antibiotic-sensitive and -resistant strains. The subpocket 1-specific inhibitors exhibit a generally higher activity than subpocket 2-specific inhibitors, and they also hold a strong selectivity between their cognate and noncognate subpockets. Dynamics and energetics analyses reveal that the SaDHQS active site prefers to interact with amphipathic and polar inhibitors by forming multiple hydrogen bonds and van der Waals packing at the complex interfaces of the two subpockets with their cognate inhibitors.

scholarly journals Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1679
Author(s):  
Vishnu Mohan ◽  
Jean P. Gaffney ◽  
Inna Solomonov ◽  
Maxim Levin ◽  
Mordehay Klepfish ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Drug Targets ◽  
Fas Ligand ◽  
Specific Activity ◽  
Matrix Metalloproteases ◽  
Ductal Adenocarcinoma ◽  
Enzyme Active Site ◽  
Induced Apoptosis

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7′s enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

scholarly journals A bacteriocin-based antimicrobial formulation to effectively disrupt the cell viability of methicillin-resistant Staphylococcus aureus (MRSA) biofilms

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Christian Kranjec ◽  
Kirill V. Ovchinnikov ◽  
Torstein Grønseth ◽  
Kumar Ebineshan ◽  
Aparna Srikantam ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Viability ◽  
Cell Damage ◽  
Penicillin G ◽  
Antibiotic Resistant ◽  
Methicillin Resistant ◽  
Lactam Antibiotic ◽  
Mrsa Strains ◽  
Therapeutic Tools

AbstractAntibiotic-resistant and biofilm-associated infections brought about by methicillin-resistant Staphylococcus aureus (MRSA) strains is a pressing issue both inside as well as outside nosocomial environments worldwide. Here, we show that a combination of two bacteriocins with distinct structural and functional characteristics, garvicin KS, and micrococcin P1, showed a synergetic antibacterial activity against biofilms produced in vitro by S. aureus, including several MRSA strains. In addition, this bacteriocin-based antimicrobial combination showed the ability to restore the sensitivity of the highly resilient MRSA strain ATCC 33591 to the β-lactam antibiotic penicillin G. By using a combination of bacterial cell metabolic assays, confocal and scanning electron microscopy, we show that the combination between garvicin KS, micrococcin P1, and penicillin G potently inhibit cell viability within S. aureus biofilms by causing severe cell damage. Together these data indicate that bacteriocins can be valuable therapeutic tools in the fight against biofilm-associated MRSA infections.

Increase in the in vitro susceptibility of Staphylococcus aureus to antimicrobial agents in the presence of Candida albicans

1979 ◽  
Vol 25 (4) ◽  
pp. 429-435 ◽  
Author(s):  
J. deRepentigny ◽  
R. Lévesque ◽  
L. G. Mathieu
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Candida Albicans ◽  
Inhibitory Activity ◽  
Antimicrobial Agents ◽  
Mixed Cultures ◽  
Alpha Toxin ◽  
In Vitro Susceptibility ◽  
Pure Cultures

In experiments with mixed cultures of Staphylococcus aureus and Candida albicans both in the absence and in the presence of 5-fluorocytosine (5-FC), we have observed that (1) there is an inhibition of S. aureus growth in mixed cultures with C. albicans in media supplemented with 1 μg/mL of 5-FC and that 5-FC has no effect on staphylococci in pure cultures; (2) this inhibition occurred with clinically isolated and laboratory strains and could be reversed by specific metabolites; (3) Staphylococcus aureus was inhibited by filtrates of C. albicans cultures treated with 5-FC and this seemed to be favored by some C. albicans filterable product which can affect the cell wall and the permeability of the staphylococcal cells since they become sensitive to 5-FC; (4) nine other commonly used antimicrobials showed an increased inhibitory activity against S. aureus in mixed cultures with C. albicans; and (5) there is a decrease in the number of precipitating antigens of S. aureus and of the activity of alpha toxin when this species was grown with both C. albicans and 5-FC. Our results indicate that the susceptibility of some species to antimicrobials could be significantly modified in the presence of other species. One cannot exclude that a similar phenomenon could happen in hosts under treatment with antibiotics against infection.

scholarly journals Transposon Disruption of the Complex I NADH Oxidoreductase Gene (snoD) in Staphylococcus aureus Is Associated with Reduced Susceptibility to the Microbicidal Activity of Thrombin-Induced Platelet Microbicidal Protein 1

2006 ◽  
Vol 188 (1) ◽  
pp. 211-222 ◽  
Author(s):  
Arnold S. Bayer ◽  
Peter McNamara ◽  
Michael R. Yeaman ◽  
Natalie Lucindo ◽  
Tiffanny Jones ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Complex I ◽  
Enzyme Inhibitor ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Gene Products ◽  
Chromosomal Dna ◽  
In Vitro Susceptibility ◽  
Ph Tolerance ◽  
Platelet Microbicidal Protein

ABSTRACT The cationic molecule thrombin-induced platelet microbicidal protein 1 (tPMP-1) exerts potent activity against Staphylococcus aureus. We previously reported that a Tn551 S. aureus transposon mutant, ISP479R, and two bacteriophage back-transductants, TxA and TxB, exhibit reduced in vitro susceptibility to tPMP-1 (tPMP-1r) compared to the parental strain, ISP479C (V. Dhawan, M. R. Yeaman, A. L. Cheung, E. Kim, P. M. Sullam, and A. S. Bayer, Infect. Immun. 65:3293-3299, 1997). In the current study, the genetic basis for tPMP-1r in these mutants was identified. GenBank homology searches using sequence corresponding to chromosomal DNA flanking Tn551 mutant strains showed that the fourth gene in the staphylococcal mnh operon (mnhABCDEFG) was insertionally inactivated. This operon was previously reported to encode a Na+/H+ antiporter involved in pH tolerance and halotolerance. However, the capacity of ISP479R to grow at pH extremes and in high NaCl concentrations (1 to 3 M), coupled with its loss of transmembrane potential (ΔΨ) during postexponential growth, suggested that the mnh gene products are not functioning as a secondary (i.e., passive) Na+/H+ antiporter. Moreover, we identified protein homologies between mnhD and the nuo genes of Escherichia coli that encode components of a complex I NADH:ubiquinone oxidoreductase. Consistent with these data, exposures of tPMP-1-susceptible (tPMP-1s) parental strains (both clinical and laboratory derived) with either CCCP (a proton ionophore which collapses the proton motive force) or pieracidin A (a specific complex I enzyme inhibitor) significantly reduced tPMP-induced killing to levels seen in the tPMP-1r mutants. To reflect the energization of the gene products encoded by the mnh operon, we have renamed the locus sno (S. aureus nuo orthologue). These novel findings indicate that disruption of a complex I enzyme locus can confer reduced in vitro susceptibility to tPMP-1 in S. aureus.

scholarly journals Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis

2015 ◽  
Vol 112 (20) ◽  
pp. 6347-6352 ◽  
Author(s):  
Bradley M. Hover ◽  
Nam K. Tonthat ◽  
Maria A. Schumacher ◽  
Kenichi Yokoyama
Keyword(s):  
Active Site ◽  
Biochemical Characterization ◽  
De Novo ◽  
Specific Binding ◽  
Molybdenum Cofactor ◽  
Enzyme Active Site ◽  
Complex Rearrangement ◽  
Cofactor Biosynthesis ◽  
Molybdenum Cofactor Biosynthesis

The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5′-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3′,8-cyclo-7,8-dihydro-guanosine 5′-triphosphate (3′,8-cH2GTP) as the product of in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, we performed biochemical characterization of 3′,8-cH2GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3′,8-cH2GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3′,8-cH2GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3′,8-cH2GTP (GTP 3′,8-cyclase), and that of MoaC is to catalyze the rearrangement of 3′,8-cH2GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. Thus, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.

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