Antibiotic-loaded nanoparticles for the treatment of intracellular methicillin-resistant Staphylococcus Aureus infections: In vitro and in vivo efficacy of a novel antibiotic.

Antimicrobial resistance is considered one of the biggest threats to public health worldwide. Methicillin-resistant S. aureus is the causative agent of a number of infections and lung colonization in people suffering from cystic fibrosis.

The design, synthesis, and inhibition of Clostridioides difficile spore germination by acyclic and bicyclic tertiary amide analogs of cholate.

Clostridioides difficile infection (CDI) is a major identifiable cause of antibiotic-associated diarrhea. In our previous study (J. Med.

A Fluorescence-Based Assay for N -Carboxyaminoimidazole Ribonucleotide Mutase.

The enzyme N -carboxylaminoinidazole ribonucleotide (N -CAIR) mutase is found in microbial de novo purine biosynthesis but is absent in humans making it an attractive antimicrobial target. N -CAIR mutase catalyzes the synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N -CAIR which is itself prepared from aminoimidazole ribonucleotide (AIR) by the enzyme N -CAIR synthetase. During our research on identifying inhibitors of N -CAIR mutase, we developed an innovative, fluorescence-based assay to measure the activity of this enzyme.

Studies on the Importance of the 7α-, and 12α- hydroxyl groups of N-Aryl-3α,7α,12α-trihydroxy-5β-cholan-24-amides on their Antigermination Activity Against a Hypervirulent Strain of Clostridioides (Clostridium) difficile.

Chenodeoxycholic acid (CDCA) is a natural germination inhibitor for C. difficile spores. In our previous study (J.

An Aniline-Substituted Bile Salt Analog Protects both Mice and Hamsters from Multiple Clostridioides difficile Strains.

Clostridioides difficile infection (CDI) is the major identifiable cause of antibiotic-associated diarrhea. The emergence of hypervirulent C. difficile strains has led to increases in both hospital- and community-acquired CDI.

Isatins Inhibit N-CAIR Synthetase by a Substrate Depletion Mechanism.

The continued rise of antibiotic-resistant infections coupled with the limited pipeline of new antimicrobials highlights the pressing need for the development of new antibacterial agents. One potential pathway for new agents is de novo purine biosynthesis as studies have shown that bacteria and lower eukaryotes synthesize purines differently than humans. Microorganisms utilize two enzymes, N-CAIR synthetase and N-CAIR mutase, to convert 5-aminoimidazole ribonucleotide (AIR) into 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) through the intermediate N-carboxy-5-aminoimidazole ribonucleotide (N-CAIR).

Physicochemical and In Vitro Evaluation of Drug Delivery of an Antibacterial Synthetic Benzophenone in Biodegradable PLGA Nanoparticles.

Due to the increasing incidents of antimicrobial-resistant pathogens, the development of new antibiotics and their efficient formulation for suitable administration is crucial. Currently, one group of promising antimicrobial compounds are the benzophenone tetra-amides which show good activity even against gram-positive, drug-resistant pathogens. These compounds suffer from poor water solubility and bioavailability.

The Design, Synthesis, and Characterizations of Spore Germination Inhibitors Effective against an Epidemic Strain of Clostridium difficile.

Clostridium difficile infections (CDI), particularly those caused by the BI/NAP1/027 epidemic strains, are challenging to treat. One method to address this disease is to prevent the development of CDI by inhibiting the germination of C. difficile spores.

Intramolecular charge-assisted hydrogen bond strength in pseudochair carboxyphosphate.

Carboxyphosphate, a suspected intermediate in ATP-dependent carboxylases, has not been isolated nor observed directly by experiment. Consequently, little is known concerning its structure, stability, and ionization state. Recently, carboxyphosphate as either a monoanion or dianion has been shown computationally to adopt a novel pseudochair conformation featuring an intramolecular charge-assisted hydrogen bond (CAHB).

Site-directed mutagenesis of catalytic residues in N(5)-carboxyaminoimidazole ribonucleotide synthetase.

N(5)-CAIR synthetase, an essential enzyme in microorganisms, converts 5-aminoimidazole ribonucleotide (AIR) and bicarbonate to N(5)-CAIR with the aid of ATP. Previous X-ray crystallographic analyses of Aspergillus clavatus N(5)-CAIR synthetase postulated that R271, H273, and K353 were important for bicarbonate binding and for catalysis. As reported here, site-directed mutagenesis of these residues revealed that R271 and H273 are, indeed, critical for bicarbonate binding and catalysis whereas all K353 mutations, even ones conservative in nature, are inactive.

Cyclam-based polymeric copper chelators for gene delivery and potential PET imaging.

A series of reducible polycationic copper chelators (RPCs) based on 1,4,8,11-tetraazacyclotetradecane (cyclam) were synthesized by Michael addition. Molecular weight of the polycations was controlled by reaction stoichiometry and reaction conditions, resulting in polymers with molecular weights ranging from 4400 to 13 800. The cyclam moieties in the polycations retained their ability to form complexes with Cu(II).

Thieno[2,3-d]pyrimidinedione derivatives as antibacterial agents.

Several thieno[2,3-d]pyrimidinediones have been synthesized and examined for antibacterial activity against a range of gram-positive and gram-negative pathogens. Two compounds displayed potent activity (2-16 mg/L) against multi-drug resistant gram-positive organisms, including methicillin resistant, vancomycin-intermediate, vancomycin-resistant Staphylococcus aureus (MRSA, VISA, VRSA) and vancomycin-resistant enterococci (VRE). Only one of these agents possessed moderate activity (16-32 mg/L) against gram-negative strains.

The ATP-grasp enzymes.

The ATP-grasp enzymes consist of a superfamily of 21 proteins that contain an atypical ATP-binding site, called the ATP-grasp fold. The ATP-grasp fold is comprised of two α+β domains that "grasp" a molecule of ATP between them and members of the family typically have an overall structural design containing three common conserved focal domains. The founding members of the family consist of biotin carboxylase, d-ala-d-ala ligase and glutathione synthetase, all of which catalyze the ATP-assisted reaction of a carboxylic acid with a nucleophile via the formation of an acylphosphate intermediate.

Examination of a synthetic benzophenone membrane-targeted antibiotic.

The enormous success of antibiotics is seriously threatened by the development of resistance to most of the drugs available on the market. Thus, novel antibiotics are needed that are less prone to bacterial resistance and are directed toward novel biological targets. Antimicrobial peptides (AMPs) have attracted considerable attention due to their unique mode of action and broad spectrum activity.

Characterization of insulin ILPR sequences for their ability to adopt a G-quadruplex structure.

A major genetic factor linked to the progression of type 1 diabetes occurs in the insulin-linked polymorphic repeat region (ILPR) located 363 bp upstream of the human insulin gene. Genetic studies have shown that individuals with class I repeats (30-60) are predisposed to the development of type 1 diabetes while individuals with longer repeats are protected. Previous research has suggested that some sequences found within the ILPR can adopt a G-quadruplex structure, and this finding has lead to speculation that G-quadruplexes may control insulin expression in certain circumstances.

Structural and functional studies of Aspergillus clavatus N(5)-carboxyaminoimidazole ribonucleotide synthetase .

N(5)-Carboxyaminoimidazole ribonucleotide synthetase (N(5)-CAIR synthetase), a key enzyme in microbial de novo purine biosynthesis, catalyzes the conversion of aminoimidazole ribonucleotide (AIR) to N(5)-CAIR. To date, this enzyme has been observed only in microorganisms, and thus, it represents an ideal target for antimicrobial drug development. Here we report the cloning, crystallization, and three-dimensional structural analysis of Aspergillus clavatus N(5)-CAIR synthetase solved in the presence of either Mg(2)ATP or MgADP and AIR.

Solution-phase parallel synthesis of novel membrane-targeted antibiotics.

The increase in the incidence of antibiotic-resistant infections is a major concern to healthcare workers and requires the development of novel antibacterial agents. Recently, we described a series of benzophenone-containing antibiotics which displayed activity against antibiotic-resistant bacteria. We have shown that these agents function by disrupting the bacterial membrane.

Design, synthesis, and structure-activity relationships of benzophenone-based tetraamides as novel antibacterial agents.

The increase in the incidence of both hospital- and community-acquired antibiotic-resistant infections is a major concern to the healthcare community. There have been only two new classes of antibiotics approved by the FDA over the past 40 years, and clearly there is a growing need for additional antimicrobial agents. In this paper, we present our work on the discovery of a class of benzophenone containing compounds that possess good activity against MRSA, VISA, VRSA, and VRE and moderate activity against E.

Identification of inhibitors of N5-carboxyaminoimidazole ribonucleotide synthetase by high-throughput screening.

The increasing risk of drug-resistant bacterial infections indicates that there is a growing need for new and effective antimicrobial agents. One promising, but unexplored area in antimicrobial drug design is de novo purine biosynthesis. Recent research has shown that de novo purine biosynthesis in microbes is different from that in humans.

Benzo(h)quinoline derivatives as G-quadruplex binding agents.

G-quadruplexes are unusual structures formed from guanine-rich sequences of nucleic acids. G-quadruplexes have been postulated to play important roles in a number of biological systems including gene regulation and the inhibition of enzyme function. Recently, our laboratory reported on the synthesis and evaluation of a triaza-cyclopentaphenanthrene compound which bound to G-quadruplexes with good affinity and selectivity.

Interrogating the mechanism of a tight binding inhibitor of AIR carboxylase.

The enzyme aminoimidazole ribonucleotide (AIR) carboxylase catalyzes the synthesis of the purine intermediate, 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). Previously, we have shown that the compound 4-nitro-5-aminoimidazole ribonucleotide (NAIR) is a slow, tight binding inhibitor of the enzyme with a Ki of 0.34 nM.

Structural analysis of the active site geometry of N5-carboxyaminoimidazole ribonucleotide synthetase from Escherichia coli.

N(5)-Carboxyaminoimidazole ribonucleotide synthetase (N(5)-CAIR synthetase) converts 5-aminoimidazole ribonucleotide (AIR), MgATP, and bicarbonate into N(5)-CAIR, MgADP, and P(i). The enzyme is required for de novo purine biosynthesis in microbes yet is not found in humans suggesting that it represents an ideal and unexplored target for antimicrobial drug design. Here we report the X-ray structures of N(5)-CAIR synthetase from Escherichia coli with either MgATP or MgADP/P(i) bound in the active site cleft.

Development of a neuroscience-oriented "methods" course for graduate students of pharmacology and toxicology.

To provide graduate students in pharmacology/toxicology exposure to, and cross-training in, a variety of relevant laboratory skills, the Duquesne University School of Pharmacy developed a "methods" course as part of the core curriculum. Because some of the participating departmental faculty are neuroscientists, this course often applied cutting-edge techniques to neuroscience-based systems, including experiments with brain G protein-coupled receptors. Techniques covered by the course include animal handling and behavioral testing, bacterial and mammalian cell culture, enzyme-linked immunosorbent assay, western blotting, receptor binding of radioligands, plasmid DNA amplification and purification, reverse transcriptase-polymerase chain reaction, gel electrophoresis, and UV-visible and fluorescence spectroscopy.

Regulation of transcription by synthetic DNA-bending agents.

Gene expression is regulated by a complex interplay between binding and the three-dimensional arrangement of transcription factors with RNA polymerase and DNA. Previous studies have supported a direct role for DNA bending and conformation in gene expression, which suggests that agents that induce bends in DNA might be able to control gene expression. To test this hypothesis, we examined the effect of triple-helix-forming oligonucleotide (TFO) bending agents on the transcription of luciferase in an in vitro transcriptional/translational system.