Anti-Restriction Gene Homologs Are Highly Represented in Methicillin-Resistant and Multidrug-Resistant Staphylococcus aureus ST239 and ST398: Implications for Resistance Gene Acquisitions.

Multidrug resistance is commonly acquired by transferring DNA from one bacterium to another. However, the mechanisms that enhance the acquisitions of foreign genes are poorly understood, as well as the dynamics of their transmission between hosts in different environments. Here, genomic approaches were applied to evaluate the enrichment of the S. aureus chromosome with resistance traits in groups of genomes with or without anti-restriction genes and to analyze some evolutionary aspects of these acquisitions. Furthermore, the role played by an anti-restriction gene in improving multiresistance in MRSA was investigated by molecular cloning. A strong association was observed between the presence of anti-restriction gene homologs and patterns of multidrug resistance. Human isolates, mainly ST239-SCCmecIII, carry ardA-H1, and from animal sources, mainly CC398, carry ardA-H2. Increased DNA transfer was observed for clones that express the ardA-H1 allele, corroborating its role in promoting gene transfer. In addition, ardA-H1 was expressed in the dsDNA format in the BMB9393 strain. The evolution of successful multidrug-resistant MRSA lineages of the ST239 and ST398 was initiated not only by the entry of the mec cassette but also by the acquisition of anti-restriction gene homologs. Understanding the mechanisms that affect DNA transfer may provide new tools to control the spread of drug resistance.

Analysis of a novel class A ß-lactamase OKP-B-6 of Klebsiella quasipneumoniae: structural characterisation and interaction with commercially available drugs.

BACKGROUND: Gram-negative and Gram-positive bacteria produce beta-lactamase as factors to overcome beta-lactam antibiotics, causing their hydrolysis and impaired antimicrobial action. Class A beta-lactamase contains the chromosomal sulfhydryl reagent variable (SHV, point mutation variants of SHV-1), LEN (Klebsiella pneumoniae strain LEN-1), and other K. pneumoniae beta-lactamase (OKP) found mostly in Klebsiella's phylogroups. The SHV known as extended-spectrum ß-lactamase can inactivate most beta-lactam antibiotics. Class A also includes the worrisome plasmid-encoded Klebsiella pneumoniae carbapenemase (KPC-2), a carbapenemase that can inactivate most beta-lactam antibiotics, carbapenems, and some beta-lactamase inhibitors. OBJECTIVES: So far, there is no 3D crystal structure for OKP-B, so our goal was to perform structural characterisation and molecular docking studies of this new enzyme. METHODS: We applied a homology modelling method to build the OKP-B-6 structure, which was compared with SHV-1 and KPC-2 according to their electrostatic potentials at the active site. Using the DockThor-VS, we performed molecular docking of the SHV-1 inhibitors commercially available as sulbactam, tazobactam, and avibactam against the constructed model of OKP-B-6. FINDINGS: From the point of view of enzyme inhibition, our results indicate that OKP-B-6 should be an extended-spectrum beta-lactamase (ESBL) susceptible to the same drugs as SHV-1. MAIN CONCLUSIONS: This conclusion advantageously impacts the clinical control of the bacterial pathogens encoding OKP-B in their genome by using any effective, broad-spectrum, and multitarget inhibitor against SHV-containing bacteria.

Design, synthesis, and biological evaluation of new thalidomide-donepezil hybrids as neuroprotective agents targeting cholinesterases and neuroinflammation.

A new series of eight multifunctional thalidomide-donepezil hybrids were synthesized based on the multi-target-directed ligand strategy and evaluated as potential neuroprotective, cholinesterase inhibitors and anti-neuroinflammatory agents against neurodegenerative diseases. A molecular hybridization approach was used for structural design by combining the N-benzylpiperidine pharmacophore of donepezil and the isoindoline-1,3-dione fragment from the thalidomide structure. The most promising compound, PQM-189 (3g), showed good AChE inhibitory activity with an IC50 value of 3.15 µM, which was predicted by docking studies as interacting with the enzyme in the same orientation observed in the AChE-donepezil complex and a similar profile of interaction. Additionally, compound 3g significantly decreased iNOS and IL-1ß levels by 43% and 39%, respectively, after 24 h of incubation with lipopolysaccharide. In vivo data confirmed the ability of 3g to prevent locomotor impairment and changes in feeding behavior elicited by lipopolysaccharide. Moreover, the PAMPA assay evidenced adequate blood-brain barrier and gastrointestinal tract permeabilities with an Fa value of 69.8%. Altogether, these biological data suggest that compound 3g can treat the inflammatory process and oxidative stress resulting from the overexpression of iNOS and therefore the increase in reactive nitrogen species, and regulate the release of pro-inflammatory cytokines such as IL-1ß. In this regard, compound PQM-189 (3g) was revealed to be a promising neuroprotective and anti-neuroinflammatory agent with an innovative thalidomide-donepezil-based hybrid molecular architecture.

Analysis of a novel class A β-lactamase OKP-B-6 of Klebsiella quasipneumoniae: structural characterisation and interaction with commercially available drugs

BACKGROUND Gram-negative and Gram-positive bacteria produce beta-lactamase as factors to overcome beta-lactam antibiotics, causing their hydrolysis and impaired antimicrobial action. Class A beta-lactamase contains the chromosomal sulfhydryl reagent variable (SHV, point mutation variants of SHV-1), LEN (Klebsiella pneumoniae strain LEN-1), and other K. pneumoniae beta-lactamase (OKP) found mostly in Klebsiella's phylogroups. The SHV known as extended-spectrum β-lactamase can inactivate most beta-lactam antibiotics. Class A also includes the worrisome plasmid-encoded Klebsiella pneumoniae carbapenemase (KPC-2), a carbapenemase that can inactivate most beta-lactam antibiotics, carbapenems, and some beta-lactamase inhibitors. OBJECTIVES So far, there is no 3D crystal structure for OKP-B, so our goal was to perform structural characterisation and molecular docking studies of this new enzyme. METHODS We applied a homology modelling method to build the OKP-B-6 structure, which was compared with SHV-1 and KPC-2 according to their electrostatic potentials at the active site. Using the DockThor-VS, we performed molecular docking of the SHV-1 inhibitors commercially available as sulbactam, tazobactam, and avibactam against the constructed model of OKP-B-6. FINDINGS From the point of view of enzyme inhibition, our results indicate that OKP-B-6 should be an extended-spectrum beta-lactamase (ESBL) susceptible to the same drugs as SHV-1. MAIN CONCLUSIONS This conclusion advantageously impacts the clinical control of the bacterial pathogens encoding OKP-B in their genome by using any effective, broad-spectrum, and multitarget inhibitor against SHV-containing bacteria.

Cinnamoyl-N-Acylhydrazone-Donepezil Hybrids: Synthesis and Evaluation of Novel Multifunctional Ligands Against Neurodegenerative Diseases.

A new series of ten multifunctional Cinnamoyl-N-acylhydrazone-donepezil hybrids was synthesized and evaluated as multifunctional ligands against neurodegenerative diseases. The molecular hybridization approach was based on the combination of 1-benzyl-4-piperidine fragment from the anti-Alzheimer AChE inhibitor donepezil (1) and the cinnamoyl subunit from curcumin (2), a natural product with remarkable antioxidant, neuroprotective and anti-inflammatory properties, using a N-acylhydrazone fragment as a spacer subunit. Compounds 4a and 4d showed moderate inhibitory activity towards AChE with IC50 values of 13.04 and 9.1 µM, respectively. In addition, compound 4a and 4d showed a similar predicted binding mode to that observed for donepezil in the molecular docking studies. On the other hand, compounds 4a and 4c exhibited significant radical scavenging activity, showing the best effects on the DPPH test and also exhibited a significant protective neuronal cell viability exposed to t-BuOOH and against 6-OHDA insult to prevent the oxidative stress in Parkinson's disease. Similarly, compound 4c was capable to prevent the ROS formation, with indirect antioxidant activity increasing intracellular GSH levels and the ability to counteract the neurotoxicity induced by both OAß1-42 and 3-NP. In addition, ADMET in silico prediction indicated that both compounds 4a and 4c did not show relevant toxic effects. Due to their above-mentioned biological properties, compounds 4a and 4c could be explored as lead compounds in search of more effective and low toxic small molecules with multiple neuroprotective effects for neurodegenerative diseases.

Design, Synthesis and Biological Evaluation of Novel Triazole N-acylhydrazone Hybrids for Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder that involves different pathogenic mechanisms. In this regard, the goal of this study was the design and synthesis of new compounds with multifunctional pharmacological activity by molecular hybridization of structural fragments of curcumin and resveratrol connected by an N-acyl-hydrazone function linked to a 1,4-disubstituted triazole system. Among these hybrid compounds, derivative 3e showed the ability to inhibit acetylcholinesterase activity, the intracellular formation of reactive oxygen species as well as the neurotoxicity elicited by Aß42 oligomers in neuronal SH-SY5Y cells. In parallel, compound 3e showed a good profile of safety and ADME parameters. Taken together, these results suggest that 3e could be considered a lead compound for the further development of AD therapeutics.

Synthesis of new lophine-carbohydrate hybrids as cholinesterase inhibitors: cytotoxicity evaluation and molecular modeling.

In this study, we synthesized nine novel hybrids derived from d-xylose, d-ribose, and d-galactose sugars connected by a methylene chain with lophine. The compounds were synthesized by a four-component reaction to afford the substituted imidazole moiety, followed by the displacement reaction between sugar derivatives with an appropriate N-alkylamino-lophine. All the compounds were found to be the potent and selective inhibitors of BuChE activity in mouse serum, with compound 9a (a d-galactose derivative) being the most potent inhibitor (IC50 = 0.17 µM). According to the molecular modeling results, all the compounds indicated that the lophine moiety existed at the bottom of the BuChE cavity and formed a T-stacking interaction with Trp231, a residue accessible exclusively in the BuChE cavity. Noteworthily, only one compound exhibited activity against AChE (8b; IC50 = 2.75 µM). Moreover, the in silico ADME predictions indicated that all the hybrids formulated in this study were drug-likely, orally available, and able to reach the CNS. Further, in vitro studies demonstrated that the two most potent compounds against BuChE (8b and 9a) had no cytotoxic effects in the Vero (kidney), HepG2 (hepatic), and C6 (astroglial) cell lines.

Design, synthesis and pharmacological evaluation of N-benzyl-piperidinyl-aryl-acylhydrazone derivatives as donepezil hybrids: Discovery of novel multi-target anti-alzheimer prototype drug candidates.

A new series of sixteen multifunctional N-benzyl-piperidine-aryl-acylhydrazones hybrid derivatives was synthesized and evaluated for multi-target activities related to Alzheimer's disease (AD). The molecular hybridization approach was based on the combination, in a single molecule, of the pharmacophoric N-benzyl-piperidine subunit of donepezil, the substituted hydroxy-piperidine fragment of the AChE inhibitor LASSBio-767, and an acylhydrazone linker, a privileged structure present in a number of synthetic aryl- and aryl-acylhydrazone derivatives with significant AChE and anti-inflammatory activities. Among them, compounds 4c, 4d, 4g and 4j presented the best AChE inhibitory activities, but only compounds 4c and 4g exhibited concurrent anti-inflammatory activity in vitro and in vivo, against amyloid beta oligomer (AßO) induced neuroinflammation. Compound 4c also showed the best in vitro and in vivo neuroprotective effects against AßO-induced neurodegeneration. In addition, compound 4c showed a similar binding mode to donepezil in both acetylated and free forms of AChE enzyme in molecular docking studies and did not show relevant toxic effects on in vitro and in vivo assays, with good predicted ADME parameters in silico. Overall, all these results highlighted compound 4c as a promising and innovative multi-target drug prototype candidate for AD treatment.

Novel series of tacrine-tianeptine hybrids: Synthesis, cholinesterase inhibitory activity, S100B secretion and a molecular modeling approach.

Tianeptine was linked to various 9-aminoalkylamino-1,2,3,4-tetrahydroacridines using EDC·HCl/HOBt to afford a series of tacrine-tianeptine hybrids. The hybrids were tested for their ability to inhibit AChE and BuChE and IC50 values in the nanomolar concentration scale were obtained. AChE molecular modeling studies of these hybrids indicated that tacrine moiety interacts in the bottom of the gorge with the catalytic active site (CAS) while tianeptine binds to peripheral anionic site (PAS). Furthermore, the compounds 2g and 2e were able to reduce the in vitro basal secretion of S100B, suggesting its therapeutic action in some cases or stages of Alzheimer's disease.

A unique SaeS allele overrides cell-density dependent expression of saeR and lukSF-PV in the ST30-SCCmecIV lineage of CA-MRSA.

ST30 (CC30)-SCCmec IV (USA1100) is one of the most common community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) lineages. ST30 isolates typically carry lukSF-PV genes encoding the Panton-Valentine leukocidin (PVL) and are responsible for outbreaks of invasive infections worldwide. In this study, twenty CC30 isolates were analyzed. All were very susceptible to non-ß-lactam antimicrobials, 18/20 harbored the lukSF-PV genes, only 1/20 exhibited agr-rnaIII dysfunction, and the majority was not able to form biofilm on inert surfaces. Analysis of lukSF-PV temporal regulation revealed that opposite to other CA-MRSA isolates, these genes were more highly expressed in early log phase than in stationary phase. This inverted lukSF-PV temporal expression was associated with a similar pattern of saeRS expression in the ST30 isolates, namely high level expression in log phase and reduced expression in stationary phase. Reduced saeRS expression in stationary phase was associated with low expression levels of the sae regulators, agr and agr-upregulator sarA, which activate the stationary phase sae-P1 promoter and overexpression of agr-RNAIII restored the levels of saeR and lukSF-PV trancripts in stationary phase. Altered SaeRS activity in the ST30 isolates was attributed to amino acid substitutions (N227S, E268K and S351T) in the HTPase_c domain of SaeS (termed SaeS(SKT)). Complementation of a USA300 saeS mutant with the saeS(SKT) and saeS alleles under the direction of the log phase sae-P3 promoter revealed that saeR and lukSF-PV transcription levels were more significantly activated by saeS(SKT) than saeS. In summary our data identify a unique saeS allele (saeS(SKT)) which appears to override cell-density dependent SaeR and PVL expression in ST30 CA-MRSA isolates. Further studies to determine the contribution of saeS(SKT) allele to the pathogenesis of infections caused by ST30 isolates are merited.