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Increasing antibiotic resistance and diminishing pharmaceutical industry investments have increased the need for molecules that can treat infections caused by dangerous pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Quorum Sensing (QS) is a signaling mechanism that regulates bacterial virulence in pathogens. A report demonstrating that the anti-inflammatory drug Diflunisal reduces MRSA virulence factors’ expression prompted us to design, synthesize and test 16 aza-analogs as inhibitors of S. aureus virulence factors controlled by the accessory gene regulator (agr) QS system. At first, we evaluated by qRT-PCR the activity of compounds on rnaIII expression, a QS related gene. Azan-7 was the most active molecule tested and it did not show cytotoxic activity in human cell lines. Moreover, we demonstrated that it did not affect bacterial proliferation. Regulation of MRSA virulence genes by Azan-7 was investigated using qRT-PCR and RNAseq. Azan-7 significantly reduced hla, psmα, hysA, agrA, cap1A, and cap1C gene expression. In silico docking demonstrated that Azan-7 binds the response regulator AgrA. This data was confirmed by electrophoretic mobility shift assay (EMSA) reporting that Azan-7 binding to AgrA protein strongly reduced the AgrA-DNA complex formation at the P3 promoter region involved in the regulation of rnaIII transcription. Azan-7 inhibited MRSA-mediated haemolysis, reduced survival of the pathogen at low pH levels, and increased macrophage killing. In addition, Azan-7 enhanced MRSA susceptibility to clindamycin both in planktonic growth and biofilm. Azan-7 did not induce resistance over 10 days in culture. It was equally active against all the AgrA MRSA subtypes encountered among clinical isolates, but it was not active against Staphylococcus epidermidis, although the AgrA proteins show an approximate 80% homology. These results demonstrate that Azan-7 inhibits the expression of MRSA virulence factors by interfering in the QS and synergizes MRSA biofilm with clindamycin, indicating the compound as a promising candidate for the treatment of MRSA infections.

A small number of fluorinated 7-phenyl-pyrroloquinolinone (7-PPyQ) derivatives was synthesized in an attempt to improve the metabolic stability of 3N-ethyl-7-PPyQ and 3N-benzoyl-7-PPyQ. The possible impacts of the fluorine-hydrogen isosterism on both biological activity and metabolic stability were evaluated. Introduction of a fluorine atom in the 2 or 3 position of the 7-phenyl ring yielded the 7-PPyQ derivatives 12, 13 and 15, which showed potent cytotoxicity (low micromolar and sub-nanomolar GI50s) both in human leukemic and solid tumor cell lines. None of them induced significant cell death in quiescent and proliferating human lymphocytes. Moreover, 12, 13 and 15 exhibited remarkable cytotoxic activity in the multidrug-resistant cell line CEMVbl100, suggesting that they are not substrates for P-glycoprotein. All compounds inhibited tubulin assembly and the binding of [3H]colchicine to tubulin, with the best activity occurring with compound 15. Mechanistic studies carried out on compound 12 indicated that it caused (a) a strong G2/M arrest; (b) apoptosis in a time- and concentration-dependent manner; (c) a significant production of ROS (in good agreement with the observed mitochondrial depolarization); (d) caspase-3 and poly (ADP-ribose) polymerase activation; and (e) a decrease in the expression of anti-apoptotic proteins. In vivo experiments in a murine syngeneic tumor model demonstrated that compounds 12 and 15 significantly reduced tumor mass at doses four times lower than that required for the reference compound combretastatin A-4 phosphate. Neither monofluorination of the 7-phenyl ring of 3N-ethyl-7-PPyQ nor replacement of the benzoyl function of 3N-benzoyl-7-PPyQ with a 2-fluorobenzoyl moiety led to any improvement in the metabolic stability.

Our aim was to identify new multi-target compounds endowed with both anti-inflammatory and anti-bacterial activities for treatment of human infections. Diflunisal, a nonsteroidal anti-inflammatory agent, has recently been repurposed for its anti-virulence properties against methicillin-resistant Staphylococcus aureus. Effective synthesis of some aza-analogs of the anti-inflammatory drug diflunisal was carried out following the route involving key oxazole intermediates to obtain o- and m-hydroxypyridinecarboxylic acid derivatives. The newly synthesized diflunisal aza-analogs did not exhibit cytotoxic activity up to 80 μM and some of them exhibited anti-inflammatory activities, decreasing the levels of pro-inflammatory cytokines and prostaglandins induced by bacterial lipopolysaccharide in human primary macrophages. Ten of the diflunisal aza-analogs were found to have interesting antibacterial activity, sensitizing S. aureus, Streptococcus pyogenes, Enterococcus faecium, and Pseudomonas aeruginosa to the antibacterial effects of beta-lactam antibiotics and protein synthesis inhibitors.

Earlier studies had confirmed that the 7-phenylpyrroloquinolinone (7-PPyQ) nucleus was an important scaffold for new chemotherapeutic drugs targeting microtubules. For wide-ranging SARs, a series of derivatives were synthesized through a robust procedure. For comparison with the reference 3-ethyl-7-PPyQ 31, the angular geometry and substituents at the 3 and 7 positions were varied to explore interactions inside the colchicine site of tubulin. Of the new compounds synthesized, potent cytotoxicity (low and sub-nanomolar GI50 values) was observed with 21 and 24, both more potent than 31, in both leukemic and solid tumor cell lines. Neither compound 21 nor 24 induced significant cell death in normal human lymphocytes, suggesting that the compounds may be selectively active against cancer cells. In particular, 24 was a potent inducer of apoptosis in the A549 and HeLa cell lines. With both compounds, induction of apoptosis was associated with dissipation of the mitochondrial transmembrane potential and production of reactive oxygen species, indicating that cells treated with the compounds followed the intrinsic pathway of apoptosis. Moreover, immunoblot analysis revealed that compound 24 even at 50 nM reduced the expression of anti-apoptotic proteins such as Bcl-2 and Mcl-1. Finally, molecular docking studies of the newly synthesized compounds demonstrate that active pyrroloquinolinone derivatives strongly bind in the colchicine site of β-tubulin.