Synthesis, characterization, and antibacterial activities of a heteroscorpionate derivative platinum complex against methicillin-resistant Staphylococcus aureus

Staphylococcus aureus is one of the species with the greatest clinical importance and greatest impact on public health. In fact, methicillin-resistant S. aureus (MRSA) is considered a pandemic pathogen, being essential to develop effective medicines and combat its rapid spread. This study aimed to foster the translation of clinical research outcomes based on metallodrugs into clinical practice for the treatment of MRSA. Bearing in mind the promising anti-Gram-positive effect of the heteroscorpionate ligand 1,1’-(2-(4-isopropylphenyl)ethane-1,1-diyl)bis(3,5-dimethyl-1H-pyrazole) (2P), we propose the coordination of this compound to platinum as a clinical strategy with the ultimate aim of overcoming resistance in the treatment of MRSA. Therefore, the novel metallodrug 2P-Pt were synthetized, fully characterized and its antibacterial effect against the planktonic and biofilm state of S. aureus evaluated. In this sense, three different strains of S. aureus were studied, one collection strain of S. aureus sensitive to methicillin and two clinical MRSA strains. To appraise the antibacterial activity, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC) were determined. Moreover, successful outcomes on the development of biofilm in a wound-like medium were obtained. The mechanism of action for 2P-Pt was proposed by measuring the MIC and MBC with EDTA (cation mediated mechanism) and DMSO (exogenous oxidative stress mechanism). Moreover, to shed light on the plausible antistaphylococcal mechanism of this novel platinum agent, additional experiments using transmission electron microscopy were carried out. 2P-Pt inhibited the growth and eradicated the three strains evaluated in the planktonic state. Another point worth stressing is the inhibition in the growth of MRSA biofilm even in a wounded medium. The results of this work support this novel agent as a promising therapeutic alternative for preventing infections caused by MRSA

Aliskiren prevents the toxic effects of peritoneal dialysis fluids during chronic dialysis in rats.

The benefits of long-term peritoneal dialysis (PD) in patients with end-stage renal failure are short-lived due to structural and functional changes in the peritoneal membrane. In this report, we provide evidence for the in vitro and in vivo participation of the renin-angiotensin-aldosterone system (RAAS) in the signaling pathway leading to peritoneal fibrosis during PD. Exposure to high-glucose PD fluids (PDFs) increases damage and fibrosis markers in both isolated rat peritoneal mesothelial cells and in the peritoneum of rats after chronic dialysis. In both cases, the addition of the RAAS inhibitor aliskiren markedly improved damage and fibrosis markers, and prevented functional modifications in the peritoneal transport, as measured by the peritoneal equilibrium test. These data suggest that inhibition of the RAAS may be a novel way to improve the efficacy of PD by preventing inflammation and fibrosis following peritoneal exposure to high-glucose PDFs

<i>In vivo</i> effect of aliskiren on p53, Bax and Bcl-2 mRNA levels in the peritoneum after daily peritoneal dialysis for 4 weeks.

<p>Twelve groups of 6 rats each one were dialyzed daily as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036268#s4" target="_blank">Methods</a> for 4 weeks in the absence (black histograms) and in the presence of aliskiren (10 and 100 mg/L). At the end of this period, peritoneal mesothelial cells (PMCs) were isolated and the mRNA levels for p53 (a), Bax (b), and Bcl-2 (c) quantified and normalized to the the β-actin mRNA levels. The dialysis fluid and the treatment for each group is indicated in the graph. Each histogram represents mean ± s.e.m. of 6 animals. *p<0.05 as compared to the vehicle group. ^#p<0.05 for the aliskiren-treated groups as compared to the same PDF in absence of aliskiren. ^$p<0.05 as compared to 10 mg/L aliskiren groups.</p

Aliskiren decreases fibrosis markers and inhibits changes in apoptosis markers induced by peritoneal dialysis fluids (PDFs) in rat peritoneal mesothelial cells (PMCs).

<p>Cells were treated for 24 h with a 1.5%-glucose PDF diluted 1∶1 in culture medium in the absence (V) or the presence of aliskiren, and the levels of mRNA for p53 (a), Bax (b), Bcl-2 (c), collagen III (d) and fibronectin (e) were determined by real-time RT-PCR. Data represent mean ± s.e.m. of 4 experiments. *p<0.05 as compared to untreated control cells (C). ^#p<0.01 as compared to cells treated with vehicle and PDF (V).</p

Aliskiren reduces the peritoneal fibrosis <i>in vivo</i> after chronic peritoneal dialysis.

<p>The groups of animals were the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036268#pone-0036268-g003" target="_blank">Fig. 3</a>. At the end of the 4 week-dialysis period, peritoneal mesothelial cells (PMCs) were isolated and the fibronectin (a) and collagen III (b) mRNA levels were determined in PMCs and normalized to the β-actin mRNA levels. The dialysis fluid used and the treatment for each group is indicated in the graph. Each histogram represents mean ± s.e.m. of 6 animals. *p<0.05 as compared to the vehicle group. ^#p<0.05 for the aliskiren-treated groups as compared to the same PDF in absence of aliskiren. ^$p<0.05 as compared to 10 mg/L aliskiren groups. c) Masson's trichrome staining of parietal peritoneum. <u>Top panel</u>. Histological sections from the vehicle, 1.5$$</sup>p<0.01 as compared to 10 mg/L aliskiren groups.</p

Aliskiren decreases the levels of inflammatory markers <i>in vivo</i> in both serum and dialysate after chronic peritoneal dialysis.

<p>The groups of animals were the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036268#pone-0036268-g003" target="_blank">Fig. 3</a>. A Peritoneal Equilibration Test (PET) was performed for 2 h at the end of the 4 weeks of dialysis. The levels of amyloid-P protein (a, b) and C-reactive protein (c, d) were determined in both serum (a, c) and dialysate (b, d) collected after 2 h dwell time. The dialysis fluid used and the treatment for each group is indicated in the graph. Each histogram represents mean ± s.e.m. of 6 animals. *p<0.05 as compared to the vehicle group. **p<0.01 as compared to the vehicle group. ^#p<0.05 for the aliskiren-treated groups as compared to the same PDF in absence of aliskiren. ^$p<0.05 as compared to 10 mg/L aliskiren groups.</p

Aliskiren decreases toxicity induced by peritoneal dialysis fluids (PDFs) in rat peritoneal mesothelial cells (PMCs).

<p>a) Effect of aliskiren on PDF-mediated reactive oxygen species (ROS) production in PMCs. Cells were treated for 8 h with a 1.5%-glucose PDF diluted 1∶1 in culture medium in the absence (V) or the presence of aliskiren. ROS production was measured using dichlorodihydrofluorescein (DCF) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036268#s4" target="_blank">Methods</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036268#s2" target="_blank">Results</a> represent mean ± s.e.m. of 4 experiments. *p<0.05 as compared to untreated control cells (C). ^#p<0.05 as compared to cells treated with vehicle and high-glucose PDF (V). b) Effect of aliskiren on phospho-p38 (p-p38) mitogen-activated protein kinase (MAPK)/p38 MAPK ratio in rat PMCs exposed to a high-glucose PDF for 24 h. PMCs were treated as above, in the absence (V) or presence of aliskiren and then both p-p38 MAPK and p38 MAPK protein levels were determined by western blot. The histograms represent a densitometric analysis of the p-p38 MAPK/p38 MAPK ratio. Data represent mean ± s.e.m. of 4 experiments.*p<0.05 as compared to C. #p<0.01 as compared to V. c) Effect of aliskiren on caspase-3 activity in rat PMCs exposed to a high-glucose PDF for 18 h. PMCs were treated as above, in the absence (V) or presence of aliskiren and then caspase-3 activity was determined (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036268#s4" target="_blank">Methods</a>). Data represent mean ± s.e.m. of 4 experiments. *p<0.05 as compared to C. ^#p<0.05 as compared to V.</p