An amphipathic cyclic tetrapeptide scaffold containing halogenated ß2,2 -amino acids with activity against multiresistant bacteria.

The present study describes the synthesis and biological studies of a small series of head-to-tail cyclic tetrapeptides of the general structure c(Lys-ß2,2 -Xaa-Lys) containing one lipophilic ß2,2 -amino acid and Lys, Gly, Ala, or Phe as the Xaa residue in the sequence. The peptides were investigated for antimicrobial activity against gram-positive and gram-negative reference strains and 30 multiresistant clinical isolates including strains with extended spectrum ß-lactamase-carbapenemase (ESBL-CARBA) production. Toxicity was determined against human red blood cells. The most potent peptides showed high activity against the gram-positive clinical isolates with minimum inhibitory concentrations of 4-8 µg/mL and low haemolytic activity. The combination of high antimicrobial activity and low toxicity shows that these cyclic tetrapeptides containing lipophilic ß2,2 -amino acids form a valuable scaffold for designing novel antimicrobial agents.

Tailored anti-biofilm activity - Liposomal delivery for mimic of small antimicrobial peptide.

The eradication of bacteria embedded in biofilms is among the most challenging obstacles in the management of chronic wounds. These biofilms are found in most chronic wounds; moreover, the biofilm-embedded bacteria are considerably less susceptible to conventional antimicrobial treatment than the planktonic bacteria. Antimicrobial peptides and their mimics are considered attractive candidates in the pursuit of novel therapeutic options for the treatment of chronic wounds and general bacterial eradication. However, some limitations linked to these membrane-active antimicrobials are making their clinical use challenging. Novel innovative delivery systems addressing these limitations represent a smart solution. We hypothesized that incorporation of a novel synthetic mimic of an antimicrobial peptide in liposomes could improve its anti-biofilm effect as well as the anti-inflammatory activity. The small synthetic mimic of an antimicrobial peptide, 7e-SMAMP, was incorporated into liposomes (~280 nm) tailored for skin wounds and evaluated for its potential activity against both biofilm formation and eradication of pre-formed biofilms. The 7e-SMAMP-liposomes significantly lowered inflammatory response in murine macrophages (~30 % reduction) without affecting the viability of macrophages or keratinocytes. Importantly, the 7e-SMAMP-liposomes completely eradicated biofilms produced by Staphylococcus aureus and Escherichia coli above concentrations of 6.25 µg/mL, whereas in Pseudomonas aeruginosa the eradication reached 75 % at the same concentration. Incorporation of 7e-SMAMP in liposomes improved both the inhibition of biofilm formation as well as biofilm eradication in vitro, as compared to non-formulated antimicrobial, therefore confirming its potential as a novel therapeutic option for bacteria-infected chronic wounds.

Amphipathic Barbiturates as Mimics of Antimicrobial Peptides and the Marine Natural Products Eusynstyelamides with Activity against Multi-resistant Clinical Isolates.

We report a series of synthetic cationic amphipathic barbiturates inspired by the pharmacophore model of small antimicrobial peptides (AMPs) and the marine antimicrobials eusynstyelamides. These N,N'-dialkylated-5,5-disubstituted barbiturates consist of an achiral barbiturate scaffold with two cationic groups and two lipophilic side chains. Minimum inhibitory concentrations of 2-8 µg/mL were achieved against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum ß-lactamase-carbapenemase production. The guanidine barbiturate 7e (3,5-di-Br) demonstrated promising in vivo antibiotic efficacy in mice infected with clinical isolates of Escherichia coli and Klebsiella pneumoniae using a neutropenic peritonitis model. Mode of action studies showed a strong membrane disrupting effect and was supported by nuclear magnetic resonance and molecular dynamics simulations. The results express how the pharmacophore model of small AMPs and the structure of the marine eusynstyelamides can be used to design highly potent lead peptidomimetics against multi-resistant bacteria.

Antimicrobial activity of amphipathic α,α-disubstituted ß-amino amide derivatives against ESBL - CARBA producing multi-resistant bacteria; effect of halogenation, lipophilicity and cationic character.

The rapid emergence and spread of multi-resistant bacteria have created an urgent need for new antimicrobial agents. We report here a series of amphipathic α,α-disubstituted ß-amino amide derivatives with activity against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum ß-lactamase - carbapenemase (ESBL-CARBA) production. A variety of halogenated aromatic side-chains were investigated to improve antimicrobial potency and minimize formation of Phase I metabolites. Net positive charge and cationic character of the derivatives had an important effect on toxicity against human cell lines. The most potent and selective derivative was the diguanidine derivative 4e with 3,5-di-brominated benzylic side-chains. Derivative 4e displayed minimum inhibitory concentrations (MIC) of 0.25-8 µg/mL against Gram-positive and Gram-negative reference strains, and 2-32 µg/mL against multi-resistant clinical isolates. Derivative 4e showed also low toxicity against human red blood cells (EC50 > 200 µg/mL), human hepatocyte carcinoma cells (HepG2: EC50 > 64 µg/mL), and human lung fibroblast cells (MRC-5: EC50 > 64 µg/mL). The broad-spectrum antimicrobial activity and low toxicity of diguanylated derivatives such as 4e make them attractive as lead compounds for development of novel antimicrobial drugs.

Metallo-ß-lactamase inhibitors by bioisosteric replacement: Preparation, activity and binding.

Bacterial resistance is compromising the use of ß-lactam antibiotics including carbapenems. The main resistance mechanism against ß-lactams is hydrolysis of the ß-lactam ring mediated by serine- or metallo-ß-lactamases (MBLs). Although several inhibitors of MBLs have been reported, none has been developed into a clinically useful inhibitor. Mercaptocarboxylic acids are among the most prominent scaffolds reported as MBL inhibitors. In this study, the carboxylate group of mercaptocarboxylic acids was replaced with bioisosteric groups like phosphonate esters, phosphonic acids and NH-tetrazoles. The influence of the replacement on the bioactivity and inhibitor binding was evaluated. A series of bioisosteres of previously reported inhibitors was synthesized and evaluated against the MBLs VIM-2, NDM-1 and GIM-1. The most active inhibitors combined a mercapto group and a phosphonate ester or acid, with two/three carbon chains connecting a phenyl group. Surprisingly, also compounds containing thioacetate groups instead of thiols showed low IC50 values. High-resolution crystal structures of three inhibitors in complex with VIM-2 revealed hydrophobic interactions for the diethyl groups in the phosphonate ester (inhibitor 2b), the mercapto bridging the two active site zinc ions, and tight stacking of the benzene ring to the inhibitor between Phe62, Tyr67, Arg228 and His263. The inhibitors show reduced enzyme activity in Escherichia coli cells harboring MBL. The obtained results will be useful for further structural guided design of MBL inhibitors.