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.

The marine natural product mimic MPM-1 is cytolytic and induces DAMP release from human cancer cell lines.

Bioprospecting contributes to the discovery of new molecules with anticancer properties. Compounds with cytolytic activity and the ability to induce immunogenic cell death can be administered as intratumoral injections with the aim to activate anti-tumor immune responses by causing the release of tumor antigens as well as damage-associated molecular patterns (DAMPs) from dying cancer cells. In the present study, we report the cytolytic and DAMP-releasing effects of a new natural product mimic termed MPM-1 that was inspired by the marine Eusynstyelamides. We found that MPM-1 rapidly killed cancer cells in vitro by inducing a necrosis-like death, which was accompanied by lysosomal swelling and perturbation of autophagy in HSC-3 (human oral squamous cell carcinoma) cells. MPM-1 also induced release of the DAMPs adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1) from Ramos (B-cell lymphoma) and HSC-3 cells, as well as cell surface expression of calreticulin in HSC-3 cells. This indicates that MPM-1 has the ability to induce immunogenic cell death, further suggesting that it may have potential as a novel anticancer compound.

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.

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.

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.

Efficient and scalable synthesis of α,α-disubstituted ß-amino amides.

A practical and efficient methodology for the preparation of 2-aminoethyl α,α-disubstituted ß-amino amides in three steps from methyl cyanoacetate has been developed. The key step in the synthesis was the chemoselective reduction of the nitrile group in presence of an amide and aryl halide functionalities. Reduction with RANEY® Nickel catalyst, either with molecular hydrogen (8-10 bar) or under transfer hydrogenation conditions, necessitated in situ protection of the resulting amines with Boc2O, whereas aryl bromide containing nitriles could be chemoselectively reduced with ZnCl2/NaBH4 without debromination. The developed protocol involved only one chromatographic purification step and can be performed at gram scale.

Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites.

Menkes disease (MD) is an X-linked recessive disorder of copper metabolism. It is caused by mutations in the ATP7A gene encoding a copper-translocating P-type ATPase, which contains six N-terminal copper-binding sites (CBS1-CBS6). Most patients die in early childhood. We investigated the functional effect of a large frameshift deletion in ATP7A (including exons 3 and 4) identified in a patient with MD with unexpectedly mild symptoms and long survival. The mutated transcript, ATP7A(Delta ex3+ex4), contains a premature termination codon after 46 codons. Although such transcripts are generally degraded by nonsense-mediated mRNA decay (NMD), it was established by real-time PCR quantification that the ATP7A(Delta ex3+ex4) transcript was protected from degradation. A combination of in vitro translation, recombinant expression, and immunocytochemical analysis provided evidence that the ATP7A(Delta ex3+ex4) transcript was protected from degradation because of reinitiation of protein translation. Our findings suggest that reinitiation takes place at two downstream internal codons. The putative N-terminally truncated proteins contain only CBS5 and CBS6. Cellular localization and copper-dependent trafficking of the major part of endogenous and recombinant ATP7A(Delta ex3+ex4) proteins were similar to the wild-type ATP7A protein. Furthermore, the ATP7A(Delta ex3+ex4) cDNA was able to rescue a yeast strain lacking the homologous gene, CCC2. In summary, we propose that reinitiation of the NMD-resistant ATP7A(Delta ex3+ex4) transcript leads to the synthesis of N-terminally truncated and at-least-partially functional Menkes proteins missing CBS1-CBS4. This finding--that a mutation that would have been assumed to be null is not--highlights the need to examine the biochemical phenotype of patients to deduce the efficacy of copper therapy.

Inter-individual variation in brain phenylalanine concentration in patients with PKU is not caused by genetic variation in the 4F2hc/LAT1 complex.

It remains a question why some patients with phenylketonuria (PKU) have high IQ and low brain phenylalanine (Phe) concentrations in spite of high blood Phe levels. One possible explanation for the low brain Phe concentrations in these patients would be a reduced transport of Phe across the blood-brain barrier. The 4F2hc/LAT1 complex has been suggested to be the most important molecular component responsible for this transport. To test the hypothesis that structural variant(s) in the genes encoding 4F2hc and LAT1 might result in a complex with reduced affinity for Phe, we have screened the two genes for sequence variants in a group of 13 PKU patients with a low ratio of brain to blood Phe concentrations. Several common sequence variants were identified, but none of these is predicted to affect the resulting protein product. Our data suggest that individual vulnerability to Phe in patients with PKU is not due to structural variants in the 4F2hc/LAT1 complex.

Pre- and postnatal diagnosis of tyrosine hydroxylase deficiency.

OBJECTIVES: Tyrosine hydroxylase (TH) is a key enzyme in the biosynthesis of dopamine, epinephrine and norepinephrine. The primary diagnosis of TH deficiency is based on the measurement of neurotransmitter metabolites and pterins in the cerebrospinal fluid, and the final diagnosis is made by detection of mutations in the TH gene. The clinical expression varies with presentations as infantile parkinsonism, L-dopa responsive spastic paraplegia, or as a progressive severe encephalopathy. Treatment with L-dopa is not always sufficient and a number of patients with poor or no response to L-dopa have recently been described. METHODS: TH is not expressed in amniotic fluid cells or in chorionic villus, so prenatal diagnosis by measurement of the enzyme activity is not possible. The only possibility of a prenatal diagnosis is by analyzing the TH gene for mutations. RESULTS: Here we describe a case of severe TH deficiency, identification of two novel mutations (p.R328W and p.T399M) and most importantly, the first prenatal diagnosis of this disease. CONCLUSIONS: The availability of prenatal diagnosis offers the parents new options. They may use the result as preparation for the birth of a child with TH deficiency, or they may decide termination of an affected pregnancy.

Identification and analysis of 21 novel disease-causing amino acid substitutions in the conserved part of ATP7A.

ATP7A encodes a copper-translocating ATPase that belongs to the large family of P-type ATPases. Eight conserved regions define the core of the P-type ATPase superfamily. We report here the identification of 21 novel missense mutations in the conserved part of ATP7A that encodes the residues p.V842-p.S1404. Using the coordinates of X-ray crystal structures of the sarcoplasmic reticulum Ca(2+)-ATPase, as determined in the presence and absence of Ca(2+), we created structural homology models of ATP7A. By mapping the substituted residues onto the models, we found that these residues are more clustered three-dimensionally than expected from the primary sequence. The location of the substituted residues in conserved regions supports the functional similarities between the two types of P-type ATPases. An immunofluorescence analysis of Menkes fibroblasts suggested that the localization of a large number of the mutated ATP7A protein variants was correct. In the absence of copper, they were located in perinuclear regions of the cells, just like the wild type. However, two of the mutated ATP7A variants showed only partly correct localization, and in five cultures no ATP7A protein could be detected. These findings suggest that although a disease-causing mutation may indicate a functional significance of the affected residue, this is not always the case.