Synthesis and in Silico Modelling of the Potential Dual Mechanistic Activity of Small Cationic Peptides Potentiating the Antibiotic Novobiocin against Susceptible and Multi-Drug Resistant Escherichia coli.

Cationic antimicrobial peptides have attracted interest, both as antimicrobial agents and for their ability to increase cell permeability to potentiate other antibiotics. However, toxicity to mammalian cells and complexity have hindered development for clinical use. We present the design and synthesis of very short cationic peptides (3-9 residues) with potential dual bacterial membrane permeation and efflux pump inhibition functionality. Peptides were designed based upon in silico similarity to known active peptides and efflux pump inhibitors. A number of these peptides potentiate the activity of the antibiotic novobiocin against susceptible Escherichia coli and restore antibiotic activity against a multi-drug resistant E. coli strain, despite having minimal or no intrinsic antimicrobial activity. Molecular modelling studies, via docking studies and short molecular dynamics simulations, indicate two potential mechanisms of potentiating activity; increasing antibiotic cell permeation via complexation with novobiocin to enable self-promoted uptake, and binding the E. coli RND efflux pump. These peptides demonstrate potential for restoring the activity of hydrophobic drugs.

Discovery of novel small molecule inhibitors of S100P with in vitro anti-metastatic effects on pancreatic cancer cells.

S100P, a calcium-binding protein, is known to advance tumor progression and metastasis in pancreatic and several other cancers. Herein is described the in silico identification of a putative binding pocket of S100P to identify, synthesize and evaluate novel small molecules with the potential to selectively bind S100P and inhibit its activation of cell survival and metastatic pathways. The virtual screening of a drug-like database against the S100P model led to the identification of over 100 clusters of diverse scaffolds. A representative test set identified a number of structurally unrelated hits that inhibit S100P-RAGE interaction, measured by ELISA, and reduce in vitro cell invasion selectively in S100P-expressing pancreatic cancer cells at 10 µM. This study establishes a proof of concept in the potential for rational design of small molecule S100P inhibitors for drug candidate development.

In Silico Structural Evaluation of Short Cationic Antimicrobial Peptides.

Cationic peptides with antimicrobial properties are ubiquitous in nature and have been studied for many years in an attempt to design novel antibiotics. However, very few molecules are used in the clinic so far, sometimes due to their complexity but, mostly, as a consequence of the unfavorable pharmacokinetic profile associated with peptides. The aim of this work is to investigate cationic peptides in order to identify common structural features which could be useful for the design of small peptides or peptido-mimetics with improved drug-like properties and activity against Gram negative bacteria. Two sets of cationic peptides (AMPs) with known antimicrobial activity have been investigated. The first reference set comprised molecules with experimentally-known conformations available in the protein databank (PDB), and the second one was composed of short peptides active against Gram negative bacteria but with no significant structural information available. The predicted structures of the peptides from the first set were in excellent agreement with those experimentally-observed, which allowed analysis of the structural features of the second group using computationally-derived conformations. The peptide conformations, either experimentally available or predicted, were clustered in an “all vs. all” fashion and the most populated clusters were then analyzed. It was confirmed that these peptides tend to assume an amphipathic conformation regardless of the environment. It was also observed that positively-charged amino acid residues can often be found next to aromatic residues. Finally, a protocol was evaluated for the investigation of the behavior of short cationic peptides in the presence of a membrane-like environment such as dodecylphosphocholine (DPC) micelles. The results presented herein introduce a promising approach to inform the design of novel short peptides with a potential antimicrobial activity.

A cellulose-based bioassay for the colorimetric detection of pathogen DNA.

Cellulose-paper-based colorimetric bioassays may be used at the point of sampling without sophisticated equipment. This study reports the development of a colorimetric bioassay based on cellulose that can detect pathogen DNA. The detection was based on covalently attached single-stranded DNA probes and visual analysis. A cellulose surface functionalized with tosyl groups was prepared by the N,N-dimethylacetamide-lithium chloride method. Tosylation of cellulose was confirmed by scanning electron microscopy, Fourier transform infrared spectroscopy and elemental analysis. Sulfhydryl-modified oligonucleotide probes complementary to a segment of the DNA sequence IS6110 of Mycobacterium tuberculosis were covalently immobilized on the tosylated cellulose. On hybridization of biotin-labelled DNA oligonucleotides with these probes, a colorimetric signal was obtained with streptavidin-conjugated horseradish peroxidase catalysing the oxidation of tetramethylbenzamidine by H2O2. The colour intensity was significantly reduced when the bioassay was subjected to DNA oligonucleotide of randomized base composition. Initial experiments have shown a sensitivity of 0.1 µM. A high probe immobilization efficiency (more than 90 %) was observed with a detection limit of 0.1 µM, corresponding to an absolute amount of 10 pmol. The detection of M. tuberculosis DNA was demonstrated using this technique coupled with PCR for biotinylation of the DNA. This work shows the potential use of tosylated cellulose as the basis for point-of-sampling bioassays.

Discovery of inhibitors of the pentein superfamily protein dimethylarginine dimethylaminohydrolase (DDAH), by virtual screening and hit analysis.

An efficient process for the discovery of inhibitors of DDAH enzymes, without the requirement for high throughput screening, is described. Physicochemical filtering of a 308,000-compound library according to drug likeness followed by reciprocal nearest neighbour selection produced a representative subset of 35,000 compounds. Virtual screening on a dual processor PC using FlexX, followed by biological screening, identified two hit series. Similarity searches of commercial databases and chemical re-synthesis of pure compounds resulted in SR445 as an inhibitor of Pseudomonas aeruginosa DDAH at 2 microM.

Disruption of methylarginine metabolism impairs vascular homeostasis.

Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both L-NMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. However, despite considerable interest in this pathway and in the role of ADMA as a cardiovascular risk factor, there is little evidence to support a causal role of ADMA in pathophysiology. Here we reveal the structure of human DDAH-1 and probe the function of DDAH-1 both by deleting the DDAH1 gene in mice and by using DDAH-specific inhibitors which, as we demonstrate by crystallography, bind to the active site of human DDAH-1. We show that loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. Our results also suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.

Inhibition of dimethylarginine dimethylaminohydrolase (DDAH) and arginine deiminase (ADI) by pentafluorophenyl (PFP) sulfonates.

A range of pentafluorophenyl (PFP) sulfonate esters derived from the reaction of PFP vinyl sulfonate and various nitrones are shown to have significant inhibitory activity against the bacterial enzymes DDAH and ADI.

Synthesis and anthelmintic properties of arylquinolines with activity against drug-resistant nematodes.

2,4-Disubstituted quinolines with additional substituents in positions 5-8 have been found to have anthelmintic properties. A number of 2,4-dimethoxy-6- or 8-arylquinolines have potent activity against the sheep nematode Haemonchus contortus, with LD99 values of the same order of magnitude as levamisole. These arylquinolines maintain their activity against levamisole-, ivermectin- and thiabendazole-resistant strains of H. contortus.

Selective substrate-based inhibitors of mammalian dimethylarginine dimethylaminohydrolase.

The enzyme DDAH metabolizes methylarginines that are inhibitors of nitric oxide synthase (NOS). Substrate-based inhibitors of mammalian DDAH have been synthesized, with optimization to give selective inhibition of DDAH with no significant direct effect on NOSs. These are the first examples of reversible DDAH inhibitors with significant activity and selectivity. In vivo administration increases plasma ADMA levels, giving proof of concept that these inhibitors can be used to probe the physiological effects of DDAH inhibition, with potential for pharmaceutical use of DDAH inhibitors in diseases where excess NO production is implicated.

Copper (II)-mediated arylation with aryl boronic acids for the N-derivatization of pyrazole libraries.

A N-derivatized 3-dimethylaminopropyloxypyrazole library was prepared using solution-phase parallel synthesis. The library was designed using physicochemical constraints designed to remove non-membrane-permeable molecules. Cupric acetate-mediated N-arylation with aryl boronic acids proceeded regioselectively to form the N-2-substituted derivatives. The presence of the 3-dimethylaminopropyloxy group was found to completely control the regioselectivity of the arylation. Presence of a dimethylaminoethyloxy or dimethylaminobutyloxy group gave a lesser degree of regioselectivity. The scope of the method as applied to library synthesis is discussed.

Demethylation of 2,4-dimethoxyquinolines: the synthesis of atanine.

The synthesis of the quinoline alkaloid atanine 6, by selective demethylation of the 2,4-dimethoxyquinoline 11 is presented. An alternative demethylation utilising a thiolate anion leads to the regioisomeric 4-hydroxyquinoline 13.

Halogenated indole-3-acetic acids as oxidatively activated prodrugs with potential for targeted cancer therapy.

Substituted indole-3-acetic acid (IAA) derivatives, plant auxins with potential for use as prodrugs in enzyme-prodrug directed cancer therapies, were oxidised with horseradish peroxidase (HRP) and toxicity against V79 Chinese hamster lung fibroblasts was determined. Rate constants for oxidation by HRP compound I were also measured. Halogenated IAAs were found to be the most cytotoxic, with typical surviving fractions of <10(-3) after incubation for 2h with 100 microM prodrug and HRP.

Reactivity toward thiols and cytotoxicity of 3-methylene-2-oxindoles, cytotoxins from indole-3-acetic acids, on activation by peroxidases.

Oxidation of indole-3-acetic acid and its derivatives by peroxidases such as that from horseradish produces many products, including 3-methylene-2-oxindoles. These have long been associated with biological activity, but their reactivity has not been characterized. We have previously demonstrated the potential value of substituted indole acetic acids and horseradish peroxidase as the basis for targeted cancer therapy, since the compounds are of low cytotoxicity until oxidized, when high cytotoxicity is observed; the combination of prodrug and enzyme depletes intracellular thiols. In this study, 3-methylene-2-oxindole and derivatives substituted in the 4-, 5-, or 6-position with methyl, F, or Cl have been synthesized and their reactivity toward representative thiol nucleophiles (glutathione, cysteine, and a cysteinyl peptide) measured using stopped-flow kinetic spectrophotometry. Rate constants were in the range approximately 2 x 10(3) to 2 x 10(4) M(-)(1) s(-)(1) at pH 7.4, 25 degrees C, implying a lifetime of a few tens of milliseconds for these methylene oxindoles in the cellular environment and diffusion distances of a few micrometers. As expected, halogen substitution decreased the rate of production of the methylene oxindoles on treatment of horseradish peroxidase. The cytotoxicities of the compounds were measured using Chinese hamster V79 fibroblast-like cells in vitro. The halogen-substituted derivatives were much more cytotoxic than the 5-methyl analogue or the parent (unsubstituted) compound, consistent with the trends in rate constant for reaction with the thiols. The results show that the cytotoxic response in the prodrug (indole acetic acid) and enzyme (horseradish peroxidase) system reflects the reactivity of methylene oxindoles toward nucleophiles much more than the rate of generation of the oxindoles, and helps explain the possible advantages of 5-fluoroindole-3-acetic acid compared to IAA as a lead compound for investigation in targeted cancer therapy.