Lipopolysaccharide (LPS), shed by gram-negative bacteria during infection and antimicrobial therapy, may lead to lethal endotoxic shock syndrome. A rational design strategy based on the presumed mechanism of antibacterial effect was adopted to design cationic antimicrobial peptides capable of binding to LPS through tandemly repeated sequences of alternating cationic and nonpolar residues. The peptides were designed to achieve enhanced antimicrobial potency due to initial bacterial membrane binding with a reduced risk of endotoxic shock. The peptides designed displayed binding affinities to LPS and lipid A (LA) in the low micromolar range and by molecular modeling were predicted to form amphipathic ␤-hairpin-like structures when they bind to LPS or LA. They also exhibited strong effects against gram-negative bacteria, with MICs in the nanomolar range, and low cytotoxic and hemolytic activities at concentrations significantly exceeding their MICs. Quantitative structure-activity relationship (QSAR) analysis of peptide sequences and their antimicrobial, cytotoxic, and hemolytic activities revealed that site-directed substitutions of residues in the hydrophobic face of the amphipathic peptides with less lipophilic residues selectively decrease the hemolytic effect without significantly affecting the antimicrobial or cytotoxic activity. On the other hand, the antimicrobial effect can be enhanced by substitutions in the polar face with more polar residues, which increase the amphipathicity of the peptide. On the basis of the QSARs, new analogs that have strong antimicrobial effects but that lack hemolytic activity can be proposed. The findings highlight the importance of peptide amphipathicity and allow a rational method that can be used to dissociate the antimicrobial and hemolytic effects of cationic peptides, which have potent antimicrobial properties, to be proposed.

We have carried out a computational structure-based design of new potent pyrrolidine carboxamide (PCAMs) inhibitors of enoyl-acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis (MTb). Three-dimensional (3D) models of InhA-PCAMx complexes were prepared by in situ modification of the crystal structure of InhA-PCAM1 (Protein Data Bank (PDB) entry code: 4U0J), the reference compound of a training set of 20 PCAMs with known experimental inhibitory potencies (IC50exp). First, we built a gas phase quantitative structure-activity relationships (QSAR) model, linearly correlating the computed enthalpy of the InhA-PCAM complex formation and the IC50exp. Further, taking into account the solvent effect and loss of inhibitor entropy upon enzyme binding led to a QSAR model with a superior linear correlation between computed Gibbs free energies (ΔΔGcom) of InhA-PCAM complex formation and IC50exp (pIC50exp = −0.1552·ΔΔGcom + 5.0448, R2 = 0.94), which was further validated with a 3D-QSAR pharmacophore model generation (PH4). Structural information from the models guided us in designing of a virtual combinatorial library (VL) of more than 17 million PCAMs. The VL was adsorption, distribution, metabolism and excretion (ADME) focused and reduced down to 1.6 million drug like orally bioavailable analogues and PH4 in silico screened to identify new potent PCAMs with predicted IC50pre reaching up to 5 nM. Combining molecular modeling and PH4 in silico screening of the VL resulted in the proposed novel potent antituberculotic agent candidates with favorable pharmacokinetic profiles.

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Truncating mutations of the gene SALL4 on chromosome 20q13.13-13.2 cause Okihiro and acro-renal-ocular syndromes. Pathogenic missense mutations within the SALL4 or SALL1 genes have not yet been reported, raising the question which phenotypic features would be associated with them. Here we describe the first missense mutation within the SALL4 gene. The mutation results in an exchange of a highly conserved zinc-coordinating Histidine crucial for zinc finger (ZF) structure within a C2H2 double ZF domain to an Arginine. Molecular modeling predicts that this exchange does not result in a loss of zinc ion binding but leads to an increased DNA-binding affinity of the domain. The index patient shows mild features of Okihiro syndrome, but in addition cranial midline defects (pituitary hypoplasia and single central incisor). This finding illustrates that the phenotypic and functional effects of SALL4 missense mutations are difficult to predict, and that other SALL4 missense mutations might lead to phenotypes not overlapping with Okihiro syndrome.

Lipid A moiety has been identified as the bioactive component of bacterial endotoxins (lipopolysaccharides). However, the molecular mechanism of biological activity of lipid A is still not fully understood. This paper contributes to understanding of the molecular mechanism of action of bacterial endotoxins by comparing molecular modelling results for two possible mechanisms with the underlying experimental data. Mechanisms of action involving specific binding of lipid A to a protein receptor as well as nonspecific intercalation into phospholipid membrane of a host cell were modelled and analysed. As the cellular receptor for endotoxin has not been identified, a model of a peptidic pseudoreceptor was proposed, based on molecular structure, symmetry of the lipid A moiety and the observed character of endotoxin-binding sites in proteins. We have studied the monomeric form of lipid A from Escherichia coli and its seven synthetic analogues with varying numbers of phosphate groups and correlated them with known biological activities determined by the Limulus assay. Gibbs free energies associated with the interaction of lipid A with the pseudoreceptor model and intercalation into phospholipid membrane calculated by molecular mechanics and molecular dynamics methods were used to compare the two possible mechanisms of action. The results suggest that specific binding of lipid A analogues to the peptidic pseudoreceptor carrying an amphipathic cationic binding pattern BHPH B (B, basic; H, hydrophobic; P, polar residue, respectively) is energetically more favourable than intercalation into the phospholipid membrane. In addition, binding affinities of lipid A analogues to the best minimum binding sequence KFSF K of the pseudoreceptor correlated with the experimental Limulus activity parameter. This correlation enabled us to rationalize the observed relationship between the number and position of the phosphate groups in the lipid A moiety and its biological activity in terms of specific ligand±receptor interactions. If lipid A±receptor interaction involves formation of phosphate-ammonium ion-pair(s) with cationic amino-acid residues, the specific mechanism of action was fully consistent with the underlying experimental data. As a consequence, recognition of lipid A variants by an amphipathic binding sequence BHPH B of a host-cell protein receptor might represent the initial and/or rate-determining molecular event of the mechanism of action of lipid A (or endotoxin). The insight into the molecular mechanism of action and the structure of the lipid A-binding pattern have potential implications for rational drug design strategies of endotoxin-neutralizing agents or binding factors.

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The outbreak of avian influenza A subtype H5N1 virus has raised a global concern for both animal as well as human health. Recently, drug resistance in H5N1 infections has been widely reported due to neuraminidase mutations. Consequently, the understanding of inhibitor-neuraminidase interactions at the molecular level represents the main goal of our study. Molecular dynamics simulations were carried out for the neuraminidase N1 in complex with six inhibitors--oseltamivir, zanamivir, peramivir, and their phosphonate analogues. Molecular dynamics trajectories were extensively analyzed in terms of important interactions between inhibitors and the enzyme target. Results show that open and closed forms (defined by the relative position of the flexible 150-loop) of neuraminidase N1 interchange during the course of 20 ns molecular dynamics simulation of the protein-inhibitor complexes. Reported free energies of closing indicate that the carboxylate inhibitors prefer the closed form more than their phosphonate analogues. This can be understood in view of the negative total charge (-1 e0) of the phosphonate inhibitors, which repels the Asp151 residue of the loop away from the inhibitor and drives the complex into the open form. Obtained results constitute new valuable information to assist further drug development of inhibitors against the H5N1 avian influenza A virus and could also inspire similar studies for other systems of the influenza family such as the 2009 influenza A (H1N1) virus.