Binding orientation and activity determinants of the antimicrobial peptide cryptdin-4 revealed by potency of mutants

Cryptdin-4 is a β-sheet antimicrobial peptide of the defensin family that is found in the immune system of mice. Several structure–activity studies of this peptide have previously been conducted, but none have been based on residue–membrane interactions as part of an overall hypothesis on the peptide's orientation in the membrane. We pursue this valuable approach by first using previously reported NMR structural data to propose a membrane-bound orientation of the peptide. Four mutants are then strategically designed to modulate membrane perturbative activity in a manner consistent with the proposed binding orientation. Membrane perturbation is evaluated using a simple fluorescence-based vesicle leakage assay using POPG to form the model membrane. Effects of peptide mutations are found to be consistent with the suggested binding orientation. This approach is successfully used to create synthetic peptides with enhanced or diminished ability to perturb membranes and also yields insights on the nature of peptide–membrane interactions.     

Structure-activity studies of 14-helical antimicrobial beta-peptides: probing the relationship between conformational stability and antimicrobial potency

Antimicrobial alpha-helical alpha-peptides are part of the host-defense mechanism of multicellular organisms and could find therapeutic use against bacteria that are resistant to conventional antibiotics. Recent work from Hamuro et al. has shown that oligomers of beta-amino acids ("beta-peptides") that can adopt an amphiphilic helix defined by 14-membered ring hydrogen bonds ("14-helix") are active against Escherichia coli [Hamuro, Y.; Schneider, J. P.; DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 12200-12201]. We have created two series of cationic 9- and 10-residue amphiphilic beta-peptides to probe the effect of 14-helix stability on antimicrobial and hemolytic activity. 14-Helix stability within these series is modulated by varying the proportions of rigid trans-2-aminocyclohexanecarboxylic acid (ACHC) residues and flexible acyclic residues. We have previously shown that a high proportion of ACHC residues in short beta-peptides encourages 14-helical structure in aqueous solution [Appella, D. H.; Barchi, J. J.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121, 2309-2310]. Circular dichroism of the beta-peptides described here reveals a broad range of 14-helix population in aqueous buffer, but this variation in helical propensity does not lead to significant changes in antibiotic activity against a set of four bacteria. Several of the 9-mers display antibiotic activity comparable to that of a synthetic magainin derivative. Among these 9-mers, hemolytic activity increases slightly with increasing 14-helical propensity, but all of the 9-mers are less hemolytic than the magainin derivative. Previous studies with conventional peptides (alpha-amino acid residues) have provided conflicting evidence on the relationship between helical propensity and antimicrobial activity. This uncertainty has arisen because alpha-helix stability can be varied to only a limited extent among linear alpha-peptides without modifying parameters important for antimicrobial activity (e.g., net charge or hydrophobicity); a much greater range of helical stability is accessible with beta-peptides. For example, it is very rare for a linear alpha-peptide to display significant alpha-helix formation in aqueous solution and manifest antibacterial activity, while the linear beta-peptides described here range from fully unfolded to very highly folded in aqueous solution. This study shows that beta-peptides can be unique tools for analyzing relationships between conformational stability and biological activity.     

Improvement of the antimicrobial potency,pharmacokinetic and pharmacodynamic properties of albicidin by incorporation of nitrogen atoms

The worrisome development and spread of multidrug-resistant bacteria demands new antibacterial agents with strong bioactivities particularly against Gram-negative bacteria. Albicidins were recently structurally characterized as highly active antibacterial natural products from the bacterium Xanthomonas albilineans. Albicidin, which effectively targets the bacterial DNA-gyrase, is a lipophilic hexapeptide mostly consisting of para amino benzoic acid units and only one α-amino acid. In this study, we report on the design and synthesis of new albicidins, containing N-atoms on each of the 5 different phenyl rings. We systematically introduced N-atoms into the aromatic backbone to monitor intramolecular H-bonds and for one derivative correlated them with a significant enhancement of the antibacterial activity and activity spectrum, particularly also towards Gram-positive bacteria. In parallel we conducted DFT calculations to find the most stable conformation of each derivative. A drastic angle-change was observed for the lead compound and shows a preferred planarity through H-bonding with the introduced N-atom at the D-fragment of albicidin. Finally, we went to the next level and conducted the first in vivo experiments with an albicidin analogue. Our lead compound was evaluated in two different mouse experiments: In the first we show a promising PK profile and the absence of toxicity and in the second very good efficiency and reduction of the bacterial titre in an E. coli infection model with FQ-resistant clinically relevant strains. These results qualify albicidins as active antibacterial substances with the potential to be developed as a drug for treatment of infections caused by Gram-negative and Gram-positive bacteria.

A systematic pyridine-scan of the albicidin molecule provides a new lead structure with improved antimicrobial properties.     

Two new simple iridoids from the ant-plant Myrmecodia tuberosa and their antimicrobial effects

Six iridoid derivatives (1–6), including two new compounds myrmecodoides A and B (1 and 2), were isolated from the ant-plant Myrmecodia tuberosa. Their structures were determined on the basis of spectroscopic data (¹H and ¹³C NMR, HSQC, HMBC, ¹H-¹H COSY, NOESY and HR-ESI-MS) and by comparison with the literature values. Among isolates, 3 and 4 exhibit weak antibacterial effect against Staphylococcus aureus subsp. aureus with MIC value of 100.0 μg/mL.     

Effects of linker amino acids on the potency and selectivity of dimeric antimicrobial peptides

Conformational flexibility induced by proline and aminocaproic acid can increase anticancer activity and antimicrobial activity of dimeric antimicrobial peptides with reduced hemolytic activity. This study will contribute to the design of efficient antimicrobial peptides.     

Determination of beta-lactam antibiotics in water by fluorescence quenching of mercurochrome, and application for simple investigation of potency

The fluorescence quenching reaction between fluorescein mercury or halogeno-fluorescein mercury compounds (fl. Hg, 2,7- or 2,4-dichloro-fl.Hg, 3',4',5',6'-tetrachloro-fl.Hg, mercurochrome) and beta-lactam antibiotics (ampicillin (AB-PC) and cephalexin (CEX] was investigated, and mercurochrome was selected for the detection of beta-lactam antibiotics; the detection limit was about 0.8 micrograms/ml. A fluorimetric assay of beta-lactam antibiotics was established by measuring the fluorescence of mercurochrome and mercurochrome-beta-lactam antibiotics solutions in weakly basic media to determine the degree of fluorescence quenching. The maximum emission wavelength of mercurochrome solution was at 544 nm with excitation at 470 nm. The calibration graphs were linear over the ranges of about 0-6 micrograms/ml beta-lactam antibiotics penicillins (AB-PC, penicillin G, sulbenicillin, amoxicillin, cyclacillin, oxacillin, hetacillin and piperacillin) and cepham antibiotics (CEX, cefazolin, cephaloglycin, cephaloridine and cefpyramide), and the relative standard deviation was 2.7% for 1.4 micrograms/ml of AB-PC (n = 5). This fluorescence quenching reaction between mercurochrome and beta-lactam antibiotics was applied in a survey of decomposition and remaining potency of beta-lactam antibiotics.     

Upgrading oxygenated Fischer-Tropsch derivatives and one-step direct synthesis of ethyl acetate from ethanol - examples of the desirability of research on simple chemical compounds transformations

Oxygenates formed as by-products of Fischer-Tropsch syntheses can be transformed into other Fischer-Tropsch derived oxygenates instead of treating them as unwanted chemicals. One-step direct synthesis of ethyl acetate from ethanol is feasible with the use of some heterogeneous catalysts. Despite their apparent simplicity, both transformations are discussed as targeted fields of research. Furthermore, the two concepts are justified due to the environmental protection. Arguments regarding the Fischer-Tropsch process are focused on the opportunities of the utilization of undesirable by-products. The effective striving for their utilization can make the oxygenates the targeted products of this process. Arguments regarding the one-step direct synthesis of ethyl acetate underline the environmental protection and sustainability as a less waste-generating method but, above all, highlight the possibility of reducing the glycerol overproduction problem. The production of ethyl acetate from bioethanol and then transesterification of fats and oils with the use of ethyl acetate allows managing all the renewable raw materials. Thus, the process enables the biosynthesis of biodiesel without glycerine by-product and potentially would result in the increase in the demand for ethyl acetate.

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Graphical Abstract .

     

A simple molecular theory of double reentrance exhibited by highly polar compounds

We show by simple model calculations that dipole-induced dipole and interchain interactions lead to a parallel alignment of molecules with strong longitudinal dipoles for intermolecular separations below a certain value. On the other hand an antiparallel alignment is favoured for larger separations because of dipole-dipole interactions. We incorporate this change in the intermolecular configuration, which naturally leads to two 'lengths' in the problem, in developing a simple McMillan type molecular theory of the double reentrance phenomenon in which the following phase sequence is observed on cooling the sample: isotropic-nematic-smectic A_d-reentrant nematic-smectic A₁. The calculated properties including the phase diagram are in broad agreement with experimental trends.     

Potentials in density functional theory and the importance of sum rules

In an ab initio approach to density functional theory one needs to know the electronic pair-density averaged over the coupling strength of the pair-interaction. As this pair-function is not available without having solved the associated N-electron problem, one has to resort to universal properties of the pair-density that are independent of specific features of the ground-state wavefunction. By exploiting these universal properties and so-called sum rules for the pair-correlation factors we derive very simple approximate spin-dependent expressions for the exchange-correlation energy per particle and for the associated potential in the Kohn-Sham equations. There is some similarity of the resulting density functionals with those obtained from the widely applied local spin density approximation (LSDA) based on electron gas theory. As the application of the latter to exceedingly inhomogeneous gases in realistic systems is very debatable, the manifest similarity seems to suggest that LSDA can consistently be justified only via the above pair-density analysis, but the justification of certain electron gas refinements may remain questionable. We shortly review similar attempts made by other authors and particularly focus on the issue of self-interaction and the “overbinding problem”. We demonstrate for the 3d- and 4d-metals that our approximation yields density of states (DOS), magnetic moments and Stoner parameters that are practically identical with respective data obtained from up-to-date LSDA- or gradient corrected (GGA-)potentials. There is also an excellent agreement of the DOS for the insulators (semi-conductors) C, Si, Ge, and GaAs. We show that our approach yields cohesive energies for these materials that are very close to the GGA-values indicating a distinct improvement over the standard LSDA-values. The calculations have been performed with the aid of the WIEN 97 computer code based on the Full Potential Linear Augmented Plane Wave (FLAPW-) method.     

Recent advances in simple valence-bond theory and the theory of hybrid bond orbitals

The history of important developments in the theory of hybrid bond orbitals and its application in valence-bond theory is reviewed. One of the salient points, the bond strength of a hybrid orbital, is defined as the value of the angular part of the orbital along the bond direction. Characteristic bond angles corresponding to maxima in the bond strength are presented for various basis sets. In order to alleviate computational difficulties in determining the bond strength for complex systems, an approximation to it, the pair-defect-sum approximation, is described. The results of an exhaustive test of the validity of this approximation are presented. Applicaitions of these ideas to coordinate chemistry are provided. Finally, the case is made for the continued viability of valence-bond theory in this age of omnipresent computer terminals.     

Ground-state properties of benzenoid hydrocarbons by simple bond orbital resonance theory approach

π-electron energies and bond orders of benzenoid hydrocarbons with up to five fused hexagons have been considered by the simple Bond Orbital Resonance Theory (BORT) approach. The corresponding ground states were determined according to four BORT models. In the first three models a diagonalisation of the Hückel-type Hamiltonian was performed in the bases of Kekulé, of Kekulé and mono-Claus and of Kekulé and Claus resonance structures, respectively. In the fourth model a simple BORT ansatz was used. According to this ansatz, the ground state is a linear combination of the positive Kekulé structures, all with equal coefficients. It was shown that π-electron energies and bond orders obtained by these models correlate much better with the PPP energies and bond orders than with the Hückel energies and bond orders. This indicates that a simple BORT approach is quite reliable in predicting the more sophisticated PPP results. Concerning the relative performance of the four BORT models, the best results were obtained with the BORT ansatz. The performance deteriorates with the expansion of the basis set. This is attributed to the fact that in these models the improvement of the basis set is not accompanied with the corresponding improvement of the Hamiltonian. Comparing the BORT-ansatz bond orders with the Pauling bond orders, it was shown that BORT-ansatz bond orders correlate much better with the PPP bond orders. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biological screening of some Turkish medicinal plant extracts for antimicrobial and toxicity activities

Screening of antibacterial activity and toxicity of 22 aqueous plant extracts from 17 Turkish plants was conducted. Antibacterial activity was performed with six bacteria including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Staphylococcus epidermidis. Extracts of Tussilago farfara leaves, Helichyrsum plicatum flowers, Solanum dulcamara aerial parts and Urtica dioica leaves gave the best inhibitory activity against S. pyogenes, S. aureus and S. epidermidis. Of the 22 plant extracts, 20 extracts displayed toxicity (LC50 was <1000 mg L(-1)) in the brine shrimp bioassay. For radish seed bioassay, two different determinations (root length and seed germination) were performed with a comparison between two concentrations (50,000 mg L(-1) and 10,000 mg L(-1)). At low concentration (10,000 mg L(-1)), S. dulcamara aerial parts and Primula vulgaris leaf extracts were observed to inhibit the root length more than the other plant extracts. Also, the most inhibitive plant extract for seed germination was obtained with S. dulcamara aerial parts.     

A simple treatment of intermolecular interactions: Synthesis of ab initio calculations and combination rules

A new technique for a simple and efficient treatment of intermolecular interactions is proposed and tested. The method is based on an approximation of the first order SCF term E_SCF⁽¹⁾ which is the most structured contribution to the total interaction. E_SCF⁽¹⁾ is represented by a site–site potential V of (exp, 1/R)-type, which accounts for the exchange plus penetration and the long range Coulomb forces (by means of a point charge model). The individual contributions to V are obtained by means of combination rules from corresponding site parameters of interacting molecules. The site parameters are consequently molecular and not intermolecular properties and can conveniently be determined by probing a molecule with appropriate test particles. Site parameters are reported for He, Ne, Ar, N₂, CO, CO₂, CS₂, and HCl. Comprisons show close agreement of V with E which in turn is close to ΔE_SCF if polarization and charge transfer effects are small.     

Comparative evaluation of bioactivity change of crystalline trypsin during compression by chemoinformatics and 2-D Fourier-transform infrared spectroscopy

The purpose of this study is to develop a method of evaluating the enzymatic activity of trypsin in a solid-state based on Fourier transform infrared (FT-IR) spectra using chemoinformatics and two-dimensional (2-D) correlation spectroscopy. Crystalline trypsin powders are compressed at 0-4000 kg cm-2 by a compression/tension tester. The enzymatic activity of trypsin is assayed by the kinetic degradation method. Spectra of 10 calibration sample sets are recorded 3 times with a FT-IR spectrometer. The maximum intensity of FT-IR spectra and enzymatic activity of trypsin decrease as the compression pressure increases. The FT-IR spectra of trypsin samples are subjected to a principal component regression (PCR). A plot of the calibration data obtained is made between the actual and predicted trypsin activity based on a two-component model with gamma2=0.909 (n=30). The regression vector is almost the same as the loading vector for PC1. On the other hand, a generalized two-dimensional (2-D) correlation spectroscopic method is applied to FT-IR spectra of compressed trypsin. The result is consistent with that of the chemoinformatics method. The FT-IR chemoinformatics method allows for solid-state quantitative analysis of the bioactivity of the bulk powder of a polypeptide drug.     

A convenient synthesis of 1,3,4-thiadiazole and 1,3,4-oxadiazole based peptidomimetics employing diacylhydrazines derived from amino acids

Synthesis of novel orthogonally protected 1,3,4-thiadiazole and 1,3,4-oxadiazole tethered dipeptide mimetics is described. Both the heterocycles are prepared via a set of diacylhydrazines derived from amino acids. 1,3,4-Thiadiazoles are synthesized by dehydrosulfurization using Lawesson’s reagent while 1,3,4-oxadiazoles are obtained by EDC mediated cyclodehydration.     

A simple optimization strategy based on Rs-minimum and information theory of chromatography

Summary A simple optimization method based on the well-known Rs-minimum method and on the information theory of FUMI Φ is proposed. Resolution (Rs), peak area and height (or width) are the only parameters necessary for the calculation of the information Φ and information flow ϑ. The most precise analysis can be selected as the chromatogram having maximal ϑ. Mobile phase composition, column length, flow rate, detection wavelength, amount of internal standard, etc. can be optimized by this method.     

Natural products as templates for bioactive compound libraries. 3*. novel heterocycles and peptidomimetics generated from anabasine by isocyanide-based multicomponent reactions

Anabasine served as a starting material for the synthesis of two series of novel, drug-like compounds. On the one hand, anabasine was subjected to a direct Chichibabin amination and elaborated into medicinally relevant, racemic 6-(piperidin-2-yl)imidazo[1,2-a]pyridines via the Groebke–Blackburn reaction. On the other hand, anabasine was used as an amine for the Ugi reaction to provide novel non-racemic anabasine-containing 2-[(2-pyridin-3-yl)piperidin-1-yl]acetamide peptidomimetics with high diastereoselectivity.