Uroš Urleb

Novel Naphthalene-N-Sulfonyl-D-Glutamic Acid Derivatives as Inhibitors of Murd, a Key Peptidoglycan Biosynthesis Enzyme.

Mur ligases have essential roles in the biosynthesis of peptidoglycan, and they represent attractive targets for the design of novel antibacterials. MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase) is the second enzyme in the series of Mur ligases, and it catalyzes the addition of D-glutamic acid (D-Glu) to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA). Because of the high binding affinity of D-Glu toward MurD, we synthesized and biochemically evaluated a series of N-substituted D-Glu derivatives as potential inhibitors of MurD from E. coli, which allowed us to explore the structure-activity relationships.The substituted naphthalene-N-sulfonyl-D-Glu inhibitors, which were synthesized as potential transition state analogues, displayed IC50 values ranging from 80 to 600 microM. In addition, the high-resolution crystal structures of MurD in complex with four novel inhibitors revealed details of the binding mode of the inhibitors within the active site of MurD. Structure-activity relationships and cocrystal structures constitute an excellent starting point for further development of novel MurD inhibitors of this structural class.

Investigation of felodipine polymorphism and its glassy state

Abstract Felodipine (ethyl methyl 4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate) was investigated by means of DSC, FTIR and X-ray diffraction and three different polymorphic forms, designated as I–III, respectively, were found. During heating, no interconversion of any polymorphic form took place. After cooling the felodipine melt (in liquid N 2 or at ambient temperature) the glassy state was formed. The existence of glassy felodipine was confirmed by spin-lattice ( T 1 ) and spin-spin ( T 2 ) relaxation time measurements on a Bruker Pulse NMR spectrometer. During the heating of glassy felodipine recrystallization took place and two additional polymorphic forms, referred to as Im and IIIm, were observed.

In Vitro Activity of LK-157, a Novel Tricyclic Carbapenem As Broad-Spectrum β-Lactamase Inhibitor

ABSTRACT LK-157 is a novel tricyclic carbapenem with potent activity against class A and class C β-lactamases. When tested against the purified TEM-1 and SHV-1 enzymes, LK-157 exhibited 50% inhibitory concentrations (IC50s) in the ranges of the clavulanic acid and tazobactam IC50s (55 nM and 151 nM, respectively). Moreover, LK-157 significantly inhibited AmpC β-lactamase (IC50, 62 nM), as LK-157 was >2,000-fold more potent than clavulanic acid and approximately 28-fold more active than tazobactam. The in vitro activities of LK-157 in combination with amoxicillin, piperacillin, ceftazidime, cefotaxime, ceftriaxone, cefepime, cefpirome, and aztreonam against an array of Ambler class A (TEM-, SHV-, CTX-M-, KPC-, PER-, BRO-, and PC-type)- and class C-producing bacterial strains derived from clinical settings were evaluated in synergism experiments and compared with those of clavulanic acid, tazobactam, and sulbactam. In vitro MICs against ESBL-producing strains (except CTX-M-containing strains) were reduced 2- to >256-fold, and those against AmpC-producing strains were reduced even up to >32-fold. The lowest MICs (≤0.025 to 1.6 μg/ml) were observed for the combination of cefepime and cefpirome with a constant LK-157 concentration of 4 μg/ml, thus raising an interest for further development. LK-157 proved to be a potent β-lactamase inhibitor, combining activity against class A and class C β-lactamases, which is an absolute necessity for use in the clinical setting due to the worldwide increasing prevalence of bacterial strains resistant to β-lactam antibiotics.

Internucleosomal DNA cleavage in apoptotic WEHI 231 cells is mediated by a chymotrypsin-like protease.

Although several lines of evidence support a role for serine proteases in apoptosis, little is known about the mechanisms involved. In the present study, we have examined the apoptosis‐inducing potential and dissected the death‐signalling pathways of N‐tosyl‐L‐phenylalanine chloromethyl ketone (TPCK) and N‐tosyl‐L‐lysine chloromethyl ketone (TLCK), inhibitors of chymotrypsin‐ and trypsin‐like proteases, respectively. Our results designate two distinct roles for serine proteases. Firstly, we show that both inhibitors induce biochemical and morphological characteristics of apoptosis, including proteolysis of poly(ADP‐ribose) polymerase 1 (PARP‐1) and inhibitor of caspase‐activated DNase (ICAD), as well as mitochondrial dysfunction, and that their action is abrogated by the caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp.fluoromethylketone (z‐VAD.fmk). These results suggest that inhibition of anti‐apoptotic serine proteases governs the onset of the caspase‐dependant apoptotic cascade. Secondly, we also demonstrate the involvement of a serine protease in the terminal stage of apoptosis. We showed that chymotrypsin‐like protease activity is required for internucleosomal DNA fragmentation in apoptotic cells. Hence, DNA fragmentation is abrogated in TPCK‐pre‐treated WEHI 231 cells undergoing apoptosis triggered either by anti‐IgM or TLCK. These results indicate that internucleosomal DNA cleavage in apoptotic cells is mediated by a chymotrypsin‐like protease.

Qualitative and quantitative analysis of oxytetracycline by near-infrared spectroscopy

Near-infrared (NIR) spectroscopy, in combination with chemometrics, enable the analysis of raw materials without time-consuming sample preparation methods. The aim of our work was to estimate critical parameters in the analytical specification of oxytetracycline, and consequently the development of a method for quantification and qualification of these parameters by NIR spectroscopy. A Karl Fischer (K.F.) titration to determine the water content, a colorimetric assay method, and Fourier transform-infrared (FT-IR) spectroscopy to identify the oxytetracycline base, were used as reference methods, respectively. Multivariate calibration was performed on NIR spectral data using principal component analysis (PCA), partial least-squares (PLS 1) and principal component regression (PCR) chemometric methods. Multivariate calibration models for NIR spectroscopy have been developed. Using PCA and the Soft Independent Modelling of Class Analogy (SIMCA) approach, we established the cluster model for the determination of sample identity. PLS 1 and PCR regression methods were applied to develop the calibration models for the determination of water content and the assay of the oxytetracycline base. Comparing the PLS and PCR regression methods we found out that the PLS is better established by NIR, especially as the spectroscopic data (NIR spectra) are highly collinear and there are many wavelengths due to non-selective wavelengths. The calibration models for NIR spectroscopy are convenient alternatives to the colorimetric method and to the K.F. method, as well as to FT-IR spectroscopy, in the routine control of incoming material.

Comparison of bacterial and phage display peptide libraries in search of target-binding motif

Genetic engineering allows modification of bacterial and bacteriophage genes, which code for surface proteins, enabling display of random peptides on the surface of these microbial vectors. Biologic peptide libraries thus formed are used for high-throughput screening of clones bearing peptides with high affinity for target proteins. There are reports of many successful affinity selections performed with phage display libraries and substantially fewer cases describing the use of bacterial display systems. In theory, bacterial display has some advantages over phage display, but the two systems have never been experimentally compared. We tested both techniques in selecting streptavidin-binding peptides from two commercially available libraries. Under similar conditions, selection of phage-displayed peptides to model protein streptavidin proved convincingly better.

The influence of magnesium stearate on the characteristics of mucoadhesive microspheres.

Microspheres containing the mucoadhesive polymer chitosan hydrochloride, with matrix polymer Eudragit RS, pipemidic acid as a model drug and agglomeration preventing agent magnesium stearate were prepared by the solvent evaporation method. The amount of magnesium stearate was varied and the following methods were used for microsphere evaluation: sieve analysis, drug content and dissolution determination, scanning electron microscopy, xray diffractometry, DSC and FTIR spectroscopy. The results showed that average particle size decreased with increasing amount of magnesium stearate used for microsphere preparation. This is probably a consequence of stabilization of the emulsion droplets with magnesium stearate. Higher pipemidic acid content in the microspheres was observed in larger particle size fractions and when higher amounts of magnesium stearate were used. It was also found that these two parameters significantly influenced the dissolution rate. The important reason for the differences in drug content in microspheres of different particle sizes is the diffusion of pipemidic acid from the acetone droplets in liquid paraffin during the preparation procedure. The physical state of pipemidic acid changed from crystalline to mostly amorphous with its incorporation in microspheres, as shown by x-ray diffractometry and differentialMicrospheres containing the mucoadhesive polymer chitosan hydrochloride, with matrix polymer Eudragit RS, pipemidic acid as a model drug and agglomeration preventing agent magnesium stearate were prepared by the solvent evaporation method. The amount of magnesium stearate was varied and the following methods were used for microsphere evaluation: sieve analysis, drug content and dissolution determination, scanning electron microscopy, x-ray diffractometry, DSC and FTIR spectroscopy. The results showed that average particle size decreased with increasing amount of magnesium stearate used for microsphere preparation. This is probably a consequence of stabilization of the emulsion droplets with magnesium stearate. Higher pipemidic acid content in the microspheres was observed in larger particle size fractions and when higher amounts of magnesium stearate were used. It was also found that these two parameters significantly influenced the dissolution rate. The important reason for the differences in drug content in microspheres of different particle sizes is the diffusion of pipemidic acid from the acetone droplets in liquid paraffin during the preparation procedure. The physical state of pipemidic acid changed from crystalline to mostly amorphous with its incorporation in microspheres, as shown by x-ray diffractometry and differential scanning calorimetry. No differences were observed in the physical state of pipemidic acid and in microsphere shape and surface between different size fractions of microspheres, prepared with different amounts of magnesium stearate. Additionally, no correlation between the physical state of the drug in different microspheres and their biopharmaceutical properties was found.

Recent Advances in Serine Protease Inhibitors as Anticoagulant Agents

The drawbacks and limitations of existing anticoagulant therapy which may result in serious adverse effects and a high mortality rate, have given rise to many anticoagulant development programmes in the last decade, focusing mainly at development of thrombin and FXa low-molecular weight inhibitors. A detailed understanding of blood coagulation pathways, functioning of the serine proteases thrombin, FXa, FVIIa and FIXa and elucidation of their crystal structures resulted in many potent compounds, among which some have entered the clinical phase or have been approved for use in clinical practice. Recently, the focus of anticoagulant research turned to inhibition of the TF:FVIIa complex, with some promising clinical candidates on the horizon. This article provides an overview of the current development status of serine protease inhibitors as anticoagulants, including new trends such as dual coagulation factor inhibitors.

Ultrasound in phage display: a new approach to nonspecific elution.

Libraries of phage-displayed random peptides are routinely used to identify target-binding peptides. Phages are commonly eluted in a nonspecific manner, especially if there are no available ligands of the particular target to use as competitors. However, the present study clearly demonstrates that nonspecific elution is not always able to break peptide-target interactions. To circumvent this we have developed an improved nonspecific elution strategy that uses ultrasound to release target-bound phages and enables selection of high-affinity clones in a single step.

Peptide inhibitors of MurD and MurE, essential enzymes of bacterial cell wall biosynthesis.

Continuous development of antibacterial compounds with novel modes of action (accompanied by rationalization of chemotherapeutic prescription) is the best way to address the growing problem of antibiotic-resistant infections. Numerous clinically important antibiotics interfere with peptidoglycan cell wall biosynthesis making this unique metabolic pathway a well validated target for antimicrobials. While nearly all of these antibiotics inhibit late stages of murein synthesis occurring on the extracellular side of plasma membrane, initial cytoplasmic steps have not been extensively exploited as drug targets. We performed affinity selection of peptides from phage-displayed libraries against two essential bacterial enzymes MurD and MurE involved in the cytoplasmic synthesis of peptidoglycan monomer. Selected peptides were found to inhibit respective target enzymes in an in vitro assay with IC(50) values of 140 microM to 1.5 mM. These peptides represent starting point for design of peptidomimetic lead compounds with the ultimate objective of small molecule chemotherapeutic development.