Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum

Nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) represents the parent compound of a novel class of broad-spectrum anti-parasitic compounds named thiazolides. NTZ is active against a wide variety of intestinal and tissue-dwelling helminths, protozoa, enteric bacteria and a number of viruses infecting animals and humans. While potent, this poses a problem in practice, since this obvious non-selectivity can lead to undesired side effects in both humans and animals. In this study, we used real time PCR to determine the in vitro activities of 29 different thiazolides (NTZ-derivatives), which carry distinct modifications on both the thiazole- and the benzene moieties, against the tachyzoite stage of the intracellular protozoan Neospora caninum. The goal was to identify a highly active compound lacking the undesirable nitro group, which would have a more specific applicability, such as in food animals. By applying self-organizing molecular field analysis (SOMFA), these data were used to develop a predictive model for future drug design. SOMFA performs self-alignment of the molecules, and takes into account the steric and electrostatic properties, in order to determine 3D-quantitative structure activity relationship models. The best model was obtained by overlay of the thiazole moieties. Plotting of predicted versus experimentally determined activity produced an r2 value of 0.8052 and cross-validation using the "leave one out" methodology resulted in a q2 value of 0.7987. A master grid map showed that large steric groups at the R2 position, the nitrogen of the amide bond and position Y could greatly reduce activity, and the presence of large steric groups placed at positions X, R4 and surrounding the oxygen atom of the amide bond, may increase the activity of thiazolides against Neospora caninum tachyzoites. The model obtained here will be an important predictive tool for future development of this important class of drugs.

Snake venom C-type lectins interacting with platelet receptors. Structure-function relationships and effects on haemostasis

Snake venoms contain components that affect the prey either by neurotoxic or haemorrhagic effects. The latter category affect haemostasis either by inhibiting or activating platelets or coagulation factors. They fall into several types based upon structure and mode of action. A major class is the snake C-type lectins or C-type lectin-like family which shows a typical folding like that in classic C-type lectins such as the selectins and mannose-binding proteins. Those in snake venoms are mostly based on a heterodimeric structure with two subunits alpha and beta, which are often oligomerized to form larger molecules. Simple heterodimeric members of this family have been shown to inhibit platelet functions by binding to GPIb but others activate platelets via the same receptor. Some that act via GPIb do so by inducing von Willebrand factor to bind to it. Another series of snake C-type lectins activate platelets by binding to GPVI while yet another series uses the integrin alpha(2)beta(1) to affect platelet function. The structure of more and more of these C-type lectins have now been, and are being, determined, often together with their ligands, casting light on binding sites and mechanisms. In addition, it is relatively easy to model the structure of the C-type lectins if the primary structure is known. These studies have shown that these proteins are quite a complex group, often with more than one platelet receptor as ligand and although superficially some appear to act as inhibitors, in fact most function by inducing thrombocytopenia by various routes. The relationship between structure and function in this group of venom proteins will be discussed.

Fabric-mechanical property relationships of trabecular bone allografts are altered by supercritical CO2 treatment and gamma sterilization

Tissue grafts are implanted in orthopedic surgery every day. In order to minimize infection risk, bone allografts are often delipidated with supercritical CO2 and sterilized prior to implantation. This treatment may, however, impair the mechanical behavior of the bone graft tissue. The goal of this study was to determine clinically relevant mechanical properties of treated/sterilized human trabecular bone grafts, e.g. the apparent modulus, strength, and the ability to absorb energy during compaction. They were compared with results of identical experiments performed previously on untreated/fresh frozen human trabecular bone from the same anatomical site (Charlebois, 2008). We tested the hypothesis that the morphology–mechanical property relationships of treated cancellous allografts are similar to those of fresh untreated bone. The morphology of the allografts was determined by μCT. Subsequently, cylindrical samples were tested in unconfined and confined compression. To account for various morphologies, the experimental data was fitted to phenomenological mechanical models for elasticity, strength, and dissipated energy density based on bone volume fraction (BV/TV) and the fabric tensor determined by MIL. The treatment/sterilization process does not appear to influence bone graft stiffness. However, strength and energy dissipation of the bone grafts were found to be significantly reduced by 36% to 47% and 66% to 81%, respectively, for a broad range of volume fraction (0.14 < BV/TV < 0.39) and degree of anisotropy (1.24 < DA < 2.18). Since the latter properties are strongly dominated by BV/TV, the clinical consequences of this reduction can be compensated by using grafts with lower porosity. The data of this study suggests that an increase of 5–10% in BV/TV is sufficient to compensate for the reduced post-yield mechanical properties of treated/sterilized bone in monotonic compression. In applications where graft stiffness needs to be matched and strength is not a concern, treated allograft with the same BV/TV as an appropriate fresh bone graft may be used.

Experimental validation of a nonlinear μ FE model based on cohesive-frictional plasticity for trabecular bone

Trabecular bone is a porous mineralized tissue playing a major load bearing role in the human body. Prediction of age-related and disease-related fractures and the behavior of bone implant systems needs a thorough understanding of its structure-mechanical property relationships, which can be obtained using microcomputed tomography-based finite element modeling. In this study, a nonlinear model for trabecular bone as a cohesive-frictional material was implemented in a large-scale computational framework and validated by comparison of μFE simulations with experimental tests in uniaxial tension and compression. A good correspondence of stiffness and yield points between simulations and experiments was found for a wide range of bone volume fraction and degree of anisotropy in both tension and compression using a non-calibrated, average set of material parameters. These results demonstrate the ability of the model to capture the effects leading to failure of bone for three anatomical sites and several donors, which may be used to determine the apparent behavior of trabecular bone and its evolution with age, disease, and treatment in the future.

Novel octreotide dicarba-analogues with high affinity and different selectivity for somatostatin receptors

A limited set of novel octreotide dicarba-analogues with non-native aromatic side chains in positions 7 and/or 10 were synthesized. Their affinity toward the ssts1-5 was determined. Derivative 4 exhibited a pan-somatostatin activity, except sst4, and derivative 8 exhibited high affinity and selectivity toward sst5. Actually, compound 8 has similar sst5 affinity (IC50 4.9 nM) to SRIF-28 and octreotide. Structure-activity relationships suggest that the Z geometry of the double-bond bridge is that preferred by the receptors. The NMR study on the conformations of these compounds in SDS(-d25) micelles solution shows that all these analogues have the pharmacophore beta-turn spanning Xaa7-D-Trp8-Lys9-Yaa10 residues. Notably, the correlation between conformation families and affinity data strongly indicates that the sst5 selectivity is favored by a helical conformation involving the C-terminus triad, while a pan-SRIF mimic activity is based mainly on a conformational equilibrium between extended and folded conformational states.

Protein and lipid binding parameters in rainbow trout (Oncorhynchus mykiss) blood and liver fractions to extrapolate from an in vitro metabolic degradation assay to in vivo bioaccumulation potential of hydrophobic organic chemicals

Binding of hydrophobic chemicals to colloids such as proteins or lipids is difficult to measure using classical microdialysis methods due to low aqueous concentrations, adsorption to dialysis membranes and test vessels, and slow kinetics of equilibration. Here, we employed a three-phase partitioning system where silicone (polydimethylsiloxane, PDMS) serves as a third phase to determine partitioning between water and colloids and acts at the same time as a dosing device for hydrophobic chemicals. The applicability of this method was demonstrated with bovine serum albumin (BSA). Measured binding constants (K(BSAw)) for chlorpyrifos, methoxychlor, nonylphenol, and pyrene were in good agreement with an established quantitative structure-activity relationship (QSAR). A fifth compound, fluoxypyr-methyl-heptyl ester, was excluded from the analysis because of apparent abiotic degradation. The PDMS depletion method was then used to determine partition coefficients for test chemicals in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (K(S9w)) and blood plasma (K(bloodw)). Measured K(S9w) and K(bloodw) values were consistent with predictions obtained using a mass-balance model that employs the octanol-water partition coefficient (K(ow)) as a surrogate for lipid partitioning and K(BSAw) to represent protein binding. For each compound, K(bloodw) was substantially greater than K(S9w), primarily because blood contains more lipid than liver S9 fractions (1.84% of wet weight vs 0.051%). Measured liver S9 and blood plasma binding parameters were subsequently implemented in an in vitro to in vivo extrapolation model to link the in vitro liver S9 metabolic degradation assay to in vivo metabolism in fish. Apparent volumes of distribution (V(d)) calculated from the experimental data were similar to literature estimates. However, the calculated binding ratios (f(u)) used to relate in vitro metabolic clearance to clearance by the intact liver were 10 to 100 times lower than values used in previous modeling efforts. Bioconcentration factors (BCF) predicted using the experimental binding data were substantially higher than the predicted values obtained in earlier studies and correlated poorly with measured BCF values in fish. One possible explanation for this finding is that chemicals bound to proteins can desorb rapidly and thus contribute to metabolic turnover of the chemicals. This hypothesis remains to be investigated in future studies, ideally with chemicals of higher hydrophobicity.

Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein

OBJECTIVE: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17alpha-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein. AIM AND METHODS: To better understand structure-function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template. RESULTS: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor. CONCLUSION: Our model will help in analyzing the genotype-phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.

Quantitative depth profiling of Ce 3+ in Pt/CeO 2 by in situ high-energy XPS in a hydrogen atmosphere

The redox property of ceria is a key factor in the catalytic activity of ceria-based catalysts. The oxidation state of well-defined ceria nanocubes in gas environments was analysed in situ by a novel combination of near-ambient pressure X-ray Photoelectron Spectroscopy (XPS) and high-energy XPS at a synchrotron X-ray source. In situ high-energy XPS is a promising new tool to determine the electronic structure of matter under defined conditions. The aim was to quantitatively determine the degree of cerium reduction in a nano-structured ceria-supported platinum catalyst as a function of the gas environment. To obtain a non-destructive depth profile at near-ambient pressure, in situ high-energy XPS analysis was performed by varying the kinetic energy of photoelectrons from 1 to 5 keV, and, thus, the probing depth. In ceria nanocubes doped with platinum, oxygen vacancies formed only in the uppermost layers of ceria in an atmosphere of 1 mbar hydrogen and 403 K. For pristine ceria nanocubes, no change in the cerium oxidation state in various hydrogen or oxygen atmospheres was observed as a function of probing depth. In the absence of platinum, hydrogen does not dissociate and, thus, does not lead to reduction of ceria.