Hepatitis C RNA-dependent RNA polymerase inhibitors: a review of structure-activity and resistance relationships; different scaffolds and mutations

Hepatitis C virus (HCV), like many other flaviviruses, is widely distributed worldwide with estimated chronically infected victims between 170 and 200 million. HCV inherent error-prone RNA-dependent RNA polymerase (RdRp) is an attractive target for medicinal chemists because of the conservative nature of NS5B nucleotide-binding site. In addition, the availability of several crystal structures for HCV RdRp paved the road for conducting rational-based drug design. At the same time, RdRp is responsible for high mutation rate and rapid development of resistance to the clinically-used therapeutics. To improve the viral response, combination therapy is regularly used. The success of co-therapy disciplines depends on targeting two different active sites. This review provides an overview about different scaffolds that target HCV RdPp with insights about their binding modes and possible induced mutant strains.     

Calpain inhibitor I reduces the activation of nuclear factor-kappaB and organ injury/dysfunction in hemorrhagic shock.

There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF-kappaB (kidney), expression of iNOS and COX-2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF-kappaB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF-kappaB to DNA and the degradation of IkappaBalpha, IkappaBbeta, and IkappaBvarepsilon. Selective inhibition of iNOS activity with L-NIL reduced the circulatory failure and liver injury, while selective inhibition of COX-2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF-kappaB and thus prevention of the expression of NFkappaB-dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX-2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.     

Modelling viral and CD4 cellular population dynamics in HIV: approaches to evaluate intervention strategies.

Computational models, such as in epidemiology, provide a powerful tool that can be used to systematically examine an array of dynamic interactions among populations as well as to evaluate altemate disease intervention strategies. The specific objectives in this study were to: a/ examine the interaction of cellular (CD4) and HIV population dynamics and evaluate the impact of the use of combination chemotherapies on viral and CD4 populations (Experiment #1), b/ demonstrate how modelling can be used to evaluate the impact of an intervention (condom use) on reducing the rate of HIV/AIDS (Experiment #2). In this study, we used state transition models and conducted simulation experiments to evaluate various alternatives for the control and/or prevention of HIV/AIDS. The result indicated that combination therapy (double or triple drug therapies) was very effective. The HIV viral population decreased rapidly and remained suppressed for years. On the other hand, the CD4 cell population increased above 400 cells per ml and was maintained above that level for many years. Mono-therapy was not as effective; although the viral load decreased rapidly, it increased to its original levels within a few months. Since condom use is one of the key interventions of HIV/AIDS, we evaluated its use in 25%, 50% and 75% of an adult, sexually active population. Increasing condom use by 50% and 75% above an estimated baseline of 25% reduced the incidence of AIDS by 53% in Blacks, 49% in Hispanics and 43% in Whites. The study shows how a cellular/molecular level model can be incorporated within a macro-epidemiologic systems dynamics model to evaluate a variety of scientific questions such as to see if cellular/molecular level interventions reduce morbidity and mortality rates in HIV.     

Synthesis and activity against HBV of novel Tetra-seconucleoside analogues of dyphlline having the acyclic chains of ACV and HBG

Selective alkylation of dyphylline (1) with (2-acetoxyethoxy)methyl bromide (2a) or 4-acetoxybutyl bromide (2b) afforded 3'-O-[(acetoxyethoxy)methyl]dyphylline (3a) and 3'-O-(4-acetoxybutyl)-dyphylline (3b), respectively. A trans esterification process rather than alkylation of the dihydroxy-propyl side chain in 1 had taken place during the reaction with 2-p-toluoyloxy)ethyl chloride (5) to afford the respective 3'-toluoyloxy derivative 7 and not the anticipated 3'-O-[(p-toluoyloxy)ethyl]-dyphylline (6). Deacylation of 3a,b and 7 afforded 4a,b and 1, respectively. Viral screening of selected compounds against HBV has been investigated.     

Extracellular NAD induces a rise in [Ca]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.     

Audit of stool analysis results to ensure the prevalence of common types of intestinal parasites in Riyadh region,Saudi Arabia

The objective of the current study was to determine the incidence of common types of parasites encountered in the Central Region of Saudi Arabia. The current study is a retrospective study which includes the results of 10427 stool sample and occult blood sample. The results obtained during last two years (2005–2007), were compared to the earlier reports on parasites in the Central as well as other regions of Saudi Arabia. Attempts were made to find out the cases of increasing and/or decreasing trend of parasite incidence and to locate any differences between the current study results and the earlier reports.Key words: Intestinal parasites, Stool, Blood, Riyadh region, Saudi Arabia     

6H,13H-Pyrazino[1,2-a;4,5-a']diindole analogs: probing the pharmacophore for allosteric ligands of muscarinic M2 receptors

A series of 6H,13H-pyrazino[1,2-a;4,5-a']diindole analogs was synthesized in order to probe the pharmacophore hypothesis for allosteric ligands of muscarinic M(2) receptors. The 3D structure of the novel ring system was determined by means of NMR spectroscopy and X-ray diffraction revealing a totally flat geometry. Low binding affinities for the [(3)H]N-methylscopolamine-occupied M(2) receptors (reflected by EC(50,diss)) indicated that the spatial arrangement of the pharmacophore elements (two aromatic rings flanked by two cationic centers) incorporated in the bisquaternary analogs 5 and 6 is unfavorable for strong ligand-receptor interactions. Due to the structural similarity of the novel compounds to neuromuscular-blocking agents, their affinities (reflected by K(i)) to the muscle type of nicotinic acetylcholine receptors were also determined. The dimethyl and diallyl analogs 5 and 6 exhibited rather high affinities to the muscle type of nicotinic acetylcholine receptors, suggesting a pronounced neuromuscular-blocking activity. Compound 5 showed a 34-fold higher affinity for the muscle type nAChR than for the allosteric site of M(2) receptors.     

Type II Fatty Acid Synthesis Is Essential for the Replication of Chlamydia trachomatis

The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC₅₀ of 0.9 μm at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis.     

Development and validation of immortalized bovine mammary epithelial cell line as an in vitro model for the study of mammary gland functions

This study aimed to develop a bovine mammary epithelial (BME) cell line model, which provides a possibility to determine functional properties of the bovine mammary gland. The primary cell culture was derived from bovine mammary gland tissues and processed enzymatically to obtain cell colonies with epithelial-like morphology. The cultures of BME cells were purified and optimally cultured at 37 °C in DMEM/F12 medium supplemented with 10% fetal bovine serum. The BME cells were identified as epithelial cell line by the evaluating the expression of keratin-18 using immunofluorescence staining. A novel gene expression system strongly enhances the expression of telomerase, has been used to immortalize BME cell line termed hTBME cell line. Interestingly, telomerase remained active even after over 60 passages of hTBME cell line, required for immortalization of BME cells. In addition, the hTBME cell line was continuously subcultured with a spontaneous epithelial-like morphology, with a great proliferation activity, and without evidence of apoptotic and necrotic effects. Further characterization showed that hTBME cell line can be continuously propagated in culture with constant chromosomal features and without tumorigenic properties. Finally, established hTBME cell line was evaluated for mammary gland specific functions. Our results demonstrated that the hTBME cell line was able to retain functional-morphological structure, and functional differentiation by expression of beta (β)-casein as in the bovine mammary gland in vivo. Taken together, our findings suggest that the established hTBME cell line can serve as a valuable tool for the study of bovine mammary gland functions.     

Polarized Cell Division of Chlamydia trachomatis

Bacterial cell division predominantly occurs by a highly conserved process, termed binary fission, that requires the bacterial homologue of tubulin, FtsZ. Other mechanisms of bacterial cell division that are independent of FtsZ are rare. Although the obligate intracellular human pathogen Chlamydia trachomatis, the leading bacterial cause of sexually transmitted infections and trachoma, lacks FtsZ, it has been assumed to divide by binary fission. We show here that Chlamydia divides by a polarized cell division process similar to the budding process of a subset of the Planctomycetes that also lack FtsZ. Prior to cell division, the major outer-membrane protein of Chlamydia is restricted to one pole of the cell, and the nascent daughter cell emerges from this pole by an asymmetric expansion of the membrane. Components of the chlamydial cell division machinery accumulate at the site of polar growth prior to the initiation of asymmetric membrane expansion and inhibitors that disrupt the polarity of C. trachomatis prevent cell division. The polarized cell division of C. trachomatis is the result of the unipolar growth and FtsZ-independent fission of this coccoid organism. This mechanism of cell division has not been documented in other human bacterial pathogens suggesting the potential for developing Chlamydia-specific therapeutic treatments.