Inhibition of norovirus 3CL protease by bisulfite adducts of transition state inhibitors

Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the US alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and α-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED₅₀ of the most effective inhibitor was 60 nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as α-ketoheterocycles and α-ketoesters.     

1,2-Benzisothiazol-3-one derivatives as a novel class of small-molecule caspase-3 inhibitors

A novel series of 1,2-benzisothiazol-3-one derivatives was synthesized and their biological activities were evaluated for inhibiting caspase-3 and -7 activities, in which some of them showed low nanomolar potency against caspase-3 in vitro and significant protection against apoptosis in a camptothecin-induced Jurkat T cells system. Among the tested compounds, compound 5i exhibited the most potent caspase-3 inhibitory activity (IC₅₀ = 1.15 nM). The molecular docking predicted the interactions and binding modes of the synthesized inhibitor in the caspase-3 active site.     

Design,synthesis and evaluation of 2,2-dimethyl-1,3-dioxolane derivatives as human rhinovirus 3C protease inhibitors

The human rhinovirus (HRV) is the most significant cause of the common cold all over the world. The maturation and replication of this virus entirely depend on the activity of a virus-encoded 3C protease. Due to the high conservation among different serotypes and the minimal homology existing between 3C protease and known mammalian enzymes, 3C protease has been regarded as an attractive target for the treatment of HRV infections. In this study, we identified a novel (4R,5R)-N⁴-(2-((3-methoxyphenyl)amino)ethyl)-2,2-dimethyl-N⁵-(naphthalen-2-yl)-1,3-dioxolane-4,5-dicarboxamide (7a) to be a HRV 3C protease inhibitor via virtual screening. Further research has been focused on the design, synthesis and in vitro biological evaluation of 7a derivatives. The studies revealed that compound 7d has an IC₅₀ value of 2.50 ± 0.7 µM against HRV 3C protease, and it thus could serve as a promising compound for the development of novel anti-rhinoviral medicines.     

Design,synthesis and characterization of novel 1,2-benzisothiazol-3(2H)-one and 1,3,4-oxadiazole hybrid derivatives: Potent inhibitors of Dengue and West Nile virus NS2B/NS3 proteases

1,2-Benzisothiazol-3(2H)-ones and 1,3,4-oxadiazoles individually have recently attracted considerable interest in drug discovery, including as antibacterial and antifungal agents. In this study, a series of functionalized 1,2-benzisothiazol-3(2H)-one—1,3,4-oxadiazole hybrid derivatives were synthesized and subsequently screened against Dengue and West Nile virus proteases. Ten out of twenty-four compounds showed greater than 50% inhibition against DENV2 and WNV proteases ([I] = 10 μM). The IC₅₀ values of compound 7n against DENV2 and WNV NS2B/NS3 were found to be 3.75 ± 0.06 and 4.22 ± 0.07 μM, respectively. The kinetics data support a competitive mode of inhibition by compound 7n. Molecular modeling studies were performed to delineate the putative binding mode of this series of compounds. This study reveals that the hybrid series arising from the linking of the two scaffolds provides a suitable platform for conducting a hit-to-lead optimization campaign via iterative structure–activity relationship studies, in vitro screening and X-ray crystallography.     

Functionalized hydroxyethylamine based peptide nanostructures as potential inhibitors of falcipain-3, an essential proteases of Plasmodium falciparum

Self-assembled peptide based nanostructures gained enough popularity due to their easy biocompatibility and numerous potential applications. An excellent model of self-assembly of hydroxyethylamine based peptide nanostructures was synthesized and characterized by DLS and TEM. Spherical nano structures of I and III were observed with particle size ～50 and ～80 nm, respectively. Further, I and III were screened against anti-malarial target, falcipain-3 (FP3), a crucial cysteine protease involved as a major hemoglobinase of Plasmodium falciparum. Interestingly, compound III completely inhibited the activity of FP3. The effective concentration (1.5 μM) of III found to be more potent than I. This biochemical result was substantiated by molecular-docking studies indicating III to be best inhibitor of FP3. This is the first report showing that bis hydroxethylamine based peptide nanostructures could be very effective inhibitor of malarial cysteine proteases.     

Asymmetric biocatalysis of S-3-amino-3-phenylpropionic acid with new isolated Methylobacterium Y1-6

β-amino acids are widely used in drug research, and S-3-amino-3-phenylpropionic acid (S-APA) is an important pharmaceutical intermediate of S-dapoxetine, which has been approved for the treatment of premature ejaculation. Chiral catalysis is an excellent method for the preparation of enantiopure compounds. In this study, we used (±)-ethyl-3-amino-3-phenylpropanoate (EAP) as the sole carbon source. Three hundred thirty one microorganisms were isolated from 30 soil samples, and 17 strains could produce S-APA. After three rounds of cultivation and identification, the strain Y1-6 exhibiting the highest enantioselective activity of S-APA was identified as Methylobacterium oryzae. The optimal medium composition contained methanol (2.5 g/L), 1,2-propanediol (7.5 g/L), soluble starch (2.5 g/L), and peptone (10 g/L); it was shaken at 220 rpm for 4–5 days at 30 °C. The optimum condition for biotransformation of EAP involved cultivation at 37 °C for 48 h with 120 mg of wet cells and 0.64 mg of EAP in 1 ml of transfer solution. Under this condition, substrate ee was 92.1% and yield was 48.6%. We then attempted to use Methylobacterium Y1-6 to catalyze the hydrolytic reaction with substrates containing 3-amino-3-phenyl-propanoate ester, N-substituted-β-ethyl-3-amino-3-phenyl-propanoate, and γ-lactam. It was found that 5 compounds with ester bonds could be stereoselectively hydrolyzed to S-acid, and 2 compounds with γ-lactam bonds could be stereoselectively hydrolyzed to (-)-γ-lactam.     

Synthesis,biological evaluation,and metabolic stability of acrylamide derivatives as novel CCR3 antagonists

Our laboratory has identified several acrylamide derivatives with potent CCR3 inhibitory activity. In the present study, we evaluated the in vitro metabolic stability (CL_int; mL/min/kg) of these compounds in human liver microsomes (HLMs), and assessed the relationship between their structures and CL_int values. Among the compounds identified, N-{(3R)-1-[(6-fluoro-2-naphthyl)methyl]pyrrolidin-3-yl}-2-[1-(2-hydroxybenzoyl)piperidin-4-ylidene]acetamide (30j) was found to be a potent inhibitor (IC₅₀ = 8.4 nM) with a high metabolic stability against HLMs.     

Cloning,over expression and functional attributes of serine proteases from Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12

Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A₁8 and Haloalkaliphilic bacterium O.M.E₁2 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6 h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50 °C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis.     

Cloning,heterologous expression and characterization of two keratinases from Stenotrophomonas maltophilia BBE11-1

The keratin-degrading strain Stenotrophomonas maltophilia BBE11-1 secretes two keratinolytic proteases, KerSMD and KerSMF. However, the genes encoding these proteases remain unknown. Here, we have isolated these two genes with a modified TAIL-PCR (thermal asymmetric interlaced PCR) method based on the N-terminal amino acid sequences of mature keratinases. These two keratinase genes encode serine proteases with PPC (bacterial pre-peptidase C-terminal) domain, which are successfully expressed with the help of pelB leader in Escherichia coli cells. Recombinant KerSMD (48 kDa) shows a better activity in feather degradation, higher thermostability and substrate specificity than KerSMF (40 kDa). KerSMD has a t_1/2 of 90 min at 50 °C and 64 min at 60 °C, and a better tolerance to surfactants SDS and triton X-100. The predicted model of KerSMD helps to explain the phenomenon of auto-catalytic C-terminal propeptide truncation, the special function of PPC domain, and the molecular weight of the C-terminal-processed mature keratinase KerSMD. This work not only provides a new way to overproduce keratinases but also helps to explore keratinases folding mechanism.     

Molecular modeling studies of atorvastatin analogues as HMGR inhibitors using 3D-QSAR,molecular docking and molecular dynamics simulations

3-Hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) is generally regarded as targets for the treatment of hypercholesterolemia. HMGR inhibitors (more commonly known as statins) are discovered as plasma cholesterol lowering molecules. In this work, 120 atorvastatin analogues were studied using a combination of molecular modeling techniques including three-dimensional quantitative structure–activity relationship (3D-QSAR), molecular docking and molecular dynamics (MD) simulation. The results show that the best CoMFA (comparative molecular field analysis) model has q² = 0.558 and r² = 0.977, and the best CoMSIA (comparative molecular similarity indices analysis) model has q² = 0.582 and r² = 0.919. Molecular docking and MD simulation explored the binding relationship of the ligand and the receptor protein. The calculation results indicated that the hydrophobic and electrostatic fields play key roles in QSAR model. After MD simulation, we found four vital residues (Lys735, Arg590, Asp690 and Asn686) and three hydrophobic regions in HMGR binding site. The calculation results show that atorvastatin analogues obtained by introduction of F atoms or gem-difluoro groups could obviously improve the inhibitory activity. The new HMGR inhibitor analogues design in this Letter had been submitted which is being currently synthesized by our laboratories.     

Potential role of cyanidin 3-glucoside (C3G) in diabetic cardiomyopathy in diabetic rats: An in vivo approach

The present study aimed to evaluate the importance of cyanidin 3-glucoside (C3G) of diabetic cardiomyopathy in diabetic rats. The rats were induced with diabetic using streptozotocin and total triglyceride (TG) and total cholesterol (TC) were determined. The range of myocardial enzymes such as aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LD) were also estimated, further, the Immuno histochemical analysis and western blot investigation were determined for the actual activity of C3G. Results indicated that the marker enzymes such as CK, LD and AST were significantly (P < 0.05) increased in STZ administered rats (DM group), while the levels of these elevated marker enzymes of cardiac injury significantly (P < 0.05) declined in the DM + C3G group, as compared to the diabetic group of rats. Additionally, a decrease in the level of TNF-alpha and interleukin-6, was noticed in the C3G treated group as compared to diabetic group. Finally, blotting analysis clearly confirmed that theC3G treatment resulted to higher level response of Bcl-2 and lower level response of caspase-3 and BAX. In conclusion, C3G a natural antioxidant may prevent cardiovascular complications by ameliorating oxidative damage, inflammation, metabolic dysfunctions and apoptosis pathways in type 2 diabetes.     

Structurally diverse low molecular weight activators of the mammalian pre-mRNA 3′ cleavage reaction

The 3′ end formation of mammalian pre-mRNA contributes to gene expression regulation by setting the downstream boundary of the 3′ untranslated region, which in many genes carries regulatory sequences. A large number of protein cleavage factors participate in this pre-mRNA processing step, but chemical tools to manipulate this process are lacking. Guided by a hypothesis that a PPM1 family phosphatase negatively regulates the 3′ cleavage reaction, we have found a variety of new small molecule activators of the in vitro reconstituted pre-mRNA 3′ cleavage reaction. New activators include a cyclic peptide PPM1D inhibitor, a dipeptide with modifications common to histone tails, abscisic acid and an improved l-arginine β-naphthylamide analog. The minimal concentration required for in vitro cleavage has been improved from 200 μM to the 200 nM–100 μM range. These compounds provide unexpected leads in the search for small molecule tools able to affect pre-mRNA 3′ end formation.     

Levels of serum pentraxin 3, IL-6, fetuin A and insulin in patients with rheumatoid arthritis

ObjectiveThe aim of this study was to investigate the relationship between disease severity and biochemical parameters such as pentraxin-3, fetuin-A, IL-6, insulin and HOMA-IR levels in patients with rheumatoid arthritis.MethodsThis study included 60 patients with RA and 20 healthy controls. Serum pentraxin-3, fetuin-A, IL-6 and insulin concentrations were measured. Also, HOMA-IR values were calculated. Disease activity was assessed with Disease Activity Score (DAS28). To evaluate quality of life, the Health Assessment Questionnaire disability index was applied.ResultsThe serum values for ESR, CRP, pentraxin-3 and fetuin-A in patients with RA were found to be higher than control subjects (p values = 0.001, 0.001, 0.000, 0.000, 0.01, 0.02, respectively). A positive correlation was evident between the DAS 28 score and IL6 levels (r = 0.263, p = 0.045). We found no correlation between the DAS28 score and HOMA-IR, the levels of pentraxin 3, fetuin A, insulin (p < 0.05). Fetuin A levels were positively correlated with cumulative steroid dose (r = 0.382, p = 0.035). A statistically significant correlation was evident between presence of cardiovascular disease and HOMA-IR values in RA patients (r = 0.437, p = 0.032).ConclusionElevated levels of pentraxin-3, fetuin-A, CRP, ESR might play a role in the pathogenesis of RA. Levels of fetuin-A, insulin HOMA-IR, pentraxin-3, CRP and ESR were not associated with clinical severity of the RA.     

Design,synthesis, and evaluation of 2-aryl-7-(3′,4′-dialkoxyphenyl)-pyrazolo[1,5-a]pyrimidines as novel PDE-4 inhibitors

Described herein is design, synthesis, and biological evaluation of novel series of 2-aryl-7-(3′,4′-dialkoxyphenyl)-pyrazolo[1,5-a]pyrimidines acting as inhibitors of type 4 phosphodiesterase (PDE4) which is known as a good target for the treatment of asthma and COPD. For this purpose, structure optimization was conducted with the aid of structure-based drug design using the known X-ray crystallography. Also, biological effects of these compounds on the target enzyme were evaluated by using in vitro assays, leading to the potent and selective PDE-4 inhibitor (IC₅₀ < 10 nM).     

A convenient synthesis and molecular modeling study of novel purine and pyrimidine derivatives as CDK2/cyclin A3 inhibitors

A series of novel purine and pyrimidine derivatives were prepared and biologically evaluated for their in vitro anti-CDK2/cyclin A3 and antitumor activities in Ehrlich ascites carcinoma (EAC) cell based assay. The novel purine derivatives 13a,b demonstrated potent inhibitor activities with IC₅₀ values of 14 ± 9 and 13 ± 9 μM, respectively. Additionally, compound 15a showed the highest potency (IC₅₀ = 10 ± 6 μM) in EAC cell based assay. Molecular modeling study, including fitting to a 3D-pharmacophore model and their docking into cyclin dependant kinase2 (CDK2) active site showed high fit values and docking scores.     

Rhodanine-based PRL-3 inhibitors blocked the migration and invasion of metastatic cancer cells

PRL-3, phosphatase of regenerating liver-3, plays a role in cancer progression through its involvement in invasion, migration, metastasis, and angiogenesis. We synthesized rhodanine derivatives, CG-707 and BR-1, which inhibited PRL-3 enzymatic activity with IC₅₀ values of 0.8 μM and 1.1 μM, respectively. CG-707 and BR-1 strongly inhibited the migration and invasion of PRL-3 overexpressing colon cancer cells without exhibiting cytotoxicity. The specificity of the inhibitors on PRL-3 phosphatase activity was confirmed by the phosphorylation recovery of known PRL-3 substrates such as ezrin and cytokeratin 8. The compounds selectively inhibited PRL-3 in comparison with other phosphatases, and CG-707 regulated epithelial-to-mesenchymal transition (EMT) marker proteins. The results of the present study reveal that rhodanine is a specific PRL-3 inhibitor and a good lead molecule for obtaining a selective PRL-3 inhibitor.     

The effect of limited hydrolysis with Neutrase and ultrafiltration on the anti-adipogenic activity of soy protein

Limited hydrolysis of isolated soy protein (ISP) with Neutrase for 4 h to obtain the hydrolysate (NH4h) revealed the ability to suppress glycerol-3-phosphate dehydrogenase (GPDH) activity and relative lipid accumulation (RLA) in 3T3-L1 cells during differentiation. Lower GPDH activity or RLA indicates higher anti-adipogenic activity. Sequentially fractionating NH4h with 30–1 kDa (kilo-daltons) molecular weight cut-off (MWCO) membranes to obtain the 1 kDa concentrate resulted in further enhancing its anti-adipogenic activity in the cells. The GPDH activity significantly decreased from 280 to 100 U/mg protein (p < 0.05). When comparing the high-performance size-exclusion chromatography (HPSEC) profiles, the most active peptide for the anti-adipogenic activity was primarily composed of peptides with molecular weight between 1300 and 2200 Da. The in vitro effect of gastrointestinal protease on the anti-adipogenic activity of 1 kDa concentrate was also investigated. The results suggested that gastrointestinal proteases have very little effect on anti-adipogenic activity of the concentrate. According to the Western immunoblot analysis, 1 kDa concentrate inhibits adipogenesis by affecting the expression of peroxisome proliferators-activated receptor γ (PPARγ) and the CCAAT/enhancer binding protein α (C/EBPα) during 3T3-L1 cells differentiation.     

Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli

An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37 °C, 100 mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1 M 3-cyanopyridine to nicotinic acid within 5 h at a cell mass concentration (fresh weight) of 500 mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use.     

Prolonged stability by cyclization: Macrocyclic phosphino dipeptide isostere inhibitors of β-secretase (BACE1)

Cyclization of recently reported linear phosphino dipeptide isostere inhibitors of BACE1 via side chain olefin metathesis yielded macrocyclic BACE1 inhibitors. The most potent compound II-P1 (IC₅₀ of 47 nM) and the corresponding linear analog I were tested for serum stability. The approach led to three times prolonged half life serum stability of 44 min for the macrocyclic inhibitor II-P1 compared to the linear compound I.     

Polymorphisms of the NOS3 gene and risk of myocardial infarction in the Tunisian population

Controversial results regarding the association of eNOS gene (NOS3) polymorphisms with myocardial infarction (MI) have been reported. This study investigated the relationship of the −786T>C (rs2070744), 894G>T (rs1799983) and 4a4b polymorphisms of the NOS3 gene with the presence of MI in the Tunisian population. In addition, we also examined the association of NOS3 gene haplotypes with MI in Tunisian subjects.A total of 303 patients with MI and 225 controls were included in the study. The 894G>T and −786T>C single nucleotide polymorphisms were analyzed by PCR-RFLP, and 4a4b polymorphism just for PCR.There was significant linkage disequilibrium between the three NOS3 polymorphisms (p < 0.0001). The genotype distribution and allele frequency of NOS3 4a4b, but not −786T>C and 894G>T, polymorphism was significantly different between MI patients and controls. The univariate logistic regression analysis showed a significant association of the 4a4b polymorphism and MI according to co-dominant, dominant and recessive models (co-dominant model OR: 4.38, 95%CI: 1.24–15.41; p = 0.021, dominant model OR: 1.66, 95%CI: 1.14–2.42); p = 0.007, and recessive model OR: 3.85, 95%CI: 1.10–13.47; p = 0.035). The multivariate analysis, adjusted for traditional cardiovascular risk factors, revealed that the NOS3 4a4a genotype was an independent predisposing factor to MI, according to the models considered. In addition, a haplotype 7 (C-T-4a), (OR = 12.05, p = 0.010) was a risk factor of MI after controlling for classical risk factors.These finding suggest that the 4a4b polymorphism of the NOS3 gene was associated with MI in Tunisian patients.