2-(Methyl(phenyl)amino)-N-(phenyloxyphenyl)acetamide structural motif representing a framework for selective SIRT2 inhibition.

The mammalian cytoplasmic protein SIRT2, a class III histone deacetylase family member, possesses NAD+-dependent lysine deacetylase/deacylase activity. Dysregulation of SIRT2 has been implicated in the pathogenesis of several diseases, including neurological and metabolic disorders and cancer; thus, SIRT2 emerges as a potential therapeutic target. Herein, we identified a series of diaryl acetamides (ST61-ST90) by the structural optimization of our hit STH2, followed by enhanced SIRT2 inhibitory potency and selectivity. Among them, ST72, ST85, and ST88 selectively inhibited SIRT2 with IC50 values of 9.97, 5.74, and 8.92 µM, respectively. Finally, the entire study was accompanied by in silico prediction of binding modes of docked compounds and the stability of SIRT2-ligand complexes. We hope our findings will provide substantial information for designing selective inhibitors of SIRT2.

In silico approach reveals N-(5-phenoxythiophen-2-yl)-2-(arylthio)acetamides as promising selective SIRT2 inhibitors: the case of structural optimization of virtual screening-derived hits.

Epigenetic modifications play an essential role in tumor suppression and promotion. Among the diverse range of epigenetic regulators, SIRT2, a member of NAD+-dependent protein deacetylates, has emerged as a crucial regulator of cellular processes, including cell cycle progression, DNA repair, and metabolism, impacting tumor growth and survival. In the present work, a series of N-(5-phenoxythiophen-2-yl)-2-(arylthio)acetamide derivatives were identified following a structural optimization of previously reported virtual screening hits, accompanied by enhanced SIRT2 inhibitory potency. Among the compounds, ST44 and ST45 selectively inhibited SIRT2 with IC50 values of 6.50 and 7.24 µM, respectively. The predicted binding modes of the two compounds revealed the success of the optimization run. Moreover, ST44 displayed antiproliferative effects on the MCF-7 human breast cancer cell line. Further, the contribution of SIRT2 inhibition in this effect of ST44 was supported by western blotting, affording an increased α-tubulin acetylation. Furthermore, molecular dynamics (MD) simulations and binding free energy calculations using molecular mechanics/generalized born surface area (MM-GBSA) method evaluated the accuracy of predicted binding poses and ligand affinities. The results revealed that ST44 exhibited a remarkable level of stability, with minimal deviations from its initial docking conformation. These findings represented a significant improvement over the virtual screening hits and may contribute substantially to our knowledge for further selective SIRT2 drug discovery.Communicated by Ramaswamy H. Sarma.

Hit evaluation results in 5-benzyl-1,3,4-thiadiazole-2-carboxamide based SIRT2-selective inhibitor with improved affinity and selectivity.

Sirtuin 2 (SIRT2), member of sirtuin family, belongs to class III histone deacetylases (HDACs) and is majorly cytosolic with occasional nuclear translocation. The enzymatic activity of SIRT2 is dependent on nicotinamide adenine dinucleotide (NAD+) and SIRT2 regulates post-translational modifications that are responsible for deacetylation of lysine residues in histone and non-histone substrates. SIRT2, thus affects most likely multiple cellular processes, such as signaling, gene expression, aging, autophagy, and has been identified as potential drug target in relation to inflammation, neurodegenerative diseases and cancer. Therefore, probing potential selective inhibitors is essential for the accurate understanding of enzyme functions. Here, we report a series of heteroaryl-2-carboxamide hybrids bearing substituted benzyl or substituted phenoxy group at the 5-position of the central heterocyclic ring. The synthesized compounds were screened against SIRT1-3 and MCF-7 human breast cancer cell line to evaluate their biological activity. The best SIRT2 inhibition profiles were displayed by ST29 (SIRT2 IC50 = 38.69 µM) and ST30 (SIRT2 IC50 = 43.29 µM) with excellent selectivity against SIRT2 over SIRT1 and SIRT3. Molecular docking study of the synthesized compounds into SIRT2 active site was performed to rationalize the remarkable SIRT2 inhibitory activity. Furthermore, we performed all-atom, explicit-solvent molecular dynamics (MD) simulations and end-point binding free energy calculations using molecular mechanics/generalized Born surface area (MM/GBSA) method to evaluate whether this design strategy was successfully deployed. The results implied that the binding poses and ligand affinities were predicted without significant loss of accuracy. Conclusively, the developed chemotypes were advocated as promising leads for SIRT2 inhibition and required further investigation for SIRT2-targeted drug discovery and development.

Indoleamine-2,3-dioxygenase-related anti-inflammatory effects of 3-aminobenzamide and infliximab in experimental colitis

SUMMARY OBJECTIVE: This study aimed to investigate the presence of indoleamine-2,3-dioxygenase and bacterial translocation after the administration of 3-aminobenzamide and infliximab in the TNBS model of rat colitis. METHODS: The study group was divided into five categories as follows: group 1: (control), group 2: colitis+saline, group 3: colitis+3-aminobenzamide, group 4: colitis+infliximab, and group 5: colitis+3-aminobenzamide+infliximab. Intestinal mesenteric cultures were incubated on specific agar media plates under aerobic and anaerobic conditions, bacterial translocation was evaluated and assessed as colony-forming units per gram of tissue. Colonic tissue samples were evaluated by Western blotting method to detect the presence of indoleamine-2,3-dioxygenase. RESULTS: The results obtained were as follows: group 1: normal gut flora; group 2: eight of nine samples had bacterial translocation, of which six of them had positive indoleamine-2,3-dioxygenase protein; group 3: five of nine samples had bacterial translocation, of which seven of them had positive indoleamine-2,3-dioxygenase; group 4: three of nine samples had bacterial translocation, of which seven of them had positive indoleamine-2,3-dioxygenase; and group 5: only one sample had exact indoleamine-2,3-dioxygenase protein. CONCLUSION: Altered expression of indoleamine-2,3-dioxygenase results in a lower bacterial translocation via infliximab compared with 3-aminobenzamide treatment. Combined treatments emphasized different approaches for the new molecules related to indoleamine-2,3-dioxygenase.

Hit-to-lead optimization on aryloxybenzamide derivative virtual screening hit against SIRT.

Sirtuins (SIRTs) are a class of nicotinamide adenine dinucleotide (NAD+)-dependent protein histone deacetylases (HDACs) that are evolutionarily conserved from bacteria to mammals. This group of enzymes catalyses the reversible deacetylation of lysine residues in the histones or non-histone substrates using NAD+ as a cosubstrate. Numerous studies have demonstrated that the aberrant enzymatic activity of SIRTs has been linked to various diseases like diabetes, cancer, and neurodegenerative disorders. Previously, we performed a pharmacophore-based virtual screening campaign and an aryloxybenzamide derivative (1) displaying SIRT1/2 inhibitory effect was identified as a hit compound. In the current study, the hit-to-lead optimization on the hit compound was explored in order to improve the SIRT binding and inhibition. Fourteen compounds, ten of which were new, have been synthesized and subjected to in vitro biological evaluation for their inhibitory activity against SIRT1-3. By the structural modifications performed, a significant improvement was observed in selective SIRT1 inhibition for ST01, ST02, and ST11 compared to that of the hit compound. The highest SIRT2 inhibitory activity was observed for ST14, which was designed according to compatibility with pharmacophore model developed for SIRT2 inhibitors and thus, providing the interactions required with key residues in SIRT2 active site. Furthermore, ST01, ST02, ST11, and ST14 were subjected to in vitro cytotoxicity assay against MCF-7 human breast cancer cell line to determine the influence of the improvement in SIRT1/2 inhibition along with the structural modifications on the cytotoxic properties of the compounds. The cytotoxicity of the compounds was found to be correlated with their SIRT inhibitory profiles indicating the effects of SIRT1/2 inhibition on cancer cell viability. Overall, this study provides structural insights for further inhibitor improvement.

Identification of small-molecule urea derivatives as novel NAMPT inhibitors via pharmacophore-based virtual screening.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the condensation of nicotinamide (NAM) with 5-phosphoribosyl-1-prophosphate (PRPP) to yield nicotinamide mononucleotide (NMN), a rate limiting enzyme in a mammalian salvage pathway of nicotinamide adenine dinucleotide (NAD+) synthesis. Recently, intracellular NAD+ has received substantial attention due to the recent discovery that several enzymes including poly(ADP-ribose) polymerases (PARPs), mono(ADP-ribose) transferases (ARTs), and sirtuins (SIRTs), use NAD+ as a substrate, suggesting that intracellular NAD+ level may regulate cytokine production, metabolism, and aging through these enzymes. NAMPT is found to be upregulated in various types of cancer, and given its importance in the NAD+ salvage pathway, NAMPT is considered as an attractive target for the development of new cancer therapies. In this study, the reported NAMPT inhibitors bearing amide, cyanoguanidine, and urea scaffolds were used to generate pharmacophore models and pharmacophore-based virtual screening studies were performed against ZINC database. Following the filtering steps, ten hits were identified and evaluated for their in vitro NAMPT inhibitory effects. Compounds GF4 (NAMPT IC50 = 2.15 ± 0.22 µM) and GF8 (NAMPT IC50 = 7.31 ± 1.59 µM) were identified as new urea-typed inhibitors of NAMPT which also displayed cytotoxic activities against human HepG2 hepatocellular carcinoma cell line with IC50 values of 15.20 ± 1.28 and 24.28 ± 6.74 µM, respectively.

Microwave-assisted synthesis and pharmacological screening of some triazolothiadiazole derivatives

In this study, twenty-two new [1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles (5a-n, 6a-h) were synthesized under microwave irradiation (MWI). The chemical structures of the compounds were elucidated by their IR, 1H-NMR, LC-MS, and elemental analysis. The compounds were tested for antinociceptive activity by using the tail clip, tail flick, hot plate, and writhing methods in mice. The varying levels of antinociceptive activity of the compounds were compared with those of aspirin. Among these compounds, compound 5g and 5j were found to be significantly more active than the other compounds and the standard in the tests. Also, inhibitory effects of the test compounds on COX-1 and COX-2 activities were investigated. DuP-697 for COX-2 and SC-560 for COX-1 were used as reference standards.

Pharmacophore modeling and virtual screening studies to identify novel selective SIRT2 inhibitors.

Sirtuins (SIRTs) are a class of NAD+-dependent protein histone deacetylases (HDACs) that catalyse the reversible deacetylation of lysine residues in the histones or non-histone substrates. Mammalian sirtuins consist of seven isoforms (SIRT1-7), which show different subcellular localizations and enzymatic functions. Among the seven human sirtuins, SIRT2 predominantly located in the cytoplasm but is enriched in the nucleus during mitosis. Its activity has been found to be modulate the pathophysiology of various diseases such as cancer, metabolic and neurodegenerative disorders. Therefore, selective SIRT2 inhibitors are of growing interest as potentially candidate therapeutic agents to treat SIRT2-driven pathologies as well as valuable tools to investigate and define the biological roles of SIRT2. Herein, in order to identify potent leads against SIRT2, a multi-step pharmacophore based-virtual screening campaign was performed and 31 predicted compounds were subjected to in vitro biological evaluation. Finally, compound 2 and 3 showing better SIRT2 inhibition potency were selected for further in vitro cytotoxic assays against a panel of three human cancer cell lines. This study will hopefully provide a basis for developing potent and selective SIRT2 inhibitors.

Design, synthesis and biological evaluation of novel 1,3-diarylpyrazoles as cyclooxygenase inhibitors, antiplatelet and anticancer agents.

With the aim of achieving new compounds possessing both anti-inflammatory and antiplatelet activities, we synthesized (E)-3-[3-(pyridin-3/4-yl)-1-(phenyl/sulfonylmethylphenyl)-1H-pyrazol-4-yl]acrylamides, and evaluated their COX-1 and COX-2 inhibitory and antiplatelet activities. Since COX-2 inhibitory and antiplatelet compounds have anticancer potential, we also screened their antiproliferative effects against three human cancer cell lines. Compounds 5n, 5p, 5s, 10d, 10g and 10i were determined as dual COX-2 inhibitor/antiplatelet compounds. Compound 10h appeared to be a compound that exhibited antiplatelet activity without inhibiting the COX enzyme. Compounds 5h, 10a and 10i were the most effective derivatives which displayed antiproliferative activity against Huh7, MCF7 and HCT116 cells. Particularly, compound 10i, as the compound exhibiting the highest cytotoxic, antiplatelet and COX-2 inhibitory activity, was remarkable.

Tryptophan degradation and neopterin levels in treated rheumatoid arthritis patients.

Increased kynurenine/tryptophan-reflects trytophan degradation-and neopterin levels have been regarded as a biochemical marker of cell-mediated immune response and inflammation. This study was designed to evaluate the usefulness of tryptophan degradation and neopterin levels in active rheumatoid arthritis patients under therapy. In this case-control study, kynurenine and tryptophan levels were determined by HPLC; neopterin and tumor necrosis factor-α levels were measured with ELISA in 32 active rheumatoid arthritis patients and 20 healthy controls. Although mean values of tryptophan, kynurenine, ratio of kynurenine to tryptophan, neopterin, and tumor necrosis factor-α levels did not show statistically significant differences between patient and control groups, neopterin levels correlated positively with kynurenine (r = 0.582, p < 0.02), kynurenine/tryptophan (r = 0.486, p < 0.05), erythrocyte sedimentation rate (r = 0.472, p < 0.05) and RF (r = 0.478, p < 0.05) in the rheumatoid arthritis group. CRP levels of the patient group correlated with kynurenine levels (r = 0.524, p < 0.03). Determination of tryptophan degradation and neopterin levels in chronic inflammatory disease may provide a better understanding of progression of the disease.

Variability of total thiol compounds, oxidative and nitrosative stress in uncomplicated pregnant women and nonpregnant women.

PURPOSE: The objective of this study was to determine the changes in total plasma thiols (homocysteine, cysteine and cysteinylglycine), lipid peroxidation and nitric oxide concentrations during normal pregnancy. METHODS: These variables were measured in 28 uncomplicated pregnant women at first, second and third trimesters and in 19 nonpregnant women. RESULTS: The mean concentrations of homocysteine, cysteine and cysteinylglycine were significantly lower in all trimesters of pregnancy compared with nonpregnant controls. There was significant elevation in serum lipid peroxidation levels of pregnant women within first and third trimesters compared with nonpregnant women. In spite of increase in mean nitric oxide levels in pregnant women, this increase did not reach statistically significant levels. CONCLUSION: This study provides information about the changes in plasma levels of many variables having important role in pregnancy complication during all trimesters in uncomplicated pregnancy compared with nonpregnant women.

Usefulness of homocysteine as a cancer marker: total thiol compounds and folate levels in untreated lung cancer patients.

BACKGROUND: Apart from being a risk factor for atherosclerotic cardiovascular diseases, the latest research suggests homocysteine as a marker for cancer. We aimed to explore the clinical utility of plasma homocysteine levels as a marker in lung cancer. PATIENTS AND METHODS: Changes in serum total thiols and folate levels were investigated in newly diagnosed untreated lung cancer patients (n = 37) and compared with healthy controls (n = 26). Fluorometric HPLC methods were used for the determination of thiols. Other parameters were determined with commercial diagnostic kits. RESULTS: Increased total homocysteine (t-Hcy), decreased total glutathione (t-GSH) and folate levels were observed in lung cancer patients compared with healthy controls. Total levels of thiols and folate did not show any significant difference between SCLC and NSCLC patients. However, there were significantly higher t-Hcy, lower t-GSH and folate levels in the advanced-stage group compared with controls. Prevalence of hyperhomocysteinemia was 65% in lung cancer patients when 12 micromol/l were taken as a cut-off value for t-Hcy levels. CONCLUSION: Homocysteine is suggested as a marker for several types of cancer, but our result did not support this hypothesis for lung cancer. Although higher homocysteine levels were observed in the present study, further investigation in the larger cancer population would clarify the importance of homocysteine as a cancer marker.

Oxidation of uric acid in rheumatoid arthritis: is allantoin a marker of oxidative stress?

Free radicals are implicated in many diseases including atherosclerosis, cancer and also in rheumatoid arthritis. Reaction of uric acid with free radicals, such as hydroxyl radical and hypochlorous acid (HOCl) results in allantoin production. In this study, we measured the serum allantoin levels, oxidation products of uric acid, as a marker of free radical generation in rheumatoid arthritis. Fasting blood samples were obtained from 21 rheumatoid patients and 15 healthy controls. In this study, the serum allantoin and uric acid levels were measured by a gas chromatography-mass spectrometry method and the ratios were calculated. The mean allantoin and uric acid levels and ratios in the patient group were 22.1 +/- 11.3, 280.5 +/- 65.0 and 8.0 +/- 3.7 microM, while in the control group they were 13.6 +/- 6.3, 278.3 +/- 53.6 and 4.9 +/- 2.1 microM, respectively. The effects of gender, age, menopausal status, duration of disease and medications on serum allantoin and uric acid levels of the patient and control groups were studied. Our results suggest that uric acid acts as a free radical scavenger and thus is converted to allantoin. Increased allantoin levels suggest the possible involvement of free radicals in rheumatoid arthritis.

The role of plasma thiol compounds and antioxidant vitamins in patients with cardiovascular diseases.

BACKGROUND: Increased levels of homocysteine (Hcy) and cysteine (Cys) are associated with risk of cardiovascular diseases (CVD). These thiol compounds can generate various free radicals and so cause endothelial dysfunction. Antioxidant vitamins are effective scavengers of reactive oxygen species (ROS) and prevent endothelial dysfunction. In this study, we investigated the plasma homocysteine, cysteine, vitamins E, C and A, and beta-carotene (BC) levels in cardiovascular patients to compare with controls. We also investigated whether there is a correlation between the plasma thiol compounds and antioxidant vitamins. METHODS: Blood samples were collected from 47 patients with cardiovascular disease (16 women and 31 men) and 21 healthy subjects (8 women and 13 men) in the overnight fasting state. Serum thiol compound and antioxidant vitamin levels were measured by high-pressure liquid chromatography (HPLC) methods. RESULTS: The plasma homocysteine and cysteine levels were significantly higher in patients than those of controls. While vitamin C (VC), vitamin A (VA) and beta-carotene levels were significantly lower in patients than in controls, vitamin E (VE) levels did not change in both groups. There is a positive correlation between homocysteine and cysteine levels (r=0.622, p=0.000) in all study population. We found that the plasma level of homocysteine was significantly correlated in negative manner with vitamins E and A levels (r=-0.260, p=0.033 and r=-0.255, p=0.036, respectively) of all study population. Plasma cysteine levels were negatively correlated with only vitamin C levels (r=-0.320, p=0.008) in all study populations. CONCLUSIONS: Our data suggest that Hcy and Cys are associated with cardiovascular disease and there is negative but weak correlation's between thiol compounds and antioxidant vitamins.

Plasma total homocysteine and cysteine levels as cardiovascular risk factors in coronary heart disease.

OBJECTIVES: As an important risk factor for coronary atherosclerosis, elevated plasma total homocysteine (t-hcy) concentration has recently received greater attention than have conventional risk factors. Though less reactive than homocysteine, cysteine (cys) is the most abundant plasma thiol and may function as an extracellular regulating factor of thiol/disulfide exchange in order to maintain an adequate redox status. An increase in the total amount of this compound may be noxious depending on environmental conditions. In the present study, the aim was to investigate changes of plasma total cysteine, homocysteine and other determinants in different types of coronary heart disease. DESIGN AND METHODS: Plasma total homocysteine (t-hcy), cysteine (t-cys), cysteinylglycine (t-cysgly), folic acid, vitamin B(12), lipid parameters, total protein, albumin and creatinine levels were studied in plasma from 68 patients with coronary heart disease and 42 healthy controls. After reduction of disulfide bonds with tri-n-buthylphosphine, plasma total thiols were assayed using high performance liquid chromatography (HPLC) and fluorescence detection following derivatization of sulfhydryl groups with 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F). Other parameters were determined by using commercial kits. RESULTS: Plasma t-hcy and t-cys levels were higher in patients (P<0.0001) than in controls, but t-cysgly was unchanged. Hcy and cys levels were correlated with age in the whole study population (r=0.49, r=0.46, P<0.01). Plasma t-hcy positively correlated with plasma t-cys (r=0.53, P<0.01) and t-cysgly (r=0.49, P<0.01) in patients, and with plasma t-cys (r=0.57, P<0.01) in controls. Postmenopausal women had higher t-cys and t-hcy levels than premenopausal women among the controls (P<0.01). Folate and vitamin B(12) levels were similar in both patients and controls. Patients with vitamin B(12) levels below normal had higher plasma t-cys and t-cysgly levels (P<0.05). Interestingly, control subjects with lower vitamin B(12) levels had lower plasma t-hcy levels (P<0.05). Plasma total cholesterol, HDL-cholesterol, LDL-cholesterol, total protein, albumin and creatinine levels in patients and controls were within the normal range, but only HDL-cholesterol levels in patients were lower than in controls (P<0.0001). Triglyceride and VLDL levels of patients were also higher than those of controls (P<0.0001). CONCLUSIONS: Higher plasma total cysteine levels are as important as higher plasma total homocysteine levels. Both parameters are intercorrelated and may act synergistically. To discern their respective roles in atherosclerotic disease, these aminothiol levels have to be considered together.