Current Enzyme Inhibition (v.11, #2)

Meet Our Co-Editor by Dimitra Hadjipavlou-Litina (75-75).

Benzenesulfonamide is versatile motif employed for the discovery in the field of medicinal and pharmaceutical chemistry. Over the years of active research, sulfonamide has evolved as important privileged pharmacophore in medicinal chemistry, encompassing a diverse range of biological activities including antiviral, carbonic anhydrase inhibitors, diuretic, and a protease inhibitor, hypoglycemic, anticancer and anti-inflammatory. On the other hand, owing to the fast development of new drugs possessing sulfonamide pharmacophore many research reports generated in a short span of time. Thus, there is a need to couple the most recent data with the prior data to comprehend the present status of benzenesulfonamide as anti-inflammatory agent. The present review highlights the molecular modeling and synthetic methods of benzenesulfonamide scaffold bearing various heterocyclic or carbocyclic rings such as thiazole, triazole, imidazole, isoxazoline, pyridine, pyranone, pyridazone, pyrimidine, pyran, indole, benzothiazole, benzotriazole, benzimidazole and benzopyran as central ring scaffold. In addition to this, the manuscript will be a guideline to medicinal chemists for rationally designing a novel benzenesulfonamide bearing compounds as COX-2 inhibitors.

Flavonoids: An Outstanding Structural Core for the Inhibition of Xanthine Oxidase Enzyme by Bijo Mathew, Jerad Suresh, Githa E. Mathew, Sherin A. Rasheed, Jobin K. Vilapurathu, P. Jayaraj (108-115).
Context: Xanthine oxidase is a key enzyme in purine metabolism with an important role in various pathological conditions including gout and oxidative stress. Many of the synthetic candidates showed remarkable activity with a greater amount of side effects. Thus the search of promising structural core like flavonoids emerged greater attention for the inhibition of xanthine oxidase.
Objective: The objective of this review is to provide a detailed knowledge of the flavonoidal class of compounds including natural and synthetic derivatives for the inhibition of xanthine oxidase. The review lays emphasis on the structure activity relationship and the scope of computational chemistry for designing flavonoids which inhibit xanthine oxidase.
Methods: This review has been constructed by the most reliable literature databases including ScienceDirect, PubMed, Bentham Science and American Chemical Society Publishers.
Results: The flavonoidal class of compounds was found to exhibit remarkable xanthine oxidase inhibitory activity. In addition, structure-activity relationships, molecular descriptors and enzyme-drug interactions of the flavonoids binding to the active site of xanthine oxidase were discussed, which is required for further rational drug design.
Conclusion: It has been concluded that molecular recognition of a flavonoidal class of compound's inhibitory activity of the xanthine oxidase enzyme mainly depends on the planar nature of benzopyran ring, hydroxyl group at C-5 and C-7 position and torsion angle formed by the C3-C2-C1'-C2'.

Anti-inflammatory Activities of Phenoxyphenyl Ureas Mediated by Inhibition of p38 Kinase by Ravindra Kulkarni, Swapna Diwyani, Stefan Laufer, Chandrshekar V. M., Achaiah G., Prashant Gurav, Prasanna Habbu (116-123).
P38 kinase has been known to be one of the important and validated targets for inflammatory diseases including rheumatoid arthritis for its vital role in production and release of proinflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin-beta (IL-1β). Pyridinyl group of our reported compounds was replaced by simple phenyl groups and compounds were synthesized and characterized. A dose of 50 mg/Kg has been administered and observed antiinflammatory activity in rats. Compounds 6h, 6i, 6j and 6o exhibited significant antiinflammatory activity at 3rd hour of carrageenan administration. Compounds 6c, 6j, 6k and others demonstrated greater than 50% p38 kinase inhibitory activity at 10 ?M.

Kinetic Data of D-Glyceraldehyde-3-Phosphate Dehydrogenase from HeLa Cells by Juliana B.B. Maurer, Fernanda Bovo, Elisa M. Gomes, Helena M.S. Loureiro, Fabiola R. Stevan, Selma F. Zawadzki-Baggio, Momoyo Nakano (124-131).
D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reversible oxidation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bis-phospho-D-glycerate, and as such participates in the glycolytic conversion of glucose to pyruvic acid in most living organisms. The glycolytic pathway plays an important role in tumor cells, but it is not clear whether the enzyme kinetics of GAPDH or their response to inhibitors, substrates, and cofactors differ between tumor cells and normal cells. To obtain a tumor-derived GAPDH sample, HeLa cells were chosen on the basis of their homogeneous differentiation pattern and ease of harvesting. We carried out experiments to investigate whether the enzyme kinetics of purified GAPDH from HeLa cells were altered in the presence of reagents containing sulfhydryl groups, divalent metal ions, and cellular metabolites such as nucleotides and coenzymes. The kinetic data were compared with data for GAPDH from normal tissue. GAPDH from HeLa cells was activated by 2-mercaptoethanol and dithioerythritol. The maximum activation was obtained at a 1 mM concentration of each reducing agent. Cupric and mercuric ions (1 mM), as well as p-chloro and phydroxymercuribenzoate (10 M), fully inhibited enzymatic activity. Among the nucleotides tested, 3-5-cyclic AMP (cAMP) was the most effective inhibitor at 30 mM concentration, with a relative activity of 22.79 (±1.76), which was significantly different (p 0.05) from that of the control, which had 100% activity in the absence of adenine nucleotide. Enzyme inhibition by adenine nucleotides appeared to be via competition with NAD+. The apparent inhibition constants (Ki) for ADP, 5-AMP, and cAMP were 2.1 mM, 1.0 mM, and 0.6 mM, respectively. GAPDH from HeLa cells was inactivated when incubated in the presence of G3P or NADH at 37 °C, and in both cases the presence of 2-mercaptoethanol protected the enzyme against inhibition. The presence of EDTA did not affect the inactivation of the enzyme by NADH, which suggested that the inactivation of HeLa GAPDH by NADH is not related to the presence of heavy metal ions. Our kinetic analysis showed that although the GAPDH of HeLa cells has a lower specific activity and stability compared with GAPDH from normal tissue, its kinetic characteristics were similar, reinforcing the key role of this enzyme in the metabolism of tumor cells.

Design and Synthesis of Some Newer Pyrimidine Hydroxamates as Histone Deacetylase Inhibitors by Jagannath Behera, Barij N. Sinha, Abhishek Kumar (132-139).
Histone Deacetylases (HDACs) are a class of enzymes that has become one of the potential, promising and well established targets for the treatment of cancer. In the present work, a series of pyrimidine hydroxamates were designed and synthesized as novel HDAC inhibitors.The compounds possess diaryl pyrimidine as cap, piperazine as linker and hydroxamate at Zn2+ binding group. These were designed with an idea to substitute diaryl group in place of single aryl cap in continuation to our earlier reported compounds from our lab. First S-methylisothiuronium hydroiodide 1 was prepared, which is reacted with monoboc piperazine 2 to form tert-butyl 4-carbamimidoylpiperazine-1- carboxylate 3, further it is treated with different substituted Chalcones (4a-k) to form 2, 4, 6- tri substituted pyrimidine (5a-k). After addition of ethylchloroacetate, the compounds formed are then treated with hydroxylamine hydrochloride to give the final compounds (7a-k). The antiproliferative activities of the synthesized compounds were evaluated by MTT assay against human breast cancer cell lines MDA-MB-231. The compounds found active were later on tested for Histone Deacetylase (HDAC) inhibitory activity. The IC50 values of the best-performing compounds were found to be in-between 9 to 38 M. The results obtained suggest that in a few cases, the modifications carried out retained the selectivity towards HDAC with increased potency, which may be associated with variation in the aryl cap region along with an increase in linker length. Out of all synthesized compounds, 2-(4-(4, 6-bis (2-hydroxyphenyl) pyrimidin-2-yl) piperazin-1-yl)-N-hydroxyacetamide (T8) showed promising inhibitory activity (MDA-MB-231= 9 M) & (HDAC = 6M). Further ADMET results also demonstrated that these synthesized compounds exhibit good absorption, permeability and penetration abilities in the human body. The ability of the diaryl cap of the compounds to accommodate both the pocket of the HDAC (B & D) may be the reason for its enzyme inhibitory activity.

An Additional Method for Analyzing the Reversible Inhibition of an Enzyme Using Acid Phosphatase as a Model by Jordan M. Baumhardt, Benjamin M. Dorsey, Craig C. McLauchlan, Marjorie A. Jones (140-146).
Using wheat germ acid phosphatase and sodium orthovanadate as a competitive inhibitor, a novel method for analyzing reversible inhibition was carried out. Our alternative approach involves plotting the initial velocity at which product is formed as a function of the ratio of substrate concentration to inhibitor concentration at a constant enzyme concentration and constant assay conditions. The concept of initial concentrations driving equilibrium leads to the chosen axes. Three apparent constants can be derived from this plot: Kmax, Kmin, and Kinflect. Kmax and Kmin represent the substrate to inhibitor concentration ratio for complete inhibition and minimal inhibition, respectively. Kinflect represents the substrate to inhibitor concentration ratio at which the enzyme-substrate complex is equal to the inhibitory complex. These constants can be interpolated from the graph or calculated using the first and second derivative of the plot. We conclude that a steeper slope and a shift of the line to the right (increased x-axis values) would indicate a better inhibitor. Since initial velocity is not a linear function of the substrate/inhibitor ratio, this means that inhibition changes more quickly with the change in the [S]/ [I] ratio. When preincubating the enzyme with substrate before the addition of inhibitor, preincubating the enzyme with inhibitor before the addition of substrate or with concurrent addition of both substrate and inhibitor, modest changes in the slopes and y-intercepts were obtained. This plot appears useful for known competitive and non-competitive inhibitors and may have general applicability.

Inhibitory effects of the Food Azo Dyes on Pig Liver Carboxylesterase Activity by Wafaa Lemerini, Zoheir Arrar, Kajima Mulengi Joseph, Zoheir Dahmani, Wassila Drici, Naima Bouazzaoui, Djamel Bendiabdellah (147-152).
The paper reports the inhibition kinetics of pig liver carboxylesterase (CE) by two food azo dyes, tartrazine (E 102) and green dye (E102-E132), using ethyl butyrate as substrate. Optimum pH and temperature for pig liver CE were found to be 8.0 and 45°C. The kinetics of CE-catalyzed hydrolysis of ethyl butyrate followed the Haldane model with an optimum substrate concentration of 30 mM. Km and Vmax values were found to be 9.44 mM and 504.62 M/min, respectively. Pig liver CE was significantly inhibited by tartrazine and green dye with IC50 values of 0.68 mM and 1.37 mM, respectively.
The kinetic analyses showed that the inhibition of CE activity by the tartrazine and green dye was uncompetitive type with KI values of 0.46 mM and 0.62 mM, respectively. From the experimental results, it was found that tartrazine is a more effective inhibitor due to lower KI and IC50 values.