Glycolytic enzymes in non-glycolytic web: functional analysis of the key players.

To survive in the tumour microenvironment, cancer cells undergo rapid metabolic reprograming and adaptability. One of the key characteristics of cancer is increased glycolytic selectivity and decreased oxidative phosphorylation (OXPHOS). Apart from ATP synthesis, glycolysis is also responsible for NADH regeneration and macromolecular biosynthesis, such as amino acid biosynthesis and nucleotide biosynthesis. This allows cancer cells to survive and proliferate even in low-nutrient and oxygen conditions, making glycolytic enzymes a promising target for various anti-cancer agents. Oncogenic activation is also caused by the uncontrolled production and activity of glycolytic enzymes. Nevertheless, in addition to conventional glycolytic processes, some glycolytic enzymes are involved in non-canonical functions such as transcriptional regulation, autophagy, epigenetic changes, inflammation, various signaling cascades, redox regulation, oxidative stress, obesity and fatty acid metabolism, diabetes and neurodegenerative disorders, and hypoxia. The mechanisms underlying the non-canonical glycolytic enzyme activities are still not comprehensive. This review summarizes the current findings on the mechanisms fundamental to the non-glycolytic actions of glycolytic enzymes and their intermediates in maintaining the tumor microenvironment.

Cellular, Biophysical and in Silico Binding Study of ß-Estradiol-6-one 6- (O-carboxy methyl Oxime) with Tubulin in Search of Antimitotic Derivative of 2-Methoxy Estradiol.

The tubulin-microtubule system is a major target for a variety of small molecules which can interfere in cell cycle progression. Therefore, it serves as a prospective to control the incessant division of cancer cells. To identify novel inhibitors of the tubulin-microtubule system, a group of estrogen derivatives has been tested with tubulin as a target since literature surveys portray coveted behaviour from the same. Out of them, ß-Estradiol-6-one 6- (O-carboxy methyl Oxime) abbreviated as Oxime, disrupts the cytoskeleton network and induces apoptosis with nuclei fragmentation. It has been revealed from the work that Oxime targets the colchicine binding site and binds tubulin in an entropy-driven manner. This suggests that structural variation might play a key role in modulating the anti-mitotic role of estrogen derivatives. Our work reveals that Oxime might serve as a lead molecule to nurture anti-cancer research, having the potential for recovery of the vast cancer population.

Antibacterial activities of sulfamethoxazolyl-azo-phenols and their Cu(II) complexes along with molecular docking properties.

Sulfamethoxazolyl-azo-phenols [SMX-N=N-C6H2(R)(R/)-OH] (1a, 2a) and their Cu(II) complexes, [Cu(SMX-N=N-C6H2(R)(R/)-O)2] (1b, 2b) (R = p-OMe, R/ = H, 1a/1b; R = p-Cl, R/ = m-CH3, 2a/2b) show antibacterial sensitivity against Gram-positive bacteria, B. subtillis; IC50: 281.47 ± 1.84 µM (1a), 126.39 ± 1.66 µM (1b), and 279.94 ± 3.15 µM (2a), 123.62 ± 1.27 µM(2b), and Gram-negative bacteria, E. coli; IC50: 204.66 ± 3.31 µM (1a) and 89.05 ± 1.48 µM (1b), 223.13 ± 2.71 µM (2a), and 98.26 ± 1.59 µM (2b). Interaction of DNA with free ligand (1a and 2a) is insignificant, while the complexes (1b and 2b) interact strongly and the binding constants are K b, 8.413 × 104 M-1 (1b) and 6.56 × 105 M-1 (2b). Optimized structures of the compounds are docked with protein structure of DHPS (E. coli) to propose the most favoured binding mode of the drugs in the active site. The in silico test of the compound helps to understand drug metabolism, drug-protein interactions, and toxicity (ADMET).

Inhibition of Porcine Pancreatic Amylase Activity by Sulfamethoxazole: Structural and Functional Aspect.

Combating Type-2 diabetes mellitus is a pivotal challenge in front of the present world. Several lines of therapy are in practice for resisting this deadly disease which often culminates with cardiovascular complexities, neuropathy and retinopathy. Among various therapies, administration of alpha glucosidase inhibitors is common and widely practiced. Sulfonylurea category of anti diabetic drug often suffers from cross reactivity with sulfamethoxazole (SMX), a common drug in use to treat a handful of microbial infections. However the specific cellular target generating postprandial hypoglycemia on SMX administration is till date unraveled. The present work has been initiated to elucidate the effects of a group of sulfonamide drugs inclusive of SMX for their amylase inhibitory role. SMX inhibits porcine pancreatic amylase (PPA) in a noncompetitive mode with an average IC50 value 0.94 mM respectively. Interaction of SMX with PPA is manifested with gradual quenching of tryptophan fluorescence with concomitant shift in lambda max value (λmax). Binding is governed by entropy driven factor (24.8 cal mol(-1) K(-1)) with unfavorable contribution from enthalpy change. SMX interferes with the activity of acarbose in a synergistic mode to reduce the effective dose of acarbose as evident from the in vitro PPA inhibition study. In summary, loss of PPA activity in presence of SMX is indicative of structural changes of PPA which is further augmented in the presence of acarbose as explained in the schematic model and docking study.

MAP2c prevents arachidonic acid-induced fibril formation of tau: Role of chaperone activity and phosphorylation.

Tau has long been associated with Alzheimer's disease, where it forms neurofibrillary tangles. Here we show for the first time by electron microscopy that MAP2c prevents arachidonic acid-induced in vitro aggregation of tau. However, phosphorylated MAP2c failed to prevent the same. Previously we reported that MAP2c possesses chaperone-like activity while tau does not (Sarkar et al., 2004, Eur J Biochem., 271(8), 1488-96). Here we demonstrate that phosphorylation severely impaired the chaperone activity of MAP2c, implying a crucial role of chaperone in preventing tau fibrillation. Additionally, the ability of MAP2c to induce microtubule polymerization was abolished completely upon phosphorylation. As tau and MAP2c possess highly homologous C-termini, we speculated that the N-terminus of MAP2c might account for its chaperone activity. Nevertheless, experiments showed that N-terminus of MAP2c alone is inactive as a chaperone. Our preliminary findings suggest that MAP2c/MAP2 could be one of the regulators maintaining tau homeostasis in the cell.

Production and characterization of thermostable alkaline protease of Bacillus subtilis (ATCC 6633) from optimized solid-state fermentation.

Proteases are the most important group of enzymes utilized commercially in various arenas of industries, such as food, detergent, leather, dairy, pharmaceutical, diagnostics, and waste management, accounting for nearly 20% of the world enzyme market. Microorganisms of specially Bacillus genera serve as a vast repository of diverse set of industrially important enzymes and utilized for the large-scale enzyme production using a fermentation technology. Approximately 30%-40% of the cost of industrial enzymes originates from the cost of the growth medium. This study is attempted to produce protease from Bacillus subtilis (ATCC 6633) after optimization of various process parameters with the aid of solid-state fermentation using a cheap nutrient source such as wheat bran. B. subtilis (ATCC 6633) produces proteases of molecular weight 36 and 20 kDa, respectively, in the fermented medium as evident from SDS zymogram. Alkaline protease activity has been detected with optimum temperature at 50 °C and is insensitive to ethylenediaminetetraacetic acid. This thermostable alkaline protease exhibits dual pH optimum at 7 and 10 with moderate pH stability at alkaline pH range. It preserves its activity in the presence of detergent such as SDS, Tween 20, and Triton X-100 and may be considered as an effective additive to detergent formulation with some industrial importance.

Optimization of Amylase Production from B. amyloliquefaciens (MTCC 1270) Using Solid State Fermentation.

Demand for microbial amylase production persists because of its immense importance in wide spectrum industries. The present work has been initiated with a goal of optimization of solid state fermentation condition for amylase using agroindustrial waste and microbial strain like B. amyloliquefaciens (MTCC 1270). In an aim to improve the productivity of amylase, fermentation has been carried out in the presence of calcium (Ca(+2)), Nitrate (NO3 (-)), and chloride ions (Cl(-)) as well as in the presence of D-inositol and mannitol. Amylase needs calcium ion for the preservation of its structure, activity and stability that proves beneficial also for amylase production using solid state fermentation. The inclusion of ions and sugars in the SSF media is promising which can be explained by the protection offered by them against thermal decay of amylase at various incubation periods at 37°C.

Production and Partial Purification of Alpha Amylase from Bacillus subtilis (MTCC 121) Using Solid State Fermentation.

Amylase is an enzyme that catalyzes the breakdown of starch into sugars and plays a pivotal role in a variety of areas like use as digestives, for the production of ethanol and high fructose corn syrup, detergents, desiring of textiles, modified starches, hydrolysis of oil-field drilling fluids, and paper recycling. In the present work, solid state fermentation (SSF) for α -amylase production has been used in lieu of submerged fermentation (SmF) due to its simple technique, low capital investment, lower levels of catabolite repression, and better product recovery. Bacillus subtilis has been well known as producer of alpha amylase and was tested using solid state fermentation for 48 hours at 37°C with wheat bran as substrate. Comparison between different fermentation hours demonstrated high yield of alpha amylase after 48 hours. This alpha amylase has optimum pH and temperature at 7.1 and 40°C, respectively. With the goal to purify alpha amylase, 30-70% (NH4)2SO4 cut concentrated the amylase activity threefold with respect to crude fermented extract. This was verified in quantitative DNS assay method as well as in zymogram gel profile. The exact molecular weight of the amylase is yet to be determined with the aid of other protein purification techniques.

Discrimination of ligands with different flexibilities resulting from the plasticity of the binding site in tubulin.

Tubulin, an α,ß heterodimer, has four distinct ligand binding sites (for paclitaxel, peloruside/laulimalide, vinca, and colchicine). The site where colchicine binds is a promising drug target for arresting cell division and has been observed to accommodate compounds that are structurally diverse but possess comparable affinity. This investigation, using two such structurally different ligands as probes (one being colchicine itself and another, TN16), aims to provide insight into the origin of this diverse acceptability to provide a better perspective for the design of novel therapeutic molecules. Thermodynamic measurements reveal interesting interplay between entropy and enthalpy. Although both these parameters are favourable for TN16 binding (ΔH < 0, ΔS > 0), but the magnitude of entropy has the determining role for colchicine binding as its enthalpic component is destabilizing (ΔH > 0, ΔS > 0). Molecular dynamics simulation provides atomistic insight into the mechanism, pointing to the inherent flexibility of the binding pocket that can drastically change its shape depending on the ligand that it accepts. Simulation shows that in the complexed states both the ligands have freedom to move within the binding pocket; colchicine can switch its interactions like a "flying trapeze", whereas TN16 rocks like a "swing cradle", both benefiting entropically, although in two different ways. Additionally, the experimental results with respect to the role of solvation entropy correlate well with the computed difference in the hydration: water molecules associated with the ligands are released upon complexation. The complementary role of van der Waals packing versus flexibility controls the entropy-enthalpy modulations. This analysis provides lessons for the design of new ligands that should balance between the "better fit" and "flexibility"', instead of focusing only on the receptor-ligand interactions.

Binding of indanocine to the colchicine site on tubulin promotes fluorescence, and its binding parameters resemble those of the colchicine analogue AC.

Indanocine, a synthetic indanone, has shown potential antiproliferative activity against several tumor types. It is different from many other microtubule-disrupting drugs, because it displays toxicity toward multidrug resistance cells. We have examined the interaction of indanocine with tubulin and determined their binding and thermodynamic parameters using isothermal titration calorimetry (ITC). Indanocine is weakly fluorescent in aqueous solution, and the binding to tubulin enhances fluorescence with a large blue shift in the emission maxima. Indanocine binds to the colchicine site of tubulin, although it bears no structural similarity with colchicine. Nevertheless, like colchicine analogue AC, indanocine is a flexible molecule in which two halves of the molecule are connected through a single bond. Also, like AC, indanocine binds to the colchicine binding site of tubulin in a reversible manner and the association reaction occurs at a faster rate compared to that of colchicine-tubulin binding. The binding kinetics was studied using stopped-flow fluorescence. The association process follows biphasic kinetics similar to that of the colchicine-tubulin interaction. The activation energy of the reaction was 10.5 +/- 0.81 kcal/mol. Further investigation using ITC revealed that the enthalpy of association of indanocine with tubulin is negative and occurs with a negative heat capacity change (DeltaC(p) = -175.1 cal mol(-1) K(-1)). The binding is unique with a simultaneous participation of both hydrophobic and hydrogen bonding forces. Finally, we conclude that even though indanocine possesses no structural similarity with colchicine, it recognizes the colchicine binding site of tubulin and its binding properties resemble those of the colchicine analogue AC.

Anti-mitotic activity of colchicine and the structural basis for its interaction with tubulin.

In this review, an attempt has been made to throw light on the mechanism of action of colchicine and its different analogs as anti-cancer agents. Colchicine interacts with tubulin and perturbs the assembly dynamics of microtubules. Though its use has been limited because of its toxicity, colchicine can still be used as a lead compound for the generation of potent anti-cancer drugs. Colchicine binds to tubulin in a poorly reversible manner with high activation energy. The binding interaction is favored entropically. In contrast, binding of its simple analogs AC or DAAC is enthalpically favored and commences with comparatively low activation energy. Colchicine-tubulin interaction, which is normally pH dependent, has been found to be independent of pH in the presence of microtubule-associated proteins, salts or upon cleavage of carboxy termini of tubulin. Biphasic kinetics of colchicines-tubulin interaction has been explained in light of the variation in the residues around the drug-binding site on beta-tubulin. Using the crystal structure of the tubulin-DAMAcolchicine complex, a detailed discussion on the pharmacophore concept that explains the variation of affinity for different colchicine site inhibitors (CSI) has been discussed.

Oxalone and lactone moieties of podophyllotoxin exhibit properties of both the B and C rings of colchicine in its binding with tubulin.

Thermodynamics of podophyllotoxin binding to tubulin and its multiple points of attachment with tubulin has been studied in detail using isothermal titration calorimetry. The calorimetric enthalpy of the association of podophyllotoxin with tubulin is negative and occurs with a negative heat capacity change (DeltaC(p) = -2.47 kJ mol(-)(1) K(-)(1)). The binding is unique with a simultaneous participation of both hydrophobic and hydrogen-bonding forces with unfavorable negative entropic contribution at higher temperature, favored with an enthalpy-entropy compensation. Interestingly, the binding of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (AC, a colchicine analogue without the B ring) with tubulin is enthalpy-favored. However, the podophyllotoxin-tubulin association depending upon the temperature of the reaction has a favorable entropic and enthalpic component, which resembles both B- and C-ring properties of colchicine. On the basis of the crystal structure of the podophyllotoxin-tubulin complex, distance calculations have indicated a possible interaction between threonine 179 of alpha-tubulin and the hydroxy group on the D ring of podophyllotoxin. To confirm the involvement of the oxalone moiety as well as the lactone ring of podophyllotoxin in tubulin binding, analogues of podophyllotoxin are synthesized with methoxy substitution at the 4' position of ring D along with its isomer and another analogue epimerized at ring E. From these results, involvement of oxalone as well as the lactone ring of the drug in a specific orientation inclusive of ring A is indicated for podophyllotoxin-tubulin binding. Therefore, podophyllotoxin, like colchicine, behaves as a bifunctional ligand having properties of both the B and C rings of colchicine by making more than one point of attachment with the protein tubulin.

Biphasic kinetics of the colchicine-tubulin interaction: role of amino acids surrounding the A ring of bound colchicine molecule.

Isotypes of vertebrate tubulin have variable amino acid sequences, which are clustered at their C-terminal ends. Isotypes bind colchicine at different on-rates and affinity constants. The kinetics of colchicine binding to purified (unfractionated) brain tubulin have been reported to be biphasic under pseudo-first-order conditions. Experiments with individual isotypes established that the presence of beta(III) in the purified tubulin is responsible for the biphasic kinetics. Because the isotypes mainly differ at the C termini, the colchicine-binding kinetics of unfractionated tubulin and the beta(III) isotype, cleaved at the C termini, have been tested under pseudo-first-order conditions. Removal of the C termini made no difference to the nature of the kinetics. Sequence alignment of different beta isotypes of tubulin showed that besides the C-terminal region, there are differences in the main body as well. To establish whether these differences lie at the colchicine-binding site or not, homology modeling of all beta-tubulin isotypes was done. We found that the isotypes differed from each other in the amino acids located near the A ring of colchicine at the colchicine-binding site on beta tubulin. While the beta(III) isotype has two hydrophilic residues (serine(242) and threonine(317)), both beta(II) and beta(IV) have two hydrophobic residues (leucine(242) and alanine(317)). beta(II) has isoleucine at position 318, while beta(III) and beta(IV) have valine at that position. Thus, these alterations in the nature of the amino acids surrounding the colchicine site could be responsible for the different colchicine-binding kinetics of the different isotypes of tubulin.

-NH-dansyl isocolchicine exhibits a significantly improved tubulin-binding affinity and microtubule inhibition in comparison to isocolchicine by binding tubulin through its A and B rings.

Structure-activity relationship studies have established that the A and C rings of colchicine comprise the minimum structural feature necessary for high affinity drug-tubulin binding. Thus, colchicine acts as a bifunctional ligand by making two points of attachment to the protein. Furthermore, analogues belonging to the iso series of colchicine are virtually inactive in binding to tubulin and inhibiting microtubule assembly. In the present study, we found that the substitution of a hydrophobic dansyl group on the B-ring side chain (C7 position) of isocolchicine reverses the structural alterations at the C ring and the newly synthesized -NH-dansyl isocolchicine restores the lost biological activity of the compound. It inhibits microtubule assembly efficiently with an IC(50) value of 10 microM and competes with [(3)H]colchicine for binding to tubulin. Moreover, although -NH-dansyl colchicine binding to tubulin involves two steps, the -NH-dansyl isocolchicine-tubulin interaction has been found to occur via a one-step process. Also, the affinity constant of the -NH-dansyl isocolchicine-tubulin interaction is roughly only 3 times lower than that of the -NH-dansyl colchicine-tubulin interaction. These results suggest that the enhanced microtubule inhibitory ability of -NH-dansyl isocolchicine is therefore related to the affinity of the drug-tubulin interaction and not to any conformational changes upon binding tubulin. We also observed that the competition of -NH-dansyl isocolchicine with [(3)H]colchicine for binding to tubulin was dependent on the tubulin concentration. In conclusion, this paper for the first time indicates that a biologically active bifuntional colchicine analogue can be designed where the drug binds tubulin through its A and B rings, while the C ring remains inactive.

The B-ring substituent at C-7 of colchicine and the alpha-C-terminus of tubulin communicate through the "tail-body" interaction.

The carboxy terminals of alphabeta-tubulins are flexible regions rich in acidic amino acid residues that play an inhibitory role in the polymerization of tubulin to microtubules. We have shown that the binding of colchicine and its B-ring analogs (with C-7 substituents) to tubulin are pH sensitive and have high activation energies. Under identical conditions, the binding of analogs without C-7 substituents is pH independent and has lower activation energy. Beta-C-terminus-truncated tubulin (alphabeta(s)) shows similar pH sensitivity and activation energy to native tubulin (alphabeta). Removal of the C-termini of both subunits of tubulin (alpha(s)beta(s)) or the binding of a basic peptide P2 to the negatively charged alpha-C-terminus of tubulin causes a colchicine-tubulin interaction independent of pH with a low activation energy. Tubulin dimer structure shows that the C-terminal alpha-tail is too far from the colchicine binding site to interact directly with the bound colchicine. Therefore, it is likely that the interaction of the alpha-C-terminus with the main body of tubulin indirectly affects the colchicine-tubulin interaction via conformational changes in the main body. We therefore conclude that in the presence of tail-body interaction, a B-ring substituent makes contact with the alpha-tubulin and induces significant conformational changes in alpha-tubulin.

MAP2 prevents protein aggregation and facilitates reactivation of unfolded enzymes.

It is well established that in addition to its functional role in cell motility, cell division and intracellular transport, cytoskeletal protein tubulin also possesses significant chaperone-like activity. In vitro studies from our laboratory showed that dimeric tubulin can prevent stress induced aggregation of substrate proteins, can resist thermal deactivation of enzymes and can also refold enzymes from their fully denatured state [Manna, T., Sarkar, T., Poddar, A., Roychowdhury, M., Das, K.P. & Bhattacharyya, B. (2001) J. Biol. Chem.276, 39742-39747]. Negative charges of the C-termini of both subunits of tubulin are essential for this chaperone-like property as the deletion of only beta-C-terminus or the binding of a 14-residue basic peptide P2 to the alpha-C-terminus completely abolishes this property [Sarkar, T., Manna, T., Bhattacharyya, S., Mahapatra, P., Poddar, A., Roy, S., Pena, J., Solana, R., Tarazona, R. & Bhattacharyya, B. (2001) Proteins Struct. Funct. Genet.44, 262-269]. Based on these results, one would expect that the microtubular proteins (MTP, tubulin with microtubular-associated proteins, i.e. MAPs bound to the C-terminus) should not possess any chaperone-like activity. To our surprise we noticed excellent chaperone-like activity of MTP. MTP prevents chemical and thermal aggregation of other proteins and can enhance the extent of refolding of fully unfolded substrate enzymes. Because MTP contains tubulin as well as several MAPs bound to the C-termini of tubulin, we fractionated and purified microtubular associated protein 2 (MAP2) and tau using phosphocellulose chromatography. Experiments with purified proteins demonstrated that it is the MAP2 of MTP that exhibits significant chaperone-like activity. This has been shown by the prevention of dithiothreitol-induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase and regain of enzymatic activity during refolding of unfolded substrates. Tau, which shares a homologous C-terminal domain with MAP2, possesses no such activity.

Antimicrotubular drugs binding to vinca domain of tubulin.

Studies on vinca domain binding drugs were done in great details by a number of workers as it is recognized as a potential target for anticancer drug development. Their structures, properties, mode of action, success and failures as potential anticancer drug have been discussed in short details in this review. Among these drugs rhizoxin and maytansine are competitive inhibitors, and bind at the vinblastine binding site of tubulin where as others are non-competitive inhibitors. Besides binding, these drugs also differ in the extent of GTP hydrolysis, GTP exchange and in the stabilization of colchicine binding site. The toxicity level of these drugs towards the host cells and the extent of efflux of drugs by the P-glycoprotein mediated pump are also discussed.

BisANS binding to tubulin: isothermal titration calorimetry and the site-specific proteolysis reveal the GTP-induced structural stability of tubulin.

Interactions of bisANS and ANS to tubulin in the presence and absence of GTP were investigated, and the binding and thermodynamic parameters were determined using isothermal titration calorimetry. Like bisANS binding to tubulin, we observed a large number of lower affinity ANS binding sites (N1 = 1.3, K1 = 3.7 x 10(5) M(-1), N2 = 10.5, K2 = 7 x 10(4)/M(-1)) in addition to 1-2 higher affinity sites. Although the presence of GTP lowers the bisANS binding to both higher and lower affinity sites (N1 = 4.3, N2 = 11.7 in absence and N1 = 1.8, N2 = 3.6 in presence of GTP), the stoichiometries of both higher and lower affinity sites of ANS remain unaffected in the presence of GTP. BisANS-induced structural changes on tubulin were studied using site-specific proteolysis with trypsin and chymotrypsin. Digestion of both alpha and beta tubulin with trypsin and chymotrypsin, respectively, has been found to be very specific in presence of GTP. GTP has dramatic effects on lowering the extent of nonspecific digestion of beta tubulin with trypsin and stabilizing the intermediate bands produced from both alpha and beta. BisANS-treated tubulin is more susceptible to both trypsin and chymotrypsin digestion. At higher bisANS concentration (>20 microM) both alpha and beta tubulins are almost totally digested with enzymes, indicating bisANS-induced unfolding or destabilization of tubulin structure. Again, the addition of GTP has remarkable effect on lowering the bisANS-induced enhanced digestion of tubulin as well as stabilizing effect on intermediate bands. These results of isothermal titration calorimetry, proteolysis and the DTNB-kinetics data clearly established that the addition of GTP makes tubulin compact and rigid and hence the GTP-induced stabilization of tubulin structure. No such destabilization of tubulin structure has been noticed with ANS, although, like bisANS, ANS possesses a large number of lower affinity binding sites. On the basis of these results, we propose that the unique structure of bisANS, which in absence of GTP can bind tubulin as a bifunctional ligand (through its two ANS moieties), is responsible for the structural changes of tubulin.