Novel drug designing approach for dual inhibitors as anti-inflammatory agents: implication of pyridine template

Compounds incorporating thiophene moiety, a pi excess five membered heterocycle, have attracted a great deal of research interest, owing to the therapeutic utility of the template as useful drug molecular scaffolding. We report the synthesis and pharmacological evaluation of thiophenes substituted with 4-methanesulfonyl benzoyl moiety at the fifth position of the ring, as possible anti-inflammatory lead candidates. The aryl sulfonyl methyl thiophene analogs AP29, AP82, and AP37, when screened for anti-inflammatory activity in carrageenin induced rat paw edema, an acute in vivo model, exhibited moderate to good activity at a dose level of 100 mg/kg body weight P.o compared to Ibuprofen. In a five day formalin induced rat paw edema, a chronic in vivo anti-inflammatory model, candidates AP29, AP82, and AP37 inhibited the disease progression by 53%, 34%, and 65%, respectively on the fifth day, at a dose level of 100 mg/kg body weight P.o compared to Rofecoxib, Ibuprofen, and Dexamethasone at therapeutic doses which gave a protection of 53.8%, 81.5%, and 81.5%, respectively. The replacement of the 4-methanesulfonyl benzoyl moiety in AP82 with the pyridine template, 3,5-dimethyl-4-methoxy-2-pyridyl function, gave rise to AP84, which was less active in the acute model, but gave 54% and 75% protection both during the first day and fifth day, respectively, in the chronic model. A dual mechanism of action is proposed for AP84, a non-steroidal drug which has exhibited remarkable activity when compared to the steroid dexamethasone. These results open up new avenues in designing novel anti-inflammatory drugs as dual inhibitors with the incorporation of a pyridine template as part of the pharmacophore.     

Macroaffinity ligand-facilitated three-phase partitioning (MLFTPP) for purification of xylanase

It is shown that eudragit S-100, a copolymer of methylacrylic acid and methylmethacrylate, undergoes three-phase partitioning. It was found that 95% eudragit S-100 could be recovered as the interfacial precipitate by using 30% (w/v) ammonium sulfate, 1:1 ratio of t-butanol to polymer solution at 40 degrees C. Three-phase partitioning of proteins uses simultaneous addition of ammonium sulfate and t-butanol to precipitate proteins in an interfacial layer separating the aqueous phase and organic solvent. Exploiting the affinity of xylanases towards eudragit S-100, it was possible to purify xylanase from Aspergillus niger; 60% recovery of activity with 95-fold purification could be obtained by this process. The purified enzyme showed A single band on SDS-PAGE. The technique shows promise to develop into a general method that could be termed "macroaffinity ligand-facilitated three-phase partitioning (MLFTPP).     

CYP1A1*2 and CYP1A1*3 polymorphisms cosegregate in the Indian population

Several ethnic groups have been genotyped for polymorphisms at the CYP1A1 gene locus that encodes the enzyme that catalyzes the initial step in the metabolism of polycyclic aromatic hydrocarbons. Two of the CYP1A1 polymorphisms, namely, CYP1A1*2 and CYP1A1*3 are reported to cosegregate among the Japanese and to a lesser extent in Caucasians, but not in people of African descent. In the absence of such information in the Indian population, the frequency of the CYP1A1*2 polymorphism was determined in this study, using DNA samples from 649 ethnic Indians who had been earlier genotyped for the CYP1A1*3 polymorphism. Analysis of the combined genotype data revealed that the two polymorphisms cosegregate in the Indian population.     

The interdomain region of dengue NS5 protein that binds to the viral helicase NS3 contains independently functional importin beta 1 and importin alpha/beta-recognized nuclear localization signals

Dengue virus NS5 protein is a multifunctional RNA-dependent RNA polymerase that is essential for virus replication. We have shown previously that the 37- amino acid interdomain spacer sequence (residues (369)X(2)KKX(14)KKKX(11)RKX(3)405) of Dengue2 NS5 contains a functional nuclear localization signal (NLS). In this study, beta-galactosidase fusion proteins carrying point mutations of the positively charged residues or truncations of the interdomain linker region (residues 369-389 or residues 386-405) were analyzed for nuclear import and importin binding activities to show that the N-terminal part of the linker region (residues 369-389, a/bNLS) is critical for nuclear localization and is recognized with high affinity by the conventional NLS-binding importin alpha/beta heterodimeric nuclear import receptor. We also show that the importin beta-binding site (residues 320-368, bNLS) adjacent to the a/bNLS, previously identified by yeast two-hybrid analysis, is functional as an NLS, recognized with high affinity by importin beta, and able to target beta-galactosidase to the nucleus. Intriguingly, the bNLS is highly conserved among Dengue and related flaviviruses, implying a general role for the region and importin beta in the infectious cycle.     

Alpha-crystallin binds to the aggregation-prone molten-globule state of alkaline protease: implications for preventing irreversible thermal denaturation

Alpha-crystallin, the major eye-lens protein with sequence homology with heat-shock proteins (HSPs), acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain more insight into its chaperoning ability, we used a protease as the model system that is known to require a propeptide (intramolecular chaperone) for its proper folding. The protease ("N" state) from Conidiobolus macrosporus (NCIM 1298) unfolds at pH 2.0 ("U" state) through a partially unfolded "I" state at pH 3.5 that undergoes transition to a molten globule-(MG) like "I(A)" state in the presence of 0.5 M sodium sulfate. The thermally-stressed I(A) state showed complete loss of structure and was prone to aggregation. Alpha-crystallin was able to bind to this state and suppress its aggregation, thereby preventing irreversible denaturation of the enzyme. The alpha-crystallin-bound I(A) state exhibited native-like secondary and tertiary structure showing the interaction of alpha-crystallin with the MG state of the protease. 8-Anilinonaphthalene sulphonate (ANS) binding studies revealed the involvement of hydrophobic interactions in the formation of the complex of alpha-crystallin and protease. Refolding of acid-denatured protease by dilution to pH 7.5 resulted in aggregation of the protein. Unfolding of the protease in the presence of alpha-crystallin and its subsequent refolding resulted in the generation of a near-native intermediate with partial secondary and tertiary structure. Our studies represent the first report of involvement of a molecular chaperone-like alpha-crystallin in the unfolding and refolding of a protease. Alpha-crystallin blocks the unfavorable pathways that lead to irreversible denaturation of the alkaline protease and keeps it in a near-native, folding-competent intermediate state.     

Three phase partitioning for extraction of oil from soybean

Three phase partitioning, a method generally used for protein separation, has been evaluated for extraction of oil from soybean. 82% oil was extracted within 1 h using this process which required simultaneous addition of t-butanol (1:1, v/v) and 30% ammonium sulphate to the soybean slurry.     

Conformation, orientation and dynamics of dodecylphosphocholine in micellar aggregate: a 3.2 ns molecular dynamics simulation study

A dodecylphosphocholine micelle of 86 monomers with 5776 water molecules has been simulated under NPT conditions for 3.2 ns using GROMACS2.0. The micelle was found to be very dynamic. Some of the C-C bonds, independent of their position in the DPC monomer, adopt gauche conformation and the trans <--> gauche transitions are quite frequent. An average of about 11% of the C-C bonds in the micelle are observed to be in the gauche conformation (i.e., |dihedral angle|< 120 degrees). The terminal methyl groups are randomly distributed all over the micelle whereas the nitrogen atom of phosphocholine headgroup atoms is restricted to the interface region. Some of the monomers were found to lie on the surface. The shape of micelle, influenced by the packing considerations, shows deviations from spherical shape. The phosphocholine headgroup is well solvated and there is no water penetration into the micelle core. The overall features of the micelle of 86 DPC monomers conforms to the lattice model of micelle proposed by Dill and Flory [Dill K A, Flory P J (1981) Proc Natl Acad Sci USA 78, 676-680] and is similar to DPC micelles of smaller aggregate sizes except for the positional preference of the C-C bonds for the gauche conformation and the trans<-->gauche transition times [Tieleman D P, van der Spoel D, Berendsen H J C (2000) J Phys Chem B 104, 6380-6388; Wymore T, Gao X F, Wong T C (1999) J Mol Struct (Theochem) 485-486, 195-210]. It appears that packing considerations play a predominant role in determining the shape and dynamics of the micelle.     

Use of growth indices from radiometric culture for quantification of sheep strains of Mycobacterium avium subsp. paratuberculosis

A simple method for using growth indices from radiometric BACTEC cultures was evaluated for the enumeration of Australian sheep strains of Mycobacterium avium subsp. paratuberculosis. The numbers of viable organisms in inocula were determined by end-point titration in BACTEC cultures. Growth indices were measured by using a BACTEC 460 machine. There was a linear relationship between the number of days taken for the cumulative growth index to reach 1,000 (dCGI1000) and log(10) inoculum size. The use of dCGI1000 was shown to be as effective as the use of growth index data from the entire growth cycle for the estimation of inoculum size. For particular isolates characterized by end-point titration, the dCGI1000 of a single BACTEC vial provided estimates of viable numbers within narrow prediction limits. Predictive relationships were also established for the enumeration of M. avium subsp. paratuberculosis from field samples by using the dCGI1000 of a single BACTEC vial, with prediction limits of +/-1 to 2 log units. Organisms from feces or contaminated soil grew more slowly than those from cultures or tissues, and separate equations were developed for enumeration from these sources.