Design and synthesis of 6,6-fused heterocyclic amides as raf kinase inhibitors

Compounds belonging to several scaffolds-quinazolines, quinolines and quinoxalines-were designed and synthesized as Raf kinase inhibitors. Scaffolds were assessed for in vitro Braf(V600E) inhibition, and overall kinase selectivity. Pharmacokinetic parameters for one of the scaffolds were also determined.     

Studies onplant aspartate transcarbamylase: Regulatory properties of the enzyme from mung bean (Phaseolus aureus) seedlings

Aspartate transcarbamylase (EC 2·1·3·2) purified from mung bean seedlings was used as a model to understand the mechanism of allosteric regulation. The enzyme exhibited homotropic interactions with carbamyl phosphate. Preincubation of the enzyme with aspartate abolished the sigmoidicity of the carbamyl phosphate saturation curve. UMP was the most potent inhibitor of the reaction and was noncompetitive with respect to aspartate. The sigmoidicity of carbamyl phosphate saturation curves increased with increase in UMP concentration. These results were analysed by an iterative least squares procedure. There was no change inV _max values with increase in the UMP concentration, although theK _0·5 values (concentration of carbamyl phosphate required to reach half maximal velocity) increased. This implied that the effect of UMP was on the binding of carbamyl phosphate only and not on the catalytic function of the enzyme. The allosteric properties of the enzyme could be explained in terms ofK system of the symmetry model. The values of the allosteric constantsn, L andc calculated for mung bean enzyme, making use of the Monod equation accounted for all the observed properties. The enzyme appeared to be a tetramer (n=4) and in the absence of ligands was predominantly in theT form (L _o= 2·25). Carbamyl phosphate bound preferentially to theR form (c= 10_?3), while UMP bound preferentially to theT form and hence these two ligands exhibited the typical heterotropic interactions as expected of antagonistic ligands.     

Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase

The crystal structure of human liver cytosolic recombinant serine hydroxymethyltransferase (hcSHMT) suggested that Ser53 and Arg 263 could participate in the reaction catalyzed by SHMT. The mutation of Arg262 (corresponding to Arg263 in hcSHMT) to "A" in sheep liver cytosolic SHMT (scSHMT) resulted in a 5-fold increase in Km for L-Ser and a 5-fold decrease in kcat compared to scSHMT. Further, in R262A SHMT-glycine complex, the peak at 343 nm (geminal diamine) was more pronounced, compared to wild-type enzyme. Stopped-flow studies showed that the rate constant for the formation of glycine-geminal diamine for R262A SHMT was also decreased. The rate of reaction, concentration of spectral intermediates, fluorescence excitation maximum of glycine geminal diamine and interaction with methoxyamine were altered in R262A SHMT. Although Arg263 in hcSHMT is located outside the PLP binding pocket, it positions Tyr73 for interaction with PLP, by forked H-bonding with the carbonyl groups of main chain residues, Asn71 and Lys72 of the other subunit of the tight dimer. Mutation of Arg262 to Ala and the consequent alteration in orientation of PLP leads to decreased catalytic efficiency. Ser53 (in hcSHMT) is in hydrogen bonding distance to one of the carboxylate oxygens of the amino acid substrate, which also interacts with Tyr83 and Arg402. Replacement of Ser53 with Cys (using 'O' software program) in the structure of hcSHMT resulted in disruption of these interactions, whereas replacement with Ala (S53A) only weakened the substrate interactions. There was a 10-fold increase in Km and 20-fold decrease in catalytic activity efficiency for S52C SHMT, whereas S52A SHMT retained 20% of the activity without change in Km for serine. These results suggest that S52 affects substrate binding and catalysis.     

Structure-function relationship in serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5'-phosphate (PLP)-dependent enzyme catalyzes the tetrahydrofolate (H(4)-folate)-dependent retro-aldol cleavage of serine to form 5,10-methylene H(4)-folate and glycine. The structure-function relationship of SHMT was studied in our laboratory initially by mutation of residues that are conserved in all SHMTs and later by structure-based mutagenesis of residues located in the active site. The analysis of mutants showed that K71, Y72, R80, D89, W110, S202, C203, H304, H306 and H356 residues are involved in maintenance of the oligomeric structure. The mutation of D227, a residue involved in charge relay system, led to the formation of inactive dimers, indicating that this residue has a role in maintaining the tetrameric structure and catalysis. E74, a residue appropriately positioned in the structure of the enzyme to carry out proton abstraction, was shown by characterization of E74Q and E74K mutants to be involved in conversion of the enzyme from an 'open' to 'closed' conformation rather than proton abstraction from the hydroxyl group of serine. K256, the residue involved in the formation of Schiffs base with PLP, also plays a crucial role in the maintenance of the tetrameric structure. Mutation of R262 residue established the importance of distal interactions in facilitating catalysis and Y82 is not involved in the formaldehyde transfer via the postulated hemiacetal intermediate but plays a role in stabilizing the quinonoid intermediate. The mutational analysis of scSHMT along with the structure of recombinant Bacillus stearothermophilus SHMT and its substrate(s) complexes was used to provide evidence for a direct transfer mechanism rather than retro-aldol cleavage for the reaction catalyzed by SHMT.     

Role of metal ion-mediated interactions in the assembly and stability of Sesbania mosaic virus T=3 and T=1 capsids

Sesbania mosaic virus (SeMV) capsids are stabilized by RNA-protein, protein-protein and calcium-mediated protein-protein interactions. The removal of calcium has been proposed to be a prerequisite for the disassembly of the virus. The crystal structure of native T=3 SeMV capsid revealed that residues D146 and D149 from one subunit and Y205, N267 and N268 of the neighboring subunit form the calcium-binding site (CBS). The CBS environment is found to be identical even in the recombinant CP-NDelta65 T=1 capsids. Here, we have addressed the role of calcium and the residues involved in calcium co-ordination in the assembly and stability of T=3 and T=1 capsids by mutational analysis. Deletion of N267 and N268 did not affect T=3 or T=1 assembly, although the capsids were devoid of calcium, suggesting that assembly does not require calcium ions. However, the stability of the capsids was reduced drastically. Site-directed mutagenesis revealed that either a single mutation (D149N) or a double mutation (D146N-D149N) of SeMV coat protein affected drastically both the assembly and stability of T=3 capsids. On the other hand, the D146N-D149N mutation in CP-NDelta65 did not affect the assembly of T=1 capsid, although their stability was reduced considerably. Since the major difference between the T=3 and T=1 capsids is the absence of the N-terminal arginine-rich motif (N-ARM) and the beta-annulus from the subunits forming the T=1 capsids, it is possible that D149 initiates the N-ARM-RNA interactions that lead to the formation of the beta-annulus, which is essential for T=3 capsid assembly.     

T=1 capsid structures of Sesbania mosaic virus coat protein mutants: determinants of T=3 and T=1 capsid assembly

Sesbania mosaic virus particles consist of 180 coat protein subunits of 29kDa organized on a T=3 icosahedral lattice. N-terminal deletion mutants of coat protein that lack 36 (CP-NDelta36) and 65 (CP-NDelta65) residues from the N terminus, when expressed in Escherichia coli, produced similar T=1 capsids of approximate diameter 20nm. In contrast to the wild-type particles, these contain only 60 copies of the truncated protein subunits (T=1). CP-NDelta65 lacks the "beta-annulus" believed to be responsible for the error-free assembly of T=3 particles. Though the CP-NDelta36 mutant has the beta-annulus segment, it does not form a T=3 capsid, presumably because it lacks an arginine-rich motif found close to the amino terminus. Both CP-NDelta36 and CP-NDelta65 T=1 capsids retain many key features of the T=3 quaternary structure. Calcium binding geometries at the coat protein interfaces in these two particles are also nearly identical. When the conserved aspartate residues that coordinate the calcium, D146 and D149 in the CP-NDelta65, were mutated to asparagine (CP-NDelta65-D146N-D149N), the subunits assembled into T=1 particles but failed to bind calcium ions. The structure of this mutant revealed particles that were slightly expanded. The analysis of the structures of these mutant capsids suggests that although calcium binding contributes substantially to the stability of T=1 particles, it is not mandatory for their assembly. In contrast, the presence of a large fraction of the amino-terminal arm including sequences that precede the beta-annulus and the conserved D149 appear to be indispensable for the error-free assembly of T=3 particles.     

Purification and kinetic mechanism of 5,10-metliyienetetrahydrofolate reductase from sheep liver

5,10-Methylenetetrahydrofolate reductase (EC 1.1.1.68) was purified from the cytosolic fraction of sheep liver by (NH₄)₂ SO₄ fractionation, acid precipitation, DEAE-Sephacel chromatography and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was established by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, ultracentrifugation and Ouchterlony immunodiffusion test. The enzyme was a dimer of molecular weight 1,66,000 ± 5,000 with a subunit molecular weight of 87,000 ±5,000. The enzyme showed hyperbolic saturation pattern with 5-methyltetrahydrofolate.K _0.5 values for 5-methyltetrahydrofolate menadione and NADPH were determined to be 132 ΜM, 2.45 ΜM and 16 ΜM. The parallel set of lines in the Lineweaver-Burk plot, when either NADPH or menadione was varied at different fixed concentrations of the other substrate; non-competitive inhibition, when NADPH was varied at different fixed concentrations of NADP; competitive inhibition, when menadione was varied at different fixed concentrations of NADP and the absence of inhibition by NADP at saturating concentration of menadione, clearly established that the kinetic mechanism of the reaction catalyzed by this enzyme was ping-pong.     

Structure and function of enzymes involved in the anaerobic degradation of L-threonine to propionate

In Escherichia coli and Salmonella typhimurium, L-threonine is cleaved non-oxidatively to propionate via 2-ketobutyrate by biodegradative threonine deaminase, 2-ketobutyrate formate-lyase (or pyruvate formate-lyase), phosphotransacetylase and propionate kinase. In the anaerobic condition, L-threonine is converted to the energy-rich keto acid and this is subsequently catabolised to produce ATP via substrate-level phosphorylation, providing a source of energy to the cells. Most of the enzymes involved in the degradation of L-threonine to propionate are encoded by the anaerobically regulated tdc operon. In the recent past, extensive structural and biochemical studies have been carried out on these enzymes by various groups. Besides detailed structural and functional insights, these studies have also shown the similarities and differences between the other related enzymes present in the metabolic network. In this paper, we review the structural and biochemical studies carried out on these enzymes.     

TIM-4 expressed on APCs induces T cell expansion and survival

TIM (T cell, Ig, mucin) proteins can regulate T cell immune responses. Tim-4 mRNA is not expressed in T cells, but exclusively in APCs. Tim-4 is a ligand for Tim-1 and Tim-4.Ig fusion protein was shown to either inhibit or expand T cells. However, the molecular basis for such opposite effects was not defined. By generating mAbs, we show that expression of Tim-4 protein is restricted to CD11c(+) and CD11b(+) cells and is up-regulated upon activation. We show that Tim-4 specifically phosphorylates Tim-1 and induces T cell expansion by enhancing cell division and reducing apoptosis. Tim-4 also induces the phosphorylation of signaling molecules LAT, Akt, and ERK1/2 in T cells. Tim-4, expressed on APCs, is a costimulatory molecule that promotes T cell expansion and survival by cross-linking Tim-1 on T cells.