Structure-based optimization of morpholino-triazines as PI3K and mTOR inhibitors

A virtual screen of our in-house database using various fingerprint techniques returned several triazine hits which were found to be mTOR inhibitors with a slight selectivity over PI3Kα. Using structure-guided lead optimization the inhibitory activity towards mTOR and PI3Kα was increased to the low nanomolar range. Exploiting shape differences in the binding-site allowed for the design of mTOR selective inhibitors. Focus on ligand efficiency ensured the inhibitors retained a low molecular weight and desirable drug-like properties.     

Antimicrobial activity of human β-defensin 4 analogs: Insights into the role of disulfide linkages in modulating activity

Human β-defensins (HBDs) are cationic antimicrobial peptides that are components of the innate immune system. They are characterized by three disulfide bridges. However, the number of cationic residues as well as the presence of lysine and arginine residues vary. In HBD4, the cationic residues occur predominantly in the N-terminal segment, unlike in HBD1–3. We have examined the antimicrobial activity of peptides spanning the N- and C-terminal segments of HBD4. We have introduced one, two and three disulfide bridges in the peptides corresponding to the N-terminal segments. Peptides corresponding to the N-terminal segment had identical sequences and variation was only in the number and spacing of cysteines and disulfide bridges. Antimicrobial activity to varying extents was observed for all the peptides. When two disulfide bridges were present, decrease in antimicrobial potency as well as sensitivity of activity to salt was observed. Enhanced antimicrobial activity was observed when three disulfide bridges were present. The antimicrobial potency was similar to HBD4 except against Escherichia coli and was attenuated in the presence of salt. While the presence of three disulfide bridges did not constrain the peptide to a rigid β-sheet, the activity was considerably more as compared to the peptides with one or two disulfide bridges. The peptides enter bacterial and fungal cells rapidly without membrane permeabilization and appear to exert their activity inside the cells rather than at the membrane.     

Antibacterial activity of linear peptides spanning the carboxy-terminal beta-sheet domain of arthropod defensins

The antibacterial activity of peptides without disulfide bridges, spanning the carboxy-terminal segment of arthropod defensins, has been investigated. Although all the peptides have net positive charges, they exhibited varying antibacterial potencies and spectra. Atomic force and fluorescence microscopic analyses indicate that the peptides exert their activity by permeabilizing the outer and inner membranes of Gram-negative bacteria such as Escherichia coli. It appears that the plasticity observed in the activity of mammalian defensins with respect to sequence, number of disulfide bridges or net positive charge, is also observed in insect defensins.     

Effect of site-directed mutagenesis of methylglyoxal-modifiable arginine residues on the structure and chaperone function of human alphaA-crystallin

We reported previously that chemical modification of human alphaA-crystallin by a metabolic dicarbonyl compound, methylglyoxal (MGO), enhances its chaperone-like function, a phenomenon which we attributed to formation of argpyrimidine at arginine residues (R) 21, 49, and 103. This structural change removes the positive charge on the arginine residues. To explore this mechanism further, we replaced these three R residues with a neutral alanine (A) residue one at a time or in combination and examined the impact on the structure and chaperone function. Measurement of intrinsic tryptophan fluorescence and near-UV CD spectra revealed alteration of the microenvironment of aromatic amino acid residues in mutant proteins. When compared to wild-type (wt) alphaA-crystallin, the chaperone function of R21A and R103A mutants increased 20% and 18% as measured by the insulin aggregation assay and increased it as much as 39% and 28% when measured by the citrate synthase (CS) aggregation assay. While the R49A mutant lost most of its chaperone function, R21A/R103A and R21A/R49A/R103A mutants had slightly better function (6-14% and 10-14%) than the wt protein in these assays. R21A and R103A mutants had higher surface hydrophobicity than wt alphaA-crystallin, but the R49A mutant had lower hydrophobicity. R21A and R103A mutants, but not the R49A mutant, were more efficient than wt protein in refolding guanidine hydrochloride-treated malate dehydrogenase to its native state. Our findings indicate that the positive charges on R21, R49, and R103 are important determinants of the chaperone function of alphaA-crystallin and suggest that chemical modification of arginine residues may play a role in protein aggregation during lens aging and cataract formation.     

Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from −1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions.     

Mechanistic aspects of uncatalysed and Os(VIII) catalysed oxidation of 5-flourouracil - An anticancer drug by alkaline diperiodatoargentate(III)

The oxidation of an anticancer drug 5-fluorouracil (5-FU) by diperiodatoargentate(III) (DPA) was carried out both in the absence and presence of osmium(VIII) catalyst in alkaline medium at 27 °C and a constant ionic strength of 0.20 mol dm⁻³ spectrophotometrically attached with HI-TECH SFA-12 stopped flow accessory. The oxidation products in both the cases were identified as fluoroketene and Ag(I). The stoichiometry is same in both cases, i.e., [5-FU]:[DPA] = 1:1. The reaction was of first order in both catalysed and uncatalysed cases, with respect to [DPA] and was less than unit order in [5-FU] and negative fraction in [alkali]. The order in Os(VIII) was unity. In both cases [Ag(H₃IO₆)₂]⁻ itself is the active species of DPA. The uncatalysed reaction in alkaline medium has been shown to proceed via a DPA-5-fluorouracil complex, which decomposes in a rate determining step to give the products. In catalysed reaction, it has been shown to proceed via a Os(VIII)-5-fluorouracil complex, which further reacts with one molecule of DPA in a rate determining step to give the products. The reaction constants involved in the different steps of the mechanisms were calculated for both the reactions. The catalytic constant (k_Cat.const.) was also calculated for catalysed reaction at different temperatures. The activation parameters with respect to slow step of the mechanisms were computed and discussed for both the cases. The thermodynamic quantities were also determined for both reactions.     

Purification and characterization of a solvent and detergent-stable novel protease from Bacillus cereus

A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28 kDa, pH and temperature optima for this protease were 10 and 60 °C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu²⁺ ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents.     

Mutations in Arabidopsis Fatty Acid Amide Hydrolase Reveal That Catalytic Activity Influences Growth but Not Sensitivity to Abscisic Acid or Pathogens

Fatty acid amide hydrolase (FAAH) terminates the endocannabinoid signaling pathway that regulates numerous neurobehavioral processes in animals by hydrolyzing N-acylethanolamines (NAEs). Recently, an Arabidopsis FAAH homologue (AtFAAH) was identified, and several studies, especially those using AtFAAH overexpressing and knock-out lines, have suggested an in vivo role for FAAH in the catabolism of NAEs in plants. We previously reported that overexpression of AtFAAH in Arabidopsis resulted in accelerated seedling growth, and in seedlings that were insensitive to exogenous NAEs but hypersensitive to abscisic acid (ABA) and hypersusceptible to nonhost pathogens. Here we show that whereas the enhanced growth and NAE tolerance of the AtFAAH overexpressing seedlings depend on the catalytic activity of AtFAAH, hypersensitivity to ABA and hypersusceptibility to nonhost pathogens are independent of its enzymatic activity. Five amino acids known to be critical for rat FAAH activity are also conserved in AtFAAH (Lys-205, Ser-281, Ser-282, Ser-305, and Arg-307). Site-directed mutation of each of these conserved residues in AtFAAH abolished its hydrolytic activity when expressed in Escherichia coli, supporting a common catalytic mechanism in animal and plant FAAH enzymes. Overexpression of these inactive AtFAAH mutants in Arabidopsis showed no growth enhancement and no NAE tolerance, but still rendered the seedlings hypersensitive to ABA and hypersusceptible to nonhost pathogens to a degree similar to the overexpression of the native AtFAAH. Taken together, our findings suggest that the AtFAAH influences plant growth and interacts with ABA signaling and plant defense through distinctly different mechanisms.     

The interaction between L1-type proteins and ankyrins - a master switch for L1-type CAM function

L1-type cell adhesion molecules (CAMs) are important mediators of neural differentiation, including axonal outgrowth and pathfinding and also of synapse formation and maintenance. In addition, their interactions with cytoskeletal components are highly conserved and regulated. How these different aspects of CAM functionality relate to each other is not well understood. Based on results from our and other laboratories we propose that ankyrin-binding to L1-type CAMs provides a master switch. The interaction with ankyrins directs L1-type adhesive proteins into different functional contexts, either ankyrin-independent functions, such as neurite outgrowth and axonal pathfinding or into ankyrin-dependent functions, such as L1’s role at axon initial segments (AIS), paranodal regions, synapses and in dendrites. The content of this Mini review was first presented in a shortened form at the 12th Mejbaum-Katzenellenbogen Seminar “Membrane Skeleton. Recent Advances and Future Research Directions”, June 15–18, 2008, Zakopane, Poland