ABSTRACT
Eukaryotic translation initiation factor 5A (eIF5A) is a small acidic protein highly conserved from archaea to mammals. eIF5A is the only protein which undergoes a unique lysine residue modification to hypusine. Hypusinylation is important for the function of eIF5A which is reported to be essential for cell viability. eIF5A promotes formation of the first peptide bond at the onset of protein synthesis. However, its function in Leishmania donovani is unclear. The present study focuses on the characterization and localization of L. donovani eIF5A protein. The eIF5A gene contains an ORF of 501×bp encoding 166 amino acid residues with a predicted molecular mass and isoelectric point of 17.8 kDa and 4.83 respectively. A phylogenetic tree analysis revealed its close proximity to trypanosomes however it is distantly located from Trichomonas vaginalis and Plasmodium falciparum. The L. donovani eIF5A was expressed as a 6× His tagged protein whose identity was confirmed by western blot and MALDI. Biophysical investigation by CD revealed the predominant presence of 49% ß sheet structure which correlated well with secondary structure prediction. To gain insight into the role of eIF5A in L. donovani, we investigated the subcellular distribution of eIF5A. A GFP-fusion of L. donovani eIF5A was found to be localized in cytoplasm as confirmed by subcellular fractionation. Our studies indicated that eIF5A is primarily localized to cytoplasm and is undetectable in nuclear fraction. The homology model of eIF5A of L. donovani was built and the resulting model showed acceptable Ramachandran statistics. The model is reliable and can be used to study eIF5A binding with its effector molecules.
Subject(s)
Leishmania donovani/enzymology , Peptide Initiation Factors/analysis , Peptide Initiation Factors/genetics , RNA-Binding Proteins/analysis , RNA-Binding Proteins/genetics , Cell Nucleus/chemistry , Circular Dichroism , Cytoplasm/chemistry , Isoelectric Point , Models, Molecular , Molecular Weight , Open Reading Frames , Peptide Initiation Factors/chemistry , Phylogeny , Protein Conformation , RNA-Binding Proteins/chemistry , Sequence Homology, Amino Acid , Subcellular Fractions/chemistry , Eukaryotic Translation Initiation Factor 5AABSTRACT
This study deals with the generation and characterization of various solid-state forms of mebendazole (MBZ), a benzimidazole antihelmentic. The drug was subjected to polymorphic screen using different solvents to explore the possibility of existence of different solid forms. Different reported polymorphic forms of MBZ, i.e. form A, B and C were found to be recrystallized from acetic acid:methanol mixture (1:1), ethyl acetate and methanol, respectively. N,N-Dimethyl acetamide (DMA) and N,N-dimethyl formamide (DMF) yielded two new solvates of MBZ. These solid-state forms were characterized by thermoanalytical (DSC, TGA, HSM), crystallographic (XRD), microscopic (optical, polarized), and spectroscopic (FTIR) techniques. Solubility studies were carried out for the solvates to identify the solubility advantage. Molecular modeling studies revealed moderately strong hydrogen bonding between the solvent molecules and MBZ.
Subject(s)
Antinematodal Agents/chemistry , Mebendazole/chemistry , Calorimetry, Differential Scanning , Chemistry, Pharmaceutical , Models, Molecular , Solubility , Solvents , Spectroscopy, Fourier Transform Infrared , Thermogravimetry , X-Ray DiffractionABSTRACT
Recent trends in drug discovery include methods to identify dual and triple activating drugs. This approach is being successfully employed in malaria, cancer, asthma, insulin resistance, etc. Molecular field analysis has been employed in correlating pharmacological data and field parameters. In this paper we introduce the concept of additivity of molecular fields to correlate molecular fields of dual activators and their pIC(50) values. PPARalpha and PPARgamma dual activators, which affect hypertriglyceridemia and hyperglycemia, have been chosen to validate the molecular field additivity concept. Three CoMFA models namely alpha-model, gamma-model and dual-model have been developed. The validity of this concept has been ascertained by (a) comparing contour maps, (b) by comparing CoMFA results with FlexX docking results and (c) by analyzing newly designed molecules.
Subject(s)
PPAR alpha/agonists , PPAR alpha/chemistry , PPAR gamma/agonists , PPAR gamma/chemistry , Benzofurans/chemistry , Binding Sites , Drug Design , Ligands , Models, Molecular , Oxadiazoles/chemistry , Oxazines/chemistry , Protein Binding , Quantitative Structure-Activity Relationship , Thiazoles/chemistryABSTRACT
Comparative molecular field analysis and comparative molecular similarity indices analysis were performed on 114 analogues of 1,2-diarylimidazole to optimize their cyclooxygenase-2 (COX-2) selective antiinflammatory activities. These studies produced models with high correlation coefficients and good predictive abilities. Docking studies were also carried out wherein these analogues were docked into the active sites of both COX-1 and COX-2 to analyze the receptor ligand interactions that confer selectivity for COX-2. The most active molecule in the series (53) adopts an orientation similar to that of SC-558 (4-[5-(4-bromophenyl)-3-trifluoromethyl-1H-1-pyrozolyl]-1-benzenesulfonamide) inside the COX-2 active site while the least active molecule (101) optimizes in a different orientation. In the active site, there are some strong hydrogen-bonding interactions observed between residues His90, Arg513, and Phe518 and the ligands. Additionally, a correlation of the quantitative structure-activity relationship data and the docking results is found to validate each other and suggests the importance of the binding step in overall drug action.
Subject(s)
Cyclooxygenase Inhibitors/chemistry , Imidazoles/chemistry , Isoenzymes/chemistry , Prostaglandin-Endoperoxide Synthases/chemistry , Binding Sites , Cyclooxygenase 1 , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Drug Design , Molecular Conformation , Quantitative Structure-Activity RelationshipABSTRACT
A variety of physico-chemical methods employ chemical denaturants to unfold proteins, and study different biophysical processes involved therein. Chemical denaturants are believed to induce unfolding by stabilizing the unfolded state of proteins over the folded state, either macroscopically or through specific interactions. In order to characterize the nature of specific interactions between proteins and denaturants, we have solved crystal structures of hen egg-white lysozyme complexed with denaturants, and report here dimethyl sulfoxide and guanidinium chloride complexes. The dimethyl sulfoxide molecules and guanidinium ions were seen to bind the protein at specific sites and were involved in characteristic interactions. They share a major binding site between them, the C site in the sugar binding cleft of the enzyme. Although the overall conformations of the complexes were very similar to the native structure, spectacular conformational changes were seen to occur locally. Temperature factors were also seen to drop dramatically in the local regions close to the denaturant binding sites. An interesting observation of the present study was the generation of a sodium ion binding site in hen egg-white lysozyme in the presence of denaturants, which was hitherto unknown in any of the other lysozyme structures solved so far. Loss of some of the crucial side chain-main chain interactions may form the initial events in lysozyme unfolding.
