18 research outputs found
Molecular modelling for the design of chimaeric biomimetic dye—ligands and their interaction with bovine heart mitochondrial malate dehydrogenase
Dye-affinity labelling of bovine heart mitochondrial malate dehydrogenase and study of the NADH-binding site
Isoenzyme- and Allozyme-Specific Inhibitors: 2,20-Dihydroxybenzophenones and Their Carbonyl N-Analogues that Discriminate between Human Glutathione Transferase A1-1 and P1-1 Allozymes
The selectivity of certain benzophenones and their carbonyl N-analogues was investigated towards the human GSTP1-1 allozymes A, B and C involved in MDR. The allozymes were purified from extracts derived from E. coli harbouring the plasmids pEXP5-CT/TOPO-TAhGSTP1* A, pOXO4-hGSTP1*B or pOXO4-hGSTP1*C. Compound screening with each allozyme activity indicated three compounds with appreciable inhibitory potencies, 12 and 13 with P1-1A 62% and 67%, 11 and 12 with P1-1C 51% and 70%, whereas that of 15 fell behind with P1-1B (41%). These findings were confirmed by IC50 values (74–125 lM). Enzyme inhibition kinetics, aided by molecular modelling and docking, revealed that there is competition with the substrate CDNB for the same binding site on the allozyme (Ki(13/ A) = 63.6 +- 3.0 lM, K (15/B) = 198.6 +- 14.3 lM, and Ki(11/ C) = 16.5 +- 2.7 lM). These data were brought into context by an in silico structural comparative analysis of the targeted proteins. Although the screened compounds showed moderate inhibitory potency against hGSTP1-1, remarkably, some of them demonstrated absolute isoenzyme and/or allozyme selectivity
Synthesis and Study of 2‑(Pyrrolesulfonylmethyl)‑N‑arylimines: A New Class of Inhibitors for Human Glutathione Transferase A1‑1
Overexpression of human GSTA1-1 in tumor cells is part of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfanyl]-1H-pyrrole. Molecular modeling revealed two locations in the enzyme H binding site: the catalytic primary one accommodating shorter and longer derivatives and the secondary one, where shorter derivatives can occupy. Derivative 9, displaying the highest inhibition and bearing a p-nitroarylimino moiety, and derivative 4, lacking this moiety, were studied kinetically. Derivative 9 binds (Ki(9) = 71 ± 4 μM) at the primary site competitively vs CDNB. Derivative 4 binds (Ki(4) = 135 ± 27 μM) at the primary and secondary sites, allowing the binding of a second molecule (4 or CDNB) leading to formation of unreactive and reactive complexes, respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative 9 may serve as a lead structure
Designer Xanthone An Inhibitor Scaffold for MDR-Involved Human Glutathione Transferase Isoenzyme A1-1
Glutathione transferases (GSTs) are cell detoxifiers involved in multiple drug resistance (MDR), hampering the effectiveness of certain anticancer drugs. To our knowledge, this is the first report on well-defined synthetic xanthones as GST inhibitors. Screening 18 xanthones revealed three derivatives bearing a bromomethyl and a methyl group (7) or two bromomethyl groups (8) or an aldehyde group (17), with high inhibition potency (>85%), manifested by low IC50 values (7: 1.59 ± 0.25 μM, 8: 5.30 ± 0.30 μM, and 17: 8.56 ± 0.14 μM) and a competitive modality of inhibition versus CDNB (Ki(7) = 0.76 ± 0.18 and Ki(17) = 1.69 ± 0.08 μM). Of them, derivative 17 readily inhibited hGSTA1-1 in colon cancer cell lysate (IC50 = 10.54 ± 2.41 μM). Furthermore, all three derivatives were cytotoxic to Caco-2 intact cells, with 17 being the least cytotoxic (LC50 = 151.3 ± 16.3 μM). The xanthone scaffold may be regarded as a pharmacophore for hGSTA1-1 and the three derivatives, especially 17, as potent precursors for the synthesis of new inhibitors and conjugate prodrugs for human GSTs
2,20-Dihydroxybenzophenones and their carbonyl N-analogues as inhibitor scaffolds for MDR-involved human glutathione transferase isoenzyme A1-1
The MDR-involved human GSTA1-1, an important isoenzyme overexpressed in several tumors leading to chemotherapeutic-resistant tumour cells, has been targeted by 2,2′-dihydroxybenzophenones and some of their carbonyl N-analogues, as its potential inhibitors. A structure-based library of the latter was built-up by a nucleophilic cleavage of suitably substituted xanthones to 2,2′-dihydroxy-benzophenones (5–9) and subsequent formation of their N-derivatives (oximes 11–13 and N-acyl hydrazones 14–16). Screening against hGSTA1-1 led to benzophenones 6 and 8, and hydrazones 14 and 16, having the highest inhibition potency (IC50 values in the range 0.18 ± 0.02 to 1.77 ± 0.10 μM). Enzyme inhibition kinetics, molecular modeling and docking studies showed that they interact primarily at the CDNB-binding catalytic site of the enzyme. In addition, the results from cytotoxicity studies with human colon adenocarcinoma cells showed low LC50 values for benzophenone 6 and its N-acyl hydrazone analogue 14 (31.4 ± 0.4 μM and 87 ± 1.9 μM, respectively), in addition to the strong enzyme inhibition profile (IC50(6) = 1,77 ± 0.10 μM; IC50(14) = 0.33 ± 0.05 μM). These structures may serve as leads for the design of new potent mono- and bi-functional inhibitors and pro-drugs against human GTSs
Structural and Evolutionary Insights within the Polysaccharide Deacetylase Gene Family of Bacillus anthracis and Bacillus cereus
Functional and folding constraints impose interdependence between interacting sites along the protein chain that are envisaged through protein sequence evolution. Studying the influence of structure in phylogenetic models requires detailed and reliable structural models. Polysaccharide deacetylases (PDAs), members of the carbohydrate esterase family 4, perform mainly metal-dependent deacetylation of O- or N-acetylated polysaccharides such as peptidoglycan, chitin and acetylxylan through a conserved catalytic core termed the NodB homology domain. Genomes of Bacillus anthracis and its relative Bacillus cereus contain multiple genes of putative or known PDAs. A comparison of the functional domains of the recently determined PDAs from B. anthracis and B. cereus and multiple amino acid and nucleotide sequence alignments and phylogenetic analysis performed on these closely related species showed that there were distinct differences in binding site formation, despite the high conservation on the protein sequence, the folding level and the active site assembly. This may indicate that, subject to biochemical verification, the binding site-forming sequence fragments are under functionally driven evolutionary pressure to accommodate and recognize distinct polysaccharide residues according to cell location, use, or environment. Finally, we discuss the suggestion of the paralogous nature of at least two genes of B. anthracis, ba0330 and ba0331, via specific differences in gene sequence, protein structure, selection pressure and available localization patterns. This study may contribute to understanding the mechanisms under which sequences evolve in their structures and how evolutionary processes enable structural variations
Functional significance of the rare rs35667974 IFIH1 gene polymorphism, associated with multiple autoimmune diseases, using a structural biological approach
Autoimmune diseases, which affect approximately 5% of human population, are a range of diseases in which the immune response to self-antigens results in damage or dysfunction of tissues. Recent genome wide association studies (GWAS) have successfully identified novel autoimmune disease-associated loci, with many of them shared by multiple disease-associated pathways but much of the genetics and pathophysiological mechanisms remain still obscure. Considering that most of the potential causal variants are still unknown, many studies showed that the missense variant rs35667974 at interferon-induced with helicase C domain 1 (IFIH1) gene is protective for type 1 diabetes (T1D), psoriasis (PS) and psoriatic arthritis (PsA). Recently, this variant was found to be also associated with ankylosing spondylitis (AS), Crohn’s disease (CD) and ulcerative colitis (UC). The IFIH1 gene encodes a cytoplasmic RNA helicase otherwise known as melanoma differentiation-associated 5 (MDA5) that recognizes viral RNA and is involved in innate immunity through recognition of viral RNA. In the present study we sought to investigate the association of the rare rs35667974 variant of IFIH1 gene, which resides in exon 14 and changes a conserved isoleucine at position #923 to valine, in the development of various autoimmune diseases and give a reason for the selectivity affecting different autoimmune diseases. Evolutionary studies and three-dimensional (3 D) homology modelling were employed on the MDA5 protein product, through its association with dsRNA, recognition factor controlling cytokine and chemokine signalling, to investigate the protective role of the MDA5 variant for certain autoimmune diseases