Molecular and catalytic characterization of the herbicide-inducible glutathione transferases from Phaseolus vulgaris

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris

Biocatalysis, Enzyme Engineering and Biotechnology

Enzymes are biocatalysts evolved in nature to achieve the speed and coordination of nearly all the chemical reactions that define cellular metabolism necessary to develop and maintain life. The application of biocatalysis is growing rapidly, since enzymes offer potential for many exciting applications in industry. The advent of whole genome sequencing projects enabled new approaches for biocatalyst development, based on specialised methods for enzyme heterologous expression and engineering. The engineering of enzymes with altered activity, specificity and stability, using sitedirected mutagenesis and directed evolution techniques are now well established. Over the last decade, enzyme immobilisation has become important in industry. New methods and techniques for enzyme immobilisation allow for the reuse of the catalysts and the development of efficient biotechnological processes. This chapter reviews advances in enzyme technology as well as in the techniques and strategies used for enzyme production, engineering and immobilisation and discuss their advantages and disadvantages

MDR-involved human glutathione transferases (hGSTs) are targets for inhibition by 2,2'-dihydroxybenzophenones and N-carbonyl analogues

Over expression of human GSTA1-1 in tumour cells is part of MDR mechanisms. Substituted 2-hydroxybenzophenones are ubiquitous in naturally occurring and synthetic compounds, exhibiting important biological activities. 2,2’-Dihydroxybenzophenones and N-carbonyl analogues, structurally, are ringopened forms of xanthone analogues which we reported recently as hGSTA1-1 inhibitors. The present study combined GST inhibition screening, in silico molecular docking and enzyme inhibition kinetics, revealing four analogues with strong inhibitory potency (IC50 = 0.18-1.8 μM) and modest cytotoxic activity for Caco2 cell line (LC50 = 35 to > 400 μM), thus being useful as lead structures for the design of new inhibitors against hGSTs

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

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

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