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Journal ArticleDOI

2,2′-Dihydroxybenzophenones and their carbonyl N-analogues as inhibitor scaffolds for MDR-involved human glutathione transferase isoenzyme A1-1

TL;DR: 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.
About: This article is published in Bioorganic & Medicinal Chemistry.The article was published on 2014-08-01 and is currently open access. It has received 15 citations till now.

Summary (4 min read)

Introduction

  • 1 o-Hydroxybenzophenone derivatives, in particular, are ubiquitous in naturally occurring and synthetic compounds.
  • It is reasonable to assume that the carbonyl and the o-hydroxyl groups are major determinants of this activity.
  • 15 Each monomer has an a/b domain and a large a-helical domain.
  • Following GST inhibition screening, in silico molecular docking and enzyme inhibition kinetics, analogues exhibiting satisfactory inhibitory potency would be regarded as ‘leads’ in designing new inhibitors and respective prodrugs for human GSTs of medical importance.

2.1.1. Synthesis

  • Benzophenones, carrying hydroxyl groups ortho-disposed to the carbonyl group, interfere in various transformations, thus, necessitating their protection and subsequent deprotection.
  • 25 Indeed, the cleavage to 5 may be accomplished, under mild conditions, using coordination complexes of 1 with transition metals (Cr, Fe, Ir)25 or using strong alkali, under either mild25 or forcing conditions.
  • The forcing conditions can be offset by the potential of any further desired functionalization on the regioselectively substituted 5–9, either on the aryl rings or on the carbonyl moiety.
  • 26 Furthermore, KOH/DMSO, acting as a superbase, has been recently found27 to effect the cross-coupling of a phenol with an aromatic halide, under mild conditions.
  • In ascending order of inhibition potency against hGSTA1-1. b Mean value of three enzyme assays (25 lM analogue; error 6 3%).

2.1.2. Structure

  • The bonds linking the carbonyl with the aryl rings appear to have the same length of ca. 1.475 ÅA 0 .
  • Analogous H bonding-related geometry features are demonstrated by oximes 11–13 and hydrazones 14–16.

2.2. Screening of the compounds and selection of ‘lead structures’ as hGSTA1-1 inhibitors

  • Before embarking into the inhibition studies the authors performed control experiments with their enzyme preparation using bromosulfophthalein (BSP) as a known hGSTA1-1 inhibitor.
  • 13,19 In designing the enzyme assay protocol for screening the compounds as potential hGSTA1-1 inhibitors, the concentration of 25 lM, falling within the 1–30 lM range, suggested in bibliography as an appropriate one for inhibitor screening,35 has been chosen.
  • A more crucial factor to be decided has been the substrate concentration, [CDNB], in the enzyme inhibition assay for the screening and IC50 calculations.
  • By inspecting the location of the most favourable conformations (i.e., low energy ones) of these compounds, docked in the hGSTA1-1 binding site, the following observations are evident.
  • Upon generation of the enzyme complex, the geometry of the structures adjusts to achieving the best fit.

2.3. Studying the modality of interaction between the selected inhibitor lead structures and hGSTA1-1

  • On the basis of the ‘cherry picking’ experiments and the low IC50 values observed, enzyme inhibition kinetics on compounds 6, 8, 14 and 16 were performed in order to clarify their binding modality towards the target hGSTA1-1, a fundamental knowledge useful in inhibitor design.
  • In all four cases, two sets of experiments were implemented, each employing either CDNB (37.5–0 980 lM) or GSH (100–2500 lM) as a variable substrate, in the presence of different steady inhibitor concentrations.

2.3.1. Study of inhibitors 6 and 14

  • When using CDNB as a variable substrate, 6 and 14 displayed purely competitive inhibition kinetics on the basis of the linearity observed for both the double reciprocal Lineweaver–Burk graphs , at various steady concentrations of 6 and 14 and their respective secondary derivatives .37,38.
  • The described kinetic model is in concert with the in silico molecular docking analysis.
  • This extra added volume forces 6 and 14 to adopt new orientations upon binding to hGTA1-1 (Fig. 4), eventually leaving not enough space for a simultaneous binding of CDNB at the same binding site.
  • The equilibrium model for this type bears the assumption that the inhibitor binds to both the free enzyme and its enzyme–GSH complex, with no possibility for product formation37,38 (the respective complexes are unreactive, ‘dead-end’).
  • The model suggests that inhibitors 6 and 14 may interact at a site other than the GSH-binding site of hGSTA1-1, that being partly the catalytic CDNB-binding site, as described earlier (Fig. 3).

2.3.2. Study of inhibitors 8 and 16

  • (d) The co-substrate GSH is depicted in magenta, the S atom 1.4 program.
  • These findings predict37,38 that 16 binds to both the free enzyme and the enzyme–CDNB complex, leading to formation of at least two complexes, enzyme–16 and enzyme–CDNB–16, respectively.
  • Using GSH as a variable substrate, both 8 and 16 showed, predictably, mixed inhibition kinetics, since the lines of the Lineweaver–Burk graph intersected the left of the reciprocal velocity axis (SM-7a for 8 and SM-7b for 16).

2.4. Studying the cytotoxic activity of the selected inhibitor lead structures with human colon adenocarcinoma cell line

  • In the course of lead structure studies, it is useful to evaluate compounds not only on the basis of target enzyme activity, but also on cell-based assays.
  • For the latter application, the human colon adenocarcinoma cell line (Caco2) is a good choice, particularly for this study, because it expresses predominantly the hGSTA1-1 isoenzyme of interest.
  • 13,41,42 Therefore, the four selected compounds, 6, 8, 14 and 16, along with two control structures, benzophenone 5 and ketoxime 11, were evaluated for their cytotoxicity against Caco2 cells.

3.1. Materials and instrumentation

  • Reagents were used as commercially purchased, while solvents were purified and dried according to standard procedures.
  • Melting points were measured on an Electrothermal IA9000 Series apparatus and are uncorrected.
  • Infrared spectra were recorded on a JASCO FT/IR-5300 spectrometer as KBr discs.
  • Analytical TLC was run on Fluka Silica Gel F254.
  • Details on the synthesis of the title hydroxybenzophenone derivatives used in the present work have been described earlier by Tsoungas et al.

3.2.1. Synthesis of benzophenones 5–10 (general method)

  • The reaction mixture is then concentrated in vacuo and the residue is treated with icewater, slowly acidified with concentrated HCl to pH 3 and exhaustively extracted with dichloromethane.
  • The combined extracts are repeatedly washed with water and brine, dried over sodium sulfate, concentrated and the residue is either directly chromatographed (silica, petroleum ether/dichloromethane 6:1) or triturated with an ether/petroleum ether mixture prior to chromatography.

3.3. Expression and purification of hGSTA1-1

  • This is based on a published method.19 Briefly, the expression of GST was induced from Escherichia coli BL21 (DE3) cells harbouring the plasmid pET101/D by addition of IPTG.
  • The cells were harvested by centrifugation (845 mg cell paste), resuspended in phosphate buffer, disrupted by sonication and the liquid phase (‘supernatant’), containing the enzyme was collected by centrifugation.
  • The GST, from the supernatant, was purified on an affinity chromatography adsorbent bearing the tripeptide glutathione immobilized to cross-linked agarose, previously epoxy-activated with bis-epoxirane (1,4-butanediol diglycidyl ether).
  • Fractions with enzyme activity were polled (specific activity 83 enzyme units per mg protein), concentrated (nitrocellulose filter, cutoff 10 kDa) and diluted by dropwise addition of glycerol to 50% (v/v) final concentration (typically 445 enzyme units per mL stock solution).
  • The enzyme solution can be stored at –20 C for several months without appreciable loss of activity.

3.4.1. Routine enzyme assay for determining hGSTA1-1 activity

  • DMSO was also added (5 lL, in place of equal volume of buffer) only for control assays of inhibition experiments with the test compounds (see below).
  • Initial velocities were determined in triplicate and were corrected for spontaneous reaction rates, when necessary.
  • One unit of enzyme activity is defined as the amount of enzyme that produces 1.0 lmol of product per minute under the assay conditions.

3.5.1. Determination of IC50 values for inhibitors 6, 8, 14 & 16

  • The assay employed was the same as that for the screening of the test compounds as GST inhibitors (see previous paragraph).
  • The observed rate was used to calculate the remaining activity (%), taking as 100% initial activity value the observed rate (approx. 0.15 DA340/min) after replacing the inhibitor by an equal volume of DMSO (5 lL).
  • The IC50 values were determined from a graph depicting remaining GST activity (%) versus inhibitor concentration.

3.7. Cytotoxicity experiments for determining LC50 values for

  • Caco-2 cells with compounds 5, 6, 8, 11, 14 & 16 Cytotoxicity was evaluated in Caco-2 cells using the MTT assay, which measures the ability of viable cells to reduce a soluble tetrazolium salt to an insoluble purple formazan precipipate.
  • After removal of the medium, each well was incubated with 0.5 mg/mL MTT (Sigma–Aldrich) in DMEM serum-free medium at 37 C for 3 h.
  • The in silico structure of hGSTA1-1 and docking of the 2,20-dihydroxybenzophenones and their Ncarbonyl analogues, also known as Modeling and docking.
  • The structure of hGSTA1-1 in complex with ethacrynic acid and its glutathione conjugate was downloaded from the Protein Data Bank (PDB code 1GSE) and prepared with the Protein Preparation Wizard45 in Maestro (Schrodinger, LLC, New York, NY).
  • All figures depicting 3D models were created using PyMOL, version 1.4 (Schrodinger, LLC).

4. Conclusions

  • 2,20-Benzophenones and N-carbonyl analogues have been investigated as inhibitors for the MDR-involved human GST isoenzyme A1–1.
  • 2,20-Dihydroxybenzophenones 6 and 8 and the N-acylhydrazone analogues 14 and 16 stood out after screening a structure-based library of candidate inhibitors.
  • All four structures showed strong hGSTA1-1 inhibition potency (IC50 values in the lower micromolar to sub-micromolar range), interacting at the CDNB-binding site of the enzyme.

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TL;DR: Some enzymes of the mercapturic acid pathway are described as the so-called “moonlighting proteins,” catalytic proteins that exert multiple biochemical or biophysical functions apart from catalysis.
Abstract: The mercapturic acid pathway is a major route for the biotransformation of xenobiotic and endobiotic electrophilic compounds and their metabolites. Mercapturic acids (N-acetyl-l-cysteine S-conjugates) are formed by the sequential action of the glutathione transferases, γ-glutamyltransferases, dipeptidases, and cysteine S-conjugate N-acetyltransferase to yield glutathione S-conjugates, l-cysteinylglycine S-conjugates, l-cysteine S-conjugates, and mercapturic acids; these metabolites constitute a "mercapturomic" profile. Aminoacylases catalyze the hydrolysis of mercapturic acids to form cysteine S-conjugates. Several renal transport systems facilitate the urinary elimination of mercapturic acids; urinary mercapturic acids may serve as biomarkers for exposure to chemicals. Although mercapturic acid formation and elimination is a detoxication reaction, l-cysteine S-conjugates may undergo bioactivation by cysteine S-conjugate β-lyase. Moreover, some l-cysteine S-conjugates, particularly l-cysteinyl-leukotrienes, exert significant pathophysiological effects. Finally, some enzymes of the mercapturic acid pathway are described as the so-called "moonlighting proteins," catalytic proteins that exert multiple biochemical or biophysical functions apart from catalysis.

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TL;DR: This review article attempts to summarize successful examples and current developments on GST engineering, highlighting in parallel the recent knowledge gained on their phylogenetic relationships, structural/catalytic features, and biotechnological applications.
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Cites background from "2,2′-Dihydroxybenzophenones and the..."

  • ...Therefore, human GSTs are exploited as molecular targets for the design of new compounds to sensitize drug-resistant tumors that overexpress GSTs [60,61,68,69] or to function as prodrugs that are activated in vivo by GSTs [70–72]....

    [...]

  • ...In medicine, GSTs are exploited in three different areas: as molecular targets for the design of anticancer drugs [60,61], as genetic diagnostic markers for a wide range of disorders [62] or as drug delivery tool [63]....

    [...]

Journal ArticleDOI
TL;DR: The thermodynamic analysis allowed us to conclude that the encapsulation process is endothermic in water and exothermically in methanol-water.
Abstract: The characterization of the inclusion complex between 2-hydroxybenzophenone (2OHBP) and β-cyclodextrin (βCD) in the solid state was performed using Fourier transform infrared spectroscopy (FTIR), powder X-ray diffractometry (PXRD), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and transmission electron microscopy (TEM). The apparent formation constant of the complex was determined by phase solubility diagrams and liquid chromatography (HPLC) at different temperatures. The formation of the inclusion complex induced slight shifts in the FTIR spectrum while by PXRD a new crystalline phase was observed. TEM studies revealed that the complex forms aggregates of nanometric size. The inclusion complex showed a higher solubility in the tested dissolution media than free 2OHBP. Moreover, the freeze-dried solid complex exhibits a higher thermal stability than the solid free drug. The thermodynamic analysis allowed us to conclude that the encapsulation process is endothermic in water and exothermic in methanol-water.

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Journal ArticleDOI
TL;DR: The outcome of the isoenzyme trilogy identified good binder leads for the investigated GSTs involved in MDR, and 5‐ or 5‐Bromo‐ or phenyl‐substituted inhibitors, having a H‐bonded oxime weakly acidic group of a small volume, are optimal candidates for binding hGSTM1‐1.
Abstract: A series of 2,2′-dihydroxybenzophenones and their carbonyl N-analogues were studied as potential inhibitors against human glutathione transferase M1-1 (hGSTM1-1) purified from recombinant E. coli. Their screening revealed an inhibition against hGSTM1-1 within a range of 0-42% (25 μM). The IC50 values for the two stronger ones, 16 and 13, were 53.5 ± 5.6 μΜ and 28.5 ± 2.5 μΜ, respectively. The results were compared with earlier ones for isoenzymes hGSTP1-1 and hGSTA1-1 involved in MDR. All but one bind more strongly to A1-1, than M1-1 and P1-1, the latter being a poor binder. An order of potency A1-1 > > M1-1 > P1-1 meritted 13, 14 and 16 as the most potent inhibitors with hGSTM1-1. Enzyme kinetics with hGSTM1-1 (Km(CDNB) 213 ± 10 μΜ and Km(GSH) 303 ± 11 μΜ) revealed a competitive modality for 16 (Ki(16) = 22.3 ± 1.1 μΜ) and a mixed one for 13 versus CDNB (Ki(13) = 33.3 ± 1.6 μM for the free enzyme and Ki(13)′ = 17.7 ± 1.7 μM for the enzyme-CDNB complex). 5- or 5′-Bromo- or phenyl-substituted (but not in combination) inhibitors, having a H-bonded oxime weakly acidic group of a small volume, are optimal candidates for binding hGSTM1-1. The outcome of the isoenzyme trilogy identified good binder leads for the investigated GSTs involved in MDR.

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Q1. What are the contributions in this paper?

In this paper, a structure-based library of 2,20-dihydroxybenzophenones and N-car carbonyl N-analogues was built up by a nucleophilic cleavage of suitably substituted xanthones to 2, 20-dhydroxy-benzones and subsequent formation of their N-derivatives ( oximes 11−13 and Nacyl hydrazones 14−16 ).