Anastasia De Luca

Rational design of an organometallic glutathione transferase inhibitor

Double trouble: A hybrid organic-inorganic (organometallic) inhibitor was designed to target glutathione transferases. The metal center is used to direct protein binding, while the organic moiety acts as the active-site inhibitor (see picture). The mechanism of inhibition was studied using a range of biophysical and biochemical methods.

Organometallic Ruthenium Inhibitors of Glutathione-S-Transferase P1-1 as Anticancer Drugs

Ruthenium–arene complexes conjugated to ethacrynic acid were prepared as part of a strategy to develop novel glutathione‐S‐transferase (GST) inhibitors with alternate modes of activity through the organometallic fragment, ultimately to provide targeted ruthenium‐based anticancer drugs. Enzyme kinetics and electrospray mass spectrometry experiments using GST P1‐1 and its cysteine‐modified mutant forms revealed that the complexes are effective enzyme inhibitors, but they also rapidly inactivate the enzyme by covalent binding at Cys 47 and, to a lesser extent, Cys 101. They are highly effective against the GST Pi‐positive A2780 and A2780cisR ovarian carcinoma cell lines, are among the most effective ruthenium complexes reported so far, and target ubiquitous GST Pi overexpressed in many cancers.

Monomer−Dimer Equilibrium in Glutathione Transferases: A Critical Re-Examination

Glutathione transferases (GSTs) are dimeric enzymes involved in cell detoxification versus many endogenous toxic compounds and xenobiotics. In addition, single monomers of GSTs appear to be involved in particular protein-protein interactions as in the case of the pi class GST that regulates the apoptotic process by means of a GST-c-Jun N-terminal kinase complex. Thus, the dimer-monomer transition of GSTs may have important physiological relevance, but many studies reached contrasting conclusions both about the modality and extension of this event and about the catalytic competence of a single subunit. This paper re-examines the monomer-dimer question in light of novel experiments and old observations. Recent papers claimed the existence of a predominant monomeric and active species among pi, alpha, and mu class GSTs at 20-40 nM dilution levels, reporting dissociation constants (K(d)) for dimeric GST of 5.1, 0.34, and 0.16 microM, respectively. However, we demonstrate here that only traces of monomers could be found at these concentrations since all these enzymes display K(d) values of <<1 nM, values thousands of times lower than those reported previously. Time-resolved and steady-state fluorescence anisotropy experiments, two-photon fluorescence correlation spectroscopy, kinetic studies, and docking simulations have been used to reach such conclusions. Our results also indicate that there is no clear evidence of the existence of a fully active monomer. Conversely, many data strongly support the idea that the monomeric form is scarcely active or fully inactive.

Studies of glutathione transferase P1-1 bound to a platinum(IV)-based anticancer compound reveal the molecular basis of its activation.

Platinum-based cancer drugs, such as cisplatin, are highly effective chemotherapeutic agents used extensively for the treatment of solid tumors. However, their effectiveness is limited by drug resistance, which, in some cancers, has been associated with an overexpression of pi class glutathione S-transferase (GST P1-1), an important enzyme in the mercapturic acid detoxification pathway. Ethacraplatin (EA-CPT), a trans-Pt(IV) carboxylate complex containing ethacrynate ligands, was designed as a platinum cancer metallodrug that could also target cytosolic GST enzymes. We previously reported that EA-CPT was an excellent inhibitor of GST activity in live mammalian cells compared to either cisplatin or ethacrynic acid. In order to understand the nature of the drug-protein interactions between EA-CPT and GST P1-1, and to obtain mechanistic insights at a molecular level, structural and biochemical investigations were carried out, supported by molecular modeling analysis using quantum mechanical/molecular mechanical methods. The results suggest that EA-CPT preferentially docks at the dimer interface at GST P1-1 and subsequent interaction with the enzyme resulted in docking of the ethacrynate ligands at both active sites (in the H-sites), with the Pt moiety remaining bound at the dimer interface. The activation of the inhibitor by its target enzyme and covalent binding accounts for the strong and irreversible inhibition of enzymatic activity by the platinum complex.

Treatment of doxorubicin-resistant MCF7/Dx cells with nitric oxide causes histone glutathionylation and reversal of drug resistance

Acquired drug resistance was found to be suppressed in the doxorubicin-resistant breast cancer cell line MCF7/Dx after pre-treatment with GSNO (nitrosoglutathione). The effect was accompanied by enhanced protein glutathionylation and accumulation of doxorubicin in the nucleus. Among the glutathionylated proteins, we identified three members of the histone family; this is, to our knowledge, the first time that histone glutathionylation has been reported. Formation of the potential NO donor dinitrosyl-diglutathionyl-iron complex, bound to GSTP1-1 (glutathione transferase P1-1), was observed in both MCF7/Dx cells and drug-sensitive MCF7 cells to a similar extent. In contrast, histone glutathionylation was found to be markedly increased in the resistant MCF7/Dx cells, which also showed a 14-fold higher amount of GSTP1-1 and increased glutathione concentration compared with MCF7 cells. These results suggest that the increased cytotoxic effect of combined doxorubicin and GSNO treatment involves the glutathionylation of histones through a mechanism that requires high glutathione levels and increased expression of GSTP1-1. Owing to the critical role of histones in the regulation of gene expression, the implication of this finding may go beyond the phenomenon of doxorubicin resistance.

A new target for gold(I) compounds: Glutathione-S-transferase inhibition by auranofin

Nowadays, gold compounds occupy a relevant position constituting a promising class of experimental anticancer metallodrugs. Several research efforts have been devoted to the investigations of the pharmacological properties of gold(I) complexes bearing phosphine ligands, such as the antiarthritic drug auranofin, that has also been shown to produce anticancer effects in vitro. In spite of the numerous studies that appeared in the literature the biological mechanisms of action of auranofin and analogues are still controversial. Here, we report on the inhibition effects of glutathione S-transferase P1-1 (GST P1-1) exerted by auranofin. The compound was able to inhibit GST P1-1 with a calculated IC(50) of 32.9±0.5μM. Interestingly, the inhibition of GST P1-1 and its cysteine mutants by the gold(I) compound is essentially the same, suggesting that probably the cysteine residues are not so essential for enzyme inactivation in contrast to other reported inhibitors. High-resolution electrospray ionisation Fourier transform ion cyclotron mass spectrometry (ESI FT-ICR MS) studies allowed characterising the binding of the compound with GST enzymes at a molecular level, confirming that similar gold binding sites may be present in the wild-type protein and its Cys mutants.

New insights into the mechanism of JNK1 inhibition by glutathione transferase P1-1.

The role played by glutathione transferase P1-1 (GSTP1-1) in modulating the c-Jun N-terminal kinase (JNK) pathway has been extensively investigated using JNK isoforms known to exert opposite effects in the cells. We have expressed isoform JNK1α2, which has been reported to transmit a pro-apoptotic signal, and we have analyzed both the phosphorylation level and the activity of this kinase in the presence of GSTP1-1. Contrary to what previous studies suggest, we found that GSTP1-1 is able to form a complex with the unphosphorylated and inactive JNK1α2 isoform, even in the absence of the substrate. We also analyzed the consequences of this interaction on the activity of both enzymes. The complex strongly reduced the extent of activation of JNK1α2 and preserved GSTP1-1 from inactivation. Unexpectedly, glutathione (GSH) exerted a negative effect on the affinity of GSTP1-1 for JNK1α2, suggesting that the intracellular levels of this thiol may allow a fine-tuning of the MAPK signaling pathway. Moreover, we found that the adduct formed by GSH and the strong GSTP1-1 inhibitor NBDHEX abolishes the interaction between GSTP1-1 and JNK1α2. These data confirm and extend at the molecular level previous evidence obtained in tumor cell lines.

Glutathione S‐transferase P1‐1 as a target for mesothelioma treatment

Malignant pleural mesothelioma is a poorly responsive tumor known to overexpress the phase II detoxification enzyme glutathione‐S‐transferase, which catalyzes the conjugation between glutathione and platinum(II)‐containing drugs. Therefore, we evaluated the effect of the strong glutathione S‐transferase inhibitor NBDHEX on human mesothelioma cell lines (MSTO‐211H, MPP89, MM‐B1 and Mero 48a) featuring the most common mesothelioma phenotypes: epithelioid and biphasic. Even though a different response to NBDHEX was observed, the molecule was very effective on all cell lines tested, triggering a sustained activation of both JNK and p38, followed by caspase activation and apoptosis. NBDHEX also caused severe oxidative stress in the MPP89 cells and, to a lesser extent, in the MMB1 cells, while it did not cause a significant redox imbalance in the other cell lines. The efficacy of the drug was found to be comparable or even higher than that of cisplatin. Moreover, it showed synergistic or additive effects when used in combination with cisplatin. In conclusion, NBDHEX was effective on mesothelioma cell lines, with IC50 values in the low micromolar range (IC50 between 1 and 4 μM). These findings indicate that NBDHEX, alone or in combination with cisplatin, is a promising new strategy for treating this rare and aggressive malignancy.

A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib

Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors.

TNFR-Associated Factor-2 (TRAF2): Not Only a Trimer

TNF receptor-associated factors (TRAFs) are characterized by an oligomeric structure that plays a fundamental role in the binding process with membrane receptors. In this work, we studied the trimer-to-monomer (T ↔ 3M) equilibrium transition of the TRAF2 C-terminal domain using both chemical (dilution/guanidinium hydrochloride) and mechanical stress (high pressure) to induce the dissociation of the native protein into subunits. The experimental results and computer simulations indicate that stable monomers exist and that their population accounts for 15% of the total TRAF2 molecules already at a physiological intracellular concentration (≈1 μM), being instead the predominant species in the nanomolar concentration range. Because the total amount of TRAF2 changes during a cell cycle, the monomer-trimer equilibrium can be crucial for regulating the activities of TRAF2 in vivo.