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Aurothiomalate (AuTM) is an FDA-approved antiarthritic gold drug with unique anticancer properties. To enhance its anticancer activity, we prepared a bioconjugate with human apoferritin (HuHf) by attaching some AuTM moieties to surface protein residues. The reaction of apoferritin with excess AuTM yielded a single adduct, that was characterized by ESI MS and ICP-OES analysis, using three mutant ferritins and trypsinization experiments. The adduct contains ~3 gold atoms per ferritin subunit, arranged in a small cluster bound to Cys90 and Cys102. MD simulations provide a plausible structural model for the cluster. The adduct was evaluated for its pharmacological properties and was found to be significantly more cytotoxic than free AuTM against A2780 cancer cells mainly due to higher gold uptake. NMR-metabolomics showed that AuTM bound to HuHf and free AuTM induced qualitatively similar changes in treated cancer cells, indicating that the effects on cell metabolism are approximately the same, in agreement with independent biochemical experiments. In conclusion, we have demonstrated here that a molecularly precise bioconjugate formed between AuTM and HuHf exhibits anticancer properties far superior to the free drug, while retaining its key mechanistic features. Evidence is provided that human ferritin can serve as an excellent carrier for this metallodrug.
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INTRODUCTION: Auranofin (AF) is a well-established, FDA-approved, antiarthritic gold drug that is currently being reevaluated for a variety of therapeutic indications through drug repurposing. AF has shown great promise as a potential anticancer agent and has been approved for a few clinical trials in cancer. The renewed interest in AF has led to extensive research into the design, preparation and biological evaluation of auranofin analogs, which may have an even better pharmacological profile than the parent drug. AREAS COVERED: This article reviews the strategies for chemical modification of the AF scaffold. Several auranofin analogs have been prepared and characterized for medical application in the field of cancer treatment over the last 20 years. Some emerging structure-function relationships are proposed and discussed. EXPERT OPINION: The chemical modification of the AF scaffold has been the subject of intense activity in recent years and this strategy has led to the preparation and evaluation of several AF analogs. The case of iodauranofin is a particularly promising example. The availability of homogeneous biological data for a group of AF derivatives allows some initial structure-function relationships to be proposed, which may inspire the design and synthesis of new and better AF analogs for cancer treatment.
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Antineoplásicos , Auranofina , Desenho de Fármacos , Neoplasias , Auranofina/farmacologia , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Relação Estrutura-Atividade , Neoplasias/tratamento farmacológico , Animais , Reposicionamento de MedicamentosRESUMO
The reactivity of the anticancer drug picoplatin (cis-amminedichlorido(2-methylpyridine)platinum(II) complex) with the model proteins hen egg white lysozyme (HEWL) and bovine pancreatic ribonuclease (RNase A) was investigated by electrospray ionisation mass spectrometry (ESI MS) and X-ray crystallography. The data were compared with those previously obtained for the adducts of these proteins with cisplatin, carboplatin and oxaliplatin under the same experimental conditions. ESI-MS data show binding of Pt to both proteins, with fragments retaining the 2-methylpyridine ligand and, possibly, a chloride ion. X-ray crystallography identifies different binding sites on the two proteins, highlighting a different behaviour of picoplatin in the absence or presence of dimethyl sulfoxide (DMSO). Metal-containing fragments bind to HEWL close to the side chains of His15, Asp18, Asp119 and both Lys1 and Glu7, whereas they bind to RNase A on the side chain of His12, Met29, His48, Asp53, Met79, His105 and His119. The data suggest that the presence of DMSO favours the loss of 2-methylpyridine and alters the ability of the Pt compound to bind to the two proteins. With both proteins, picoplatin appears to behave similarly to cisplatin and carboplatin when dissolved in DMSO, whereas it behaves more like oxaliplatin in the absence of the coordinating solvent. This study provides important insights into the pharmacological profile of picoplatin and supports the conclusion that coordinating solvents should not be used to evaluate the biological activities of Pt-based drugs.
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Muramidase , Compostos Organoplatínicos , Ribonuclease Pancreático , Muramidase/química , Muramidase/metabolismo , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismo , Animais , Cristalografia por Raios X , Compostos Organoplatínicos/química , Compostos Organoplatínicos/metabolismo , Bovinos , Ligação Proteica , Sítios de Ligação , Modelos Moleculares , Galinhas , Espectrometria de Massas por Ionização por Electrospray , Dimetil Sulfóxido/química , Carboplatina/química , Carboplatina/metabolismoRESUMO
The reaction of Pt-based anticancer agents with arsenic trioxide affords robust complexes known as arsenoplatins. The prototype of this family of anticancer compounds is arsenoplatin-1 (AP-1) that contains an As(OH)2 fragment linked to a Pt(II) moiety derived from cisplatin. Crystallographic and spectrometric studies of AP-1 binding to a B-DNA double helix dodecamer are presented here, in comparison with cisplatin and transplatin. Results reveal that AP-1, cisplatin and transplatin react differently with the DNA model system. Notably, in the AP-1/DNA systems, the Pt-As bond can break down with time and As-containing fragments can be released. These results have implications for the understanding of the mechanism of action of arsenoplatins.
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Antineoplásicos , Trióxido de Arsênio/análogos & derivados , DNA de Forma B , Cisplatino/química , Fator de Transcrição AP-1/metabolismo , Antineoplásicos/química , DNA/químicaRESUMO
Many efforts have been made in the last few decades to selectively transport antitumor agents to their potential target sites with the aim to improve efficacy and selectivity. Indeed, this aspect could greatly improve the beneficial effects of a specific anticancer agent especially in the case of orphan tumors like the triple negative breast cancer. A possible strategy relies on utilizing a protective leaving group like alizarin as the Pt(II) ligand to reduce the deactivation processes of the pharmacophore enacted by Pt resistant cancer cells. In this study a new series of neutral mixed-ligand Pt(II) complexes bearing alizarin and a variety of diamine ligands were synthesized and spectroscopically characterized by FT-IR, NMR and UV-Vis analyses. Three Pt(II) compounds, i.e., 2b, 6b and 7b, emerging as different both in terms of structural properties and cytotoxic effects (not effective, 10.49 ± 1.21 µM and 24.5 ± 1.5 µM, respectively), were chosen for a deeper investigation of the ability of alizarin to work as a selective carrier. The study comprises the in vitro cytotoxicity evaluation against triple negative breast cancer cell lines and ESI-MS interaction studies relative to the reaction of the selected Pt(II) complexes with model proteins and DNA fragments, mimicking potential biological targets. The results allow us to suggest the use of complex 6b as a prospective anticancer agent worthy of further investigations.
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Antraquinonas , Antineoplásicos , Neoplasias de Mama Triplo Negativas , Humanos , Antineoplásicos/química , Linhagem Celular Tumoral , DNA/química , Ligantes , Estudos Prospectivos , Espectroscopia de Infravermelho com Transformada de Fourier , FemininoRESUMO
Solution interactions of three organomercury compounds, i.e., methylmercury chloride, thimerosal and phenylmercury acetate, with a group of biochemically relevant proteins, namely cytochrome c (Cyt c), ribonuclease A (RNase A), carbonic anhydrase I (hCA I), superoxide dismutase (SOD), and serum albumin (HSA), were investigated using an established ESI MS approach. Temporal analysis of sample aliquots provided insight into the binding kinetics, while comparative analysis of the obtained mass spectra disclosed adduct formation of each mercurial with the tested proteins and the relative abundance of the species. The three organomercurials bind, exclusively and tightly, to free cysteine residues as no binding was observed in the case of proteins lacking such groups. hCA I, SOD and HSA formed distinct mercury adducts, preserving the Hg bound alkyl/aryl ligands; yet, the three organomercurials displayed significant differences in reactivity in relation to their chemical structure. The investigation was then extended to analyze the reactions with the C-terminal dodecapeptide of the enzyme human thioredoxin reductase, which contains a characteristic selenol-thiol moiety: tight Hg binding was observed. Notably, this peptide was able to remove effectively and completely the alkyl/aryl ligands of the three tested organomercurials; this behavior may be relevant to the detoxification mechanism of organomercurials in mammals. Finally, a competition experiment was carried out to establish whether protein bound mercury centers may be displaced by other competing metals. Interestingly, and quite unexpectedly, we observed that a protein bound mercury fragment may be partially displaced from its coordination site in hCA I by the medicinal gold compound auranofin.
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Mercúrio , Compostos Organomercúricos , Animais , Humanos , Compostos Organomercúricos/metabolismo , Peptídeos , Ouro , Superóxido Dismutase , Mamíferos/metabolismoRESUMO
Medicinal gold compounds, a novel class of potential anticancer drugs, are believed to produce their pharmacological effects mainly through direct gold binding to protein targets at the level of solvent exposed cysteine (or selenocysteine) residues. We have explored therein the reactions of a panel of seven representative gold compounds with the cysteine protease cathepsin B according to an established ESI MS approach. Detailed information on the mode of protein binding of these gold compounds is gained; notably, quite distinct patterns of cathepsin B metalation have emerged from these studies. It is shown that panel gold compounds interact preferentially, often exclusively, with the free cysteine located in the active site of the enzyme.
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Catepsina B , Compostos de Ouro , Compostos de Ouro/química , Compostos de Ouro/farmacologia , Catepsina B/química , Cisteína/química , Espectrometria de Massas por Ionização por Electrospray , Proteínas/químicaRESUMO
The possibility of using selectively incorporated 19F nuclei for NMR spectroscopic studies has retrieved increasing interest in recent years. The high gyromagnetic ratio of 19F and its absence in native biomolecular systems make this nucleus an interesting alternative to standard 1H NMR spectroscopy. Here we show how we can attach a label, carrying a 19F atom, to protein tyrosines, through the use of a specific three component Mannich-type reaction. To validate the efficacy and the specificity of the approach, we tested it on two selected systems with the aid of ESI MS measurements.
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Proteínas , Tirosina , Proteínas/química , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular/métodosRESUMO
NMR metabolomics is a powerful tool to characterise the changes in cancer cell metabolism elicited by anticancer drugs. Here, the large metabolic alterations produced by two cytotoxic gold carbene compounds in A2780 ovarian cancer cells are described and discussed in comparison to auranofin, in the frame of the available mechanistic knowledge.
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Gold compounds form a new class of promising anticancer agents with innovative modes of action. It is generally believed that anticancer gold compounds, at variance with clinically established platinum drugs, preferentially target proteins rather than nucleic acids. The reactions of several gold compounds with a few model proteins have been systematically explored in recent years through ESI MS measurements to reveal adduct formation and identify the main features of those reactions. Here, we focus our attention on a group of five gold compounds of remarkable medicinal interest, i.e., Auranofin, Au(NHC)Cl, [Au(NHC)2]PF6, Aubipyc, and Auoxo6, and on their reactions with four different biomolecular targets, i.e., the proteins HEWL, hCA I, HSA and the C-terminal dodecapeptide of the enzyme thioredoxin reductase. Complete ESI MS data are available for those reactions due to previous experimental work conducted in our laboratory. From the comparative analysis of the ESI MS reaction profiles, some characteristic trends in the metallodrug-protein reactivity may be identified as detailed below. The main features are described and analyzed in this review. Overall, all these observations are broadly consistent with the concept that cytotoxic gold drugs preferentially target cancer cell proteins, with a remarkable selectivity for the cysteine and selenocysteine proteome. These interactions typically result in severe damage to cancer cell metabolism and profound alterations in the redox state, leading to eventual cancer cell death.
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Antineoplásicos , Compostos de Ouro , Compostos de Ouro/farmacologia , Ouro/química , Auranofina/farmacologia , Auranofina/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Tiorredoxina Dissulfeto RedutaseRESUMO
The globally widespread perfluorooctanoic acid (PFOA) is a concerning environmental contaminant, with a possible toxic long-term effects on the environment and human health The development of sensible, rapid, and low-cost detection systems is a current change in modern environmental chemistry. In this context, two triamine-based chemosensors, L1 and L2, containing a fluorescent pyrene unit, and their Zn(II) complexes are proposed as fluorescent probes for the detection of PFOA in aqueous media. Binding studies carried out by means of fluorescence and NMR titrations highlight that protonated forms of the receptors can interact with the carboxylate group of PFOA, thanks to salt bridge formation with the ammonium groups of the aliphatic chain. This interaction induces a decrease in the fluorescence emission of pyrene at neutral and slightly acidic pH values. Similarly, emission quenching has also been observed upon coordination of PFOA by the Zn(II) complexes of the receptors. These results evidence that simple polyamine-based molecular receptors can be employed for the optical recognition of harmful pollutant molecules, such as PFOA, in aqueous media.
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Fluorocarbonos , Poliaminas , Humanos , Poliaminas/química , Caprilatos , PirenosRESUMO
The reaction of the cytotoxic compound dirhodium tetraacetate with a B-DNA double helical dodecamer was studied by X-ray crystallography and mass spectrometry. The structure of the dirhodium/DNA adduct reveals a dimetallic center binding to an adenine via axial coordination. Complementary information has been gained through ESI MS measurements. Comparison between the present data and those previously obtained for cisplatin indicates that the two metallodrugs react with this DNA dodecamer in a significantly different fashion.
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DNA de Forma B , Cristalografia por Raios X , DNA/química , Espectrometria de MassasRESUMO
Herein we describe a method for the efficient production (â¼90% fluorination) of 5-F-Trp human H ferritin via the selective incorporation of 19F into the side chain of W93 using 5-fluoroindole as the fluorinated precursor of the amino acid. Human H ferritin is a nanocage composed of 24 identical subunits, each containing a single Trp belonging to a loop exposed on the external surface of the protein nanocage. This makes 5-F-Trp a potential probe for the study of intermolecular interactions in solution by exploiting its intrinsic fluorescence. More interestingly, albeit the large size of the cage (12 nm external diameter, â¼500 kDa molecular mass) we observe a broad but well defined NMR 19F resonance that can be used for the dual purpose of detecting solution intermolecular interactions via chemical shift perturbation mapping and monitoring the uptake of ferritin by cells treated with ferritin-based drug carriers, the latter being an application area of increasing importance.
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Apoferritinas , Ferritinas , Humanos , Ferritinas/química , Aminoácidos/química , Espectroscopia de Ressonância Magnética , Portadores de FármacosRESUMO
A panel of four novel gold(I) complexes, inspired by the clinically established gold drug auranofin (1-Thio-ß-D-glucopyranosatotriethylphosphine gold-2,3,4,6-tetraacetate), was prepared and characterized. All these compounds feature the replacement of the triethylphosphine ligand of the parent compound auranofin with a trimethylphosphite ligand. The linear coordination around the gold(I) center is completed by Cl-, Br-, I- or by the thioglucose tetraacetate ligand (SAtg). The in-solution behavior of these gold compounds as well as their interactions with some representative model proteins were comparatively analyzed through 31PNMR and ESI-MS measurements. Notably, all panel compounds turned out to be stable in aqueous media, but significant differences with respect to auranofin were disclosed in their interactions with a few leading proteins. In addition, the cytotoxic effects produced by the panel compounds toward A2780, A2780R and SKOV-3 ovarian cancer cells were quantitated and found to be in the low micromolar range, since the IC50 of all compounds was found to be between 1 µM and 10 µM. Notably, these novel gold complexes showed large and similar inhibition capabilities towards the key enzyme thioredoxin reductase, again comparable to those of auranofin. The implications of these results for the discovery of new and effective gold-based anticancer agents are discussed.
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Antineoplásicos , Neoplasias Ovarianas , Fosfitos , Humanos , Feminino , Auranofina/farmacologia , Auranofina/química , Ouro/química , Linhagem Celular Tumoral , Ligantes , Antineoplásicos/farmacologia , Antineoplásicos/químicaRESUMO
Biologics are emerging as the most important class of drugs and are used to treat a large variety of pathologies. Most of biologics are proteins administered in large amounts, either by intramuscular injection or by intravenous infusion. Asparaginase is a large tetrameric protein assembly, currently used against acute lymphoblastic leukemia. Here, a gadolinium(III)-DOTA derivative has been conjugated to asparaginase, and its relaxation properties have been investigated to assess its efficiency as a possible theranostic agent. The field-dependent 1H longitudinal relaxation measurements of water solutions of gadolinium(III)-labeled asparaginase indicate a very large increase in the relaxivity of this paramagnetic protein complex with respect to small gadolinium chelates, opening up the possibility of its use as an MRI contrast agent.
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Asparaginase , Meios de Contraste , Gadolínio , Imageamento por Ressonância Magnética/métodos , QuelantesRESUMO
Gold compounds have a long tradition in medicine and offer many opportunities for new therapeutic applications. Herein, we evaluated the lead compound Auranofin and five related gold(I) complexes as possible inhibitors of SARS-CoV-2 Main Protease (SARS-CoV-2 Mpro), a validated drug target for the COVID-19 disease. The investigational panel of gold compounds included Auranofin; three halido analogues, i.e., Au(PEt3)Cl, Au(PEt3)Br, and Au(PEt3)I; and two gold carbene complexes, i.e., Au(NHC)Cl and [Au(NHC)2]PF6. Notably, all these gold compounds, with the only exception of [Au(NHC)2]PF6, turned out to be potent inhibitors of the catalytic activity of SARS-CoV-2 Mpro: the measured Ki values were in the range 2.1-0.4 µM. The reactions of the various gold compounds with SARS-CoV-2 Mpro were subsequently investigated through electrospray ionization (ESI) mass spectrometry (MS) upon a careful optimization of the experimental conditions; the ESI MS spectra provided clear evidence for the formation of tight metallodrug-protein adducts and for the coordination of well defined gold-containing fragments to the SARS-CoV-2 Mpro, again with the only exception of [Au(NHC)2]PF6, The metal-protein stoichiometry was unambiguously determined for the resulting species. The crystal structures of the metallodrug- Mpro adducts were solved in the case of Au(PEt3)Br and Au(NHC)Cl. These crystal structures show that gold coordination occurs at the level of catalytic Cys 145 in the case of Au(NHC)Cl and at the level of both Cys 145 and Cys 156 for Au(PEt3)Br. Tight coordination of gold atoms to functionally relevant cysteine residues is believed to represent the true molecular basis of strong enzyme inhibition.
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Auranofina , Tratamento Farmacológico da COVID-19 , Humanos , Auranofina/farmacologia , Proteínas Virais/química , SARS-CoV-2 , Compostos de Ouro/farmacologia , Cisteína , Ouro/farmacologiaRESUMO
Pd compounds form a promising class of experimental anticancer drug candidates whose mechanism of action is still largely unknown; in particular, a few organopalladium compounds seem very attractive. To gain mechanistic insight into medicinal palladium compounds, we have explored here - through ESI MS analysis - the interactions of four organopalladium agents (1-4) - showing remarkable in vitro antiproliferative properties - with a few representative model proteins, i.e., lysozyme (HEWL), ribonuclease A (RNase), and carbonic anhydrase (hCAI). The tested panel included three Pd allyl compounds with one or two carbene ligands and a palladacyclopentadienyl complex. Notably, the Pd allyl compounds turned out to manifest, on the whole, a modest tendency to react with the above proteins. Only complex 3 produced small amounts of characteristic adducts with hCAI bearing either one or two Pd allyl groups. In contrast, the palladacyclopentadienyl complex 4 manifested a greater and peculiar reactivity with all the above proteins generating invariably protein adducts with a mass increase of +256 Da where a butadienyl group - with no associated Pd - is attached to the proteins. Afterwards, we extended our investigations to the C-terminal dodecapeptide of thioredoxin reductase bearing the -Cys-Sec- reactive motif. In this latter case adducts were formed with all tested Pd compounds; however, complex 4 manifested towards this dodecapeptide a type of reactivity deeply different from that observed with HEWL, RNase A and hCAI. The mechanistic implications of these findings are discussed.
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A novel gold(I) complex inspired by the known medicinal inorganic compounds auranofin and thimerosal, namely ethylthiosalicylate(triethylphosphine)gold(I) (AFETT hereafter), was synthesized and characterised and its structure was resolved through X-ray diffraction. The solution behavior of AFETT and its interactions with two biologically relevant proteins (i.e. human serum albumin and haemoglobin) and with a synthetic dodecapeptide reproducing the C-terminal portion of thioredoxin reductase were comparatively analyzed through 31P NMR and ESI-MS. Remarkable binding properties toward these biomolecules were disclosed. Moreover, the cytotoxic effects produced by AFETT on two ovarian cancer cell lines (A2780 and A2780 R) and one colorectal cancer cell line (HCT116) were analyzed and found to be strong and nearly superimposable to those of auranofin. Interestingly, for both compounds, the ability to induce downregulation of vimentin expression in A2780 R cells was evidenced. Despite its close similarity to auranofin, AFETT is reported to exhibit some peculiar and distinctive features such as a lower lipophilicity, an increased water solubility and a faster reactivity towards the selected target biomolecules. These differences might confer to AFETT significant pharmaceutical and therapeutic advantages over auranofin itself.
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Antineoplásicos , Neoplasias Ovarianas , Antineoplásicos/química , Auranofina/química , Auranofina/farmacologia , Linhagem Celular Tumoral , Feminino , Ouro/química , HumanosRESUMO
Pt-Based drugs play a very important role in current cancer treatments; yet, their cellular and mechanistic aspects are not fully understood. NMR metabolomics provides a powerful tool to investigate the metabolic perturbations induced by Pt drugs in cancer cells and decipher their meaning in relation to the presumed molecular mechanisms. We have carried out a systematic and comparative 1H NMR metabolomics study to analyze the responses of A2780 human ovarian cancer cells to the main clinically established Pt drugs, i.e., cisplatin, carboplatin and oxaliplatin. Notably, NMR analysis revealed some moderate and consistent changes in the metabolomic profiles of A2780 cells treated with the 3 Pt drugs with respect to controls, but only very small differences among them. Beyond alterations at the level of nucleic acid precursors, the observed changes highlight in all cases the induction of a significant endoplasmic reticulum stress. Owing to the clinical relevance of platinum resistance, the behavior of a cisplatin resistant A2780 cancer cell line upon cisplatin treatment was also evaluated.
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Antineoplásicos , Neoplasias Ovarianas , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Compostos Organoplatínicos/farmacologia , Compostos Organoplatínicos/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismoRESUMO
Today colorectal cancer (CRC) is one of the leading causes of cancer death worldwide. This disease is poorly chemo-sensitive toward the existing medical treatments so that new and more effective therapeutic agents are urgently needed and intensely sought. Platinum drugs, oxaliplatin in particular, were reported to produce some significant benefit in CRC treatment, triggering the general interest of medicinal chemists and oncologists for metal-based compounds as candidate anti-CRC drugs. Within this frame, gold compounds and, specifically, the established antiarthritic drug auranofin with its analogs, form a novel group of promising anticancer agents. Owing to its innovative mechanism of action and its favorable pharmacological profile, auranofin together with its derivatives are proposed here as novel experimental agents for CRC treatment, capable of overcoming resistance to platinum drugs. Some encouraging results in this direction have already been obtained. A few recent studies demonstrate that the action of auranofin may be further potentiated through the preparation of suitable pharmaceutical formulations capable of protecting the gold pharmacophore from unselective reactivity or through the design of highly synergic drug combinations. The perspectives of the research in this field are outlined.