Chemistry, antiproliferative properties, tumor selectivity, and molecular mechanisms of novel gold(III) compounds for cancer treatment: a systematic study

Abstract  

The antiproliferative properties of a group of 13 structurally diverse gold(III) compounds, including six mononuclear gold(III) complexes, five dinuclear oxo-bridged gold(III) complexes, and two organogold(III) compounds, toward several human tumor cell lines were evaluated in vitro using a systematic screening strategy. Initially all compounds were tested against a panel of 12 human tumor cell lines, and the best performers were tested against a larger 36-cell-line panel. Very pronounced antiproliferative properties were highlighted in most cases, with cytotoxic potencies commonly falling in the low micromolar—and even nanomolar—range. Overall, good-to-excellent tumor selectivity was established for at least seven compounds, making them particularly attractive for further pharmacological evaluation. Compare analysis suggested that the observed antiproliferative effects are caused by a variety of molecular mechanisms, in most cases “DNA-independent,” and completely different from those of platinum drugs. Remarkably, some new biomolecular systems such as histone deacetylase, protein kinase C/staurosporine, mammalian target of rapamycin/rapamycin, and cyclin-dependent kinases were proposed for the first time as likely biochemical targets for the gold(III) species investigated. The results conclusively qualify gold(III) compounds as a promising class of cytotoxic agents, of outstanding interest for cancer treatment, while providing initial insight into their modes of action.     

Gold(III) Compounds as New Family of Anticancer Drugs

Gold(III) complexes are emerging as a new class of metal complexes with outstanding cytotoxic properties and are presently being evaluated as potential antitumor agents. This renewed interest is the result of recent studies in which various gold(III) complexes have been shown to be stable under physiological conditions and to manifest relevant antiproliferative properties against selected human tumor cell lines. The pharmacological investigation of some representative gold(III) complexes has been extended to consider their effects on the cell cycle and to reveal induction of apoptosis. Remarkably, preliminary studies suggest that the interactions in vitro of gold(Ill) complexes with calf thymus DNA are weak whereas significant binding to model proteins takes place. Our findings imply that the mechanism of action of cytotoxic gold(Ill) complexes might be substantially different from that of clinically established platinum compounds.     

Mechanistic studies on two dinuclear organogold(III) compounds showing appreciable antiproliferative properties and a high redox stability

Two dinuclear oxo-bridged organogold(III) compounds, namely [(N,N,C)(2)Au(2)(μ-O)][PF(6)](2) (with N,N,CH = 6-(1-methylbenzyl)-2,2'-bipyridine, Au(2)O1; or 6-(1,1-dimethylbenzyl)-2,2'-bipyridine, Au(2)O2), were previously prepared and characterised. Their solution chemistry under physiological-like conditions has been investigated here as well as their in vitro antiproliferative properties. Notably, these compounds reveal a marked redox stability even in the presence of effective biological reductants such as ascorbic acid and glutathione. The two dinuclear gold(iii) compounds were evaluated for cytotoxic actions against a representative panel of 12 human tumor cell lines, in comparison to respective mononuclear parent compounds [(N,N,C)AuOH][PF(6)], and appreciable biological activity could be highlighted. The reactions of Au(2)O1 and Au(2)O2 with a few model proteins were studied and the ability to form metallodrug-protein adducts monitored through ESI MS methods. Typical adducts were identified where the protein is associated to monometallic gold fragments; in these adducts gold remains in the oxidation state +3 and conserves its organic ligand. A direct comparison of the biological profiles of these binuclear organogold(III) compounds with those previously reported for a series of dinuclear oxo-bridged complexes [(N,N)(2)Au(2)(μ-O)(2)][PF(6)](2) (N,N = 6(6')-substituted 2,2'-bipyridines) named Auoxo's was carried out. It emerges that the greater cytotoxicity of the latter is mainly due to the greater oxidising power of their gold(III) centres and to propensity to generate gold(i) species; in contrast, the here described bimetallic organogold(III) complexes manifest a far higher redox stability in the biological milieu coupled to lower, but still significant, antiproliferative properties. Different molecular mechanisms are thus hypothesised for these two classes of dinuclear gold(III) agents.     

Proteomic analysis of ovarian cancer cell responses to cytotoxic gold compounds

Platinum-based chemotherapy is the primary treatment for human ovarian cancer. Overcoming platinum resistance has become a critical issue in the current chemotherapeutic strategies of ovarian cancer as drug resistance is the main reason for treatment failure. Cytotoxic gold compounds hold great promise to reach this goal; however, their modes of action are still largely unknown. To shed light on the underlying molecular mechanisms, we performed 2-DE and MS analysis to identify differential protein expression in a cisplatin-resistant human ovarian cancer cell line (A2780/R) following treatment with two representative gold compounds, namely Auranofin and Auoxo6. It is shown that Auranofin mainly acts by altering the expression of Proteasome proteins while Auoxo6 mostly modifies proteins related to mRNA splicing, trafficking and stability. We also found that Thioredoxin-like protein 1 expression is greatly reduced after treatment with both gold compounds. These results are highly indicative of the likely sites of action of the two tested gold drugs and of the affected cellular functions. The implications of the obtained results are thoroughly discussed in the frame of current knowledge on cytotoxic gold agents.     

Organogold(III) compounds as experimental anticancer agents: chemical and biological profiles

In the last few years gold(III) complexes have attracted growing attention in the medicinal chemistry community as candidate anticancer agents. In particular some organogold(III) compounds manifested quite attractive pharmacological behaviors in preclinical studies. Here we compare the chemical and biological properties of the novel organogold(III) complex [Au(bipy^dmb?H)(NH(CO)CH₃)][PF₆] (Aubipy^aa) with those of its parent compounds [Au(bipy^dmb?H)(OH)][PF₆] (Aubipy^c) and [Au₂(bipy^dmb?H)₂)(μ?O)][PF₆]₂ (Au2bipy^c), previously synthesized and characterized. The three study compounds were comparatively assessed for their antiproliferative actions against HCT-116 cancer cells, revealing moderate cytotoxic effects. Proapoptotic and cell cycle effects were also monitored. Afterward, to gain additional mechanistic insight, the three gold compounds were challenged against the model proteins HEWL, RNase A and cytochrome c and reactions investigated through UV–Vis and ESI–MS analysis. A peculiar and roughly invariant protein metalation profile emerges in the three cases consisting of protein binding of {Au(bipy^dmb?H)} moieties. The implications of these results are discussed in the frame of current knowledge on anticancer gold compounds.     

2D-DIGE analysis of ovarian cancer cell responses to cytotoxic gold compounds

Cytotoxic gold compounds hold today great promise as new pharmacological agents for treatment of human ovarian carcinoma; yet, their mode of action is still largely unknown. To shed light on the underlying molecular mechanisms, we performed 2D-DIGE analysis to identify differential protein expression in a cisplatin-sensitive human ovarian cancer cell line (A2780/S) following treatment with two representative gold(iii) complexes that are known to be potent antiproliferative agents, namely AuL12 and Au(2)Phen. Software analysis using DeCyder was performed and few differentially expressed protein spots were visualized between the three examined settings after 24 h exposure to the cytotoxic compounds, implying that cellular damage at least during the early phases of exposure is quite limited and selective, reflecting the attempts of the cell to repair damage and to survive the insult. The potential of novel proteomic methods to disclose mechanistic details of cytotoxic metallodrugs is herein further highlighted. Different patterns of proteomic changes were highlighted for the two metallodrugs with only a few perturbed protein spots in common. Using MALDI-TOF MS and ESI-Ion trap MS/MS, several differentially expressed proteins were identified. Two of these were validated by western blotting: Ubiquilin-1, responsible for inhibiting degradation of proteins such as p53 and NAP1L1, a candidate marker identified in primary tumors. Ubiquilin-1 resulted over-expressed following both treatments and NAP1L1 was down-expressed in AuL12-treated cells in comparison with control and with Au(2)Phen-treated cells. In conclusion, we performed a comprehensive analysis of proteins regulated by AuL12 and Au(2)Phen, providing a useful insight into their mechanisms of action.     

Gold complexes as prospective metal-based anticancer drugs

Medical and therapeutic value of gold has been recognized thousands of years ago, but its rational use in medicine has not begun until the early 1920s. Cisplatin is one of the first metal-containing compounds with anti-cancer activity discovered in the 1960s. Despite the fact that cisplatin treatment is efficient for several types of solid tumors, its effectiveness is limited by toxic side effects and tumor resistance that often leads to the occurrence of secondary malignancies. Since gold(III) is isoelectronic with platinum(II) and tetracoordinate gold(III) complexes have the same square-planar geometries as cisplatin, the anticancer activity of gold(III) compounds has been investigated. Previous studies suggested that, in contrast to cisplatin, gold complexes target proteins but not DNA. Recently, we have investigated gold(III) dithiocarbamates for their anticancer activity and showed that their primary target is the proteasome. Treatment of human breast tumor-bearing nude mice with a gold(III) dithiocarbamate complex resulted in significant inhibition of tumor growth, associated with proteasome inhibition and massive apoptosis induction in vivo. Better understanding of physiological processing of gold compounds will provide a rational basis for their further development into novel anticancer drugs.     

Effects of chronic treatment with sodium tetrachloroaurate(III) in mice and membrane models

Gold is a nonessential element with a variety of applications in medicine. A few gold(I) compounds are used in the clinics for treatment of rheumatoid arthritis and of discoid lupus. Some novel gold(III) compounds are under evaluation as anticancer agents. It is known that gold compounds generally produce toxic effects on the kidneys and characteristic lesions in the brain. However, information concerning the neurotoxicity of gold derivatives in humans as well as in experimental toxicology is rather scarce. For this reason we tried to shed some further light on this aspect of gold neurotoxicity by chronic treatment of mice with sodium tetrachloroaurate(III) in order to observe possible biophysical and morphological alterations that may occur in the brain. Chronic gold treatment resulted in a markedly decreased expression of metallothioneins and of glial fibrillary acidic protein in astrocytes of different brain areas. To examine its effects on cell membranes, interactions of sodium tetrachloroaurate(III) with molecular models were also evaluated. The models consisted in bilayers built-up of classes of phospholipids located in the outer and inner monolayers of biological membranes. Structural perturbation of cell membrane models was observed only at concentrations 10(5) times higher than those detected in the brains of animals after three months' treatment. These results show that toxic effects on animal brain upon treatment with sodium tetrachloroaurate develop with difficulty and may be observed only at high doses.     

Reactions of gold(III) complexes with serum albumin.

The reactions of a few representative gold(III) complexes -[Au(ethylenediamine)2]Cl3, [Au(diethylentriamine)Cl]Cl2, [Au(1,4,8,11-tetraazacyclotetradecane)](ClO4)2Cl, [Au(2,2',2'-terpyridine)Cl]Cl2, [Au(2,2'-bipyridine)(OH)2][PF6] and the organometallic compound [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)][PF6]- with BSA were investigated by the joint use of various spectroscopic methods and separation techniques. Weak metal-protein interactions were revealed for the [Au(ethylenediamine)2]3+ and [Au(1,4,8,11-tetraazacyclotetradecane)]3+ species, whereas progressive reduction of the gold(III) centre was observed in the cases of [Au(2,2'-bipyridine)(OH)2]+ and [Au(2,2',2'-terpyridine)Cl]2+. In contrast, tight metal-protein adducts are formed when BSA is reacted with either [Au(diethylentriamine)Cl]2+ and [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)]+. Notably, binding of the latter complex to serum albumin results in the appearance of characteristic CD bands in the visible spectrum. It is suggested that adduct formation for both of these gold(III) complexes occurs through coordination at the level of surface histidines. Stability of these gold(III) complexes/serum albumin adducts was tested under physiologically relevant conditions and found to be appreciable. Metal binding to the protein is tight; complete detachment of the metal from the protein has been achieved only after the addition of excess potassium cyanide. The implications of the present results for the pharmacological activity of these novel cytotoxic agents are discussed.     

Exploring the biochemical mechanisms of cytotoxic gold compounds: a proteomic study

We have recently shown that a group of structurally diverse gold compounds are highly cytotoxic toward a panel of 36 human tumor cell lines through a variety of biochemical mechanisms. A classic proteomic approach is exploited here to gain deeper insight into those mechanisms. This investigation is focused on Auoxo6, a novel binuclear gold(III) complex, and auranofin, a clinically established gold(I) antiarthritic drug. First, the 72-h cytotoxicity profiles of Auoxo6 and auranofin were determined against A2780 human ovarian carcinoma cells. Subsequently, protein extraction from gold-treated A2780 cells sensitive to cisplatin and 2D gel electrophoresis separation were carried out according to established procedures. Notably, both metallodrugs caused relatively modest changes in protein expression in comparison with controls as only 11 out of approximately 1,300 monitored spots showed appreciable quantitative changes. Very remarkably, six altered proteins were in common between the two treatments. Eight altered proteins were identified by mass spectrometry; among them was ezrin, a protein associated with the cytoskeleton and involved in apoptosis. Interestingly, two altered proteins, i.e., peroxiredoxins 1 and 6, are known to play crucial roles in the cell redox metabolism. Increased cleavage of heterogeneous ribonucleoprotein H was also evidenced, consistent with caspase 3 activation. Overall, the results of the present proteomic study point out that the mode of action of Auoxo6 is strictly related to that of auranofin, that the induced changes in protein expression are limited and selective, that both gold compounds trigger caspase 3 activation and apoptosis, and that a few affected proteins are primarily involved in cell redox homeostasis.     

Proteomic characterization of the cytotoxic mechanism of gold (III) porphyrin 1a, a potential anticancer drug

There has been increasing interest in the potential applications of gold (III) complexes as anticancer drugs with higher cytotoxicity and fewer side effects than existing metal anticancer drugs. Our previous findings demonstrated that gold (III) porphyrin 1a preferentially induced apoptosis in a cancer cell line (SUNE1). In this study, we identified differentially expressed proteins related to the drug's cytotoxic action by comparing the protein alterations induced by gold (III) porphyrin 1a and cisplatin treatments. Several clusters of altered proteins were identified, including cellular structure and stress-related chaperone proteins, proteins involved in reactive oxygen species and enzyme proteins, translation factors, proteins that mediate cell proliferation or differentiation, and proteins participating in the internal degradation systems. Our results indicated that multiple factors leading to apoptosis were involved in drug cytotoxicity in SUNE1 cells. The balance between pro-apoptotic and anti-apoptotic signals determined the final fate of cancer cells.     

The structure-based design, synthesis and biological evaluation of DNA-binding bisintercalating bisanthrapyrazole anticancer compounds

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. In order to find stronger DNA binding and more potent cytotoxic compounds, a series of ester-coupled bisanthrapyrazole derivatives of 7-chloro-2-[2-[(2-hydroxyethyl)methylamino]ethyl]anthra[1,9-cd]pyrazol-6(2H)-one (AP9) were designed and evaluated by molecular docking techniques. Because the anthrapyrazoles are unable to be reductively activated like doxorubicin and other anthracyclines, they should not be cardiotoxic like the anthracyclines. Based on the docking scores of a series of bisanthrapyrazoles with different numbers of methylene linkers (n) that were docked into an X-ray structure of double-stranded DNA, five bisanthrapyrazoles (n=1-5) were selected for synthesis and physical and biological evaluation. The synthesized compounds were evaluated for DNA binding and bisintercalation by measuring the DNA melting temperature increase, for growth inhibitory effects on the human erythroleukemic K562 cell line, and for DNA topoisomerase IIalpha-mediated cleavage of DNA and inhibition of DNA topoisomerase IIalpha decatenation activities. The results suggest that the bisanthrapyrazoles with n=2-5 formed bisintercalation complexes with DNA. In conclusion, a novel group of bisintercalating anthrapyrazole compounds have been designed, synthesized and biologically evaluated as possible anticancer agents.     

Structural and solution chemistry, protein binding and antiproliferative profiles of gold(I)/(III) complexes bearing the saccharinato ligand

A series of new gold(I) and gold(III) complexes based on the saccharinate (sac) ligand, namely M[Au(sac)₂] (with M being Na⁺, K⁺ or NH₄⁺), [(PTA)Au(sac)], K[Au(sac)₃Cl] and Na[Au(sac)₄], were synthesized and characterized, and some aspects of their biological profile investigated. Spectrophotometric analysis revealed that these gold compounds, upon dissolution in aqueous media, at physiological pH, manifest a rather favourable balance between stability and reactivity. Their reactions with the model proteins cytochrome c and lysozyme were monitored by mass spectrometry to predict their likely interactions with protein targets. In the case of disaccharinato gold(I) complexes, cytochrome c adducts bearing four coordinated gold(I) ions were preferentially formed in high yield. In contrast, [(PTA)Au(sac)] (PTA = 1,3,5-triaza-7-phosphaadamantane) turned out to be poorly effective, only producing a mono-metalated adduct in very low amount. In turn, the gold(III) saccharinate derivatives were less reactive than their gold(I) analogues: K[Au(sac)₃Cl] and Na[Au(sac)₄] caused moderate protein metalation, again with evidence of formation of tetragold adducts. Finally, the above mentioned gold compounds were challenged against the reference human tumor cell line A2780S and its cisplatin resistant subline A2780R and their respective cytotoxic profiles determined. [(PTA)Au(sac)] turned out to be highly cytotoxic whereas moderate cytotoxicities were observed for the gold(III) complexes and only modest activities for disaccharinato gold(I) complexes. The implications of these results are thoroughly discussed in the light of current knowledge on gold based drugs.     

Enhancement of cytotoxicity of bleomycin by dithiocarbamates: formation of bis(dithiocarbamato) cu(II).

Properties of the reactions of dithiocarbamates and their Cu(II) or Fe(III) complexes with Ehrlich cells were determined and related to their effects on the inhibition of cell proliferation caused by bleomycin and Cu bleomycin. In complete culture medium containing Eagle's minimal essential medium plus Earles salts and 2.5% fetal calf serum, dimethyl- and diethyldithiocarbamates and their copper complexes inhibit cell proliferation and cause cell death. The copper complexes are more effective agents. Ferric tris-diethyldithiocarbamate is also a cytotoxic species. In contrast, when cells are exposed to dimethyldithiocarbamate or its copper complex in Ringer's buffer under metal-restricted condition, washed, and then placed in complete medium, the copper complex is much more active in inhibiting cell growth. The difference is magnified when dihydroxyethyldithiocarbamate and N-methylglucamine dithiocarbamate and their copper complexes are compared in complete media. Incubation of bleomycin or copper bleomycin with Ehrlich cells in Ringer's buffer with or without dimethyldithiocarbamate or bis-dimethyldithiocarbamato Cu(II) leads to no enhancement of cytotoxicity from combinations of agents, except when the two copper complexes are present. Diethyl- or dimethyldithiocarbamate readily extracts copper from Cu(II)bleomycin and iron from Fe(III)bleomycin when ethylacetate is present to remove the tris-dithiocarbamato Fe(III) complex from aqueous solution. When bis-dimethyldithiocarbamato Cu(II) is incubated with Ehrlich cells, copper is released from the complex and bound to high molecular weight and metallothionein fractions. A reductive mode of dissociation of the copper complexes in cells is supported by ESR experiments. Reactions of diethyl- and dimethyldithiocarbamato Cu(II) with thiol compounds demonstrates one possible mechanism of reduction of these complexes.     

Interactions of selected gold(III) complexes with calf thymus DNA

DNA represents the primary target for platinum antitumor metal complexes and is the probable target for newly developed cytotoxic gold(III) complexes. To test this hypothesis the reactions with calf thymus DNA of five representative gold(III) complexes--namely [Au(en)(2)]Cl(3), [Au(dien)Cl]Cl(2), [Au(cyclam)](ClO(4))(2)Cl, [Au(terpy)Cl]Cl(2) and [Au(phen)Cl(2)]Cl--were analyzed in vitro through various physicochemical techniques including circular dichroism, absorption spectroscopy, DNA melting, and ultradialysis. It is shown that all tested complexes interact with DNA and modify significantly its solution behavior. The solution conformation of DNA is affected to variable extents by the individual complexes as shown by CD titration experiments. Notably, in all cases, the gold(III) chromophore is not largely perturbed by addition of calf thymus DNA ruling out occurrence of gold(III) reduction. Ultradialysis experiments point out that the binding affinity of the various complexes for the DNA double helix is relatively low; in most cases the gold(III)/DNA interaction is electrostatic in nature and reversible. The implications of these findings for the mechanism of action of antitumor gold(III) complexes are discussed.     

Mercury, silver, and gold inhibition of selenium-accelerated cysteine oxidation

In vivo, cysteine in proteins or glutathione is the major amino acid involved in sulfhydryl oxidation-reduction reactions. An in vitro model of cysteine oxidation accelerated by selenium compounds was used to study the interaction of selenocystine and sodium selenite with metal ions. The interaction of metal ions with selenium compounds inhibited cysteine oxidation. The ionic forms of three toxic soft-acid metals, mercury, silver, and gold, were the most effective inhibitors. The antiarthritic gold drugs, aurothiomalate and aurothioglucose, were of particular interest as they inhibit the activity of selenium-glutathione peroxidase. The effect of gold ligands on gold(I) inhibition of selenocystine-accelerated cysteine oxidation was tested. Sodium cyanide partially reversed inhibition and potassium iodide had no effect. Inhibition of selenium-accelerated oxidation-reduction reactions by soft-acid metal ions may be of biological relevance during toxicities or during antiarthritic gold therapy.     

Ploidity and cell cycle progression during treatment with gold chloride, auranofin and sodium aurothiomalate. Studies on a human epithelial cell line and its sub-strains made resistant to the antiproliferative effects of these gold compounds

The possible influence of gold(III) chloride and the two gold(I)-containing anti-arthritic drugs, auranofin and sodium aurothiomalate, on cellular ploidity and cell cycle progression was investigated on cultured human epithelial cells. Four different cell lines were used: the parent line (HE) and three sub-strains which previously had acquired resistance to the antiproliferative effects of either 350 mumol gold chloride/l culture medium (HEAu350), 2 mumol auranofin/l (HEAF) or 300 mumol sodium aurothiomalate/l (HEMyo). DNA-histograms were obtained by flow cytometry examinations during a 9-days' exposure to either of these gold-containing compounds and concentrations. The HE, HEAF and HEMyo cells had similar ploidities, close to tetraploid. The HEAu350 cells had altered ploidity to distinct tetraploid. The distribution of the resistant cells with the cell cycle phases was not different from that of untreated HE cells. The HE cells, when treated with auranofin or sodium aurothiomalate, accumulated in the G2-phase of the cell cycle. In addition, a new cedecoploid peak appeared. No such changes were observed on gold chloride exposure or in HE controls grown without drug supplement. The effects of auranofin and sodium aurothiomalate on cell cycle progression of the HE cells possibly indicate a tendency to polyploidity, and furthermore that inhibition of cellular mitosis is one mechanism of the antiproliferative effect common to the two drugs.     

Selected gold compounds cause pronounced inhibition of Falcipain 2 and effectively block P. falciparum growth in vitro

A number of structurally diverse gold compounds were evaluated as possible inhibitors of Falcipain 2 (Fp2), a cysteine protease from P. falciparum that is a validated target for the development of novel antimalarial drugs. Remarkably, most tested compounds caused pronounced but reversible inhibition of Fp2 with K_i values falling in the micromolar range. Enzyme inhibition is basically ascribed to gold binding to catalytic active site cysteine. The same gold compounds were then tested for their ability to inhibit P. falciparum growth in vitro; important parasite growth inhibition was indeed observed. However, careful analysis of the two sets of data failed to establish any direct correlation between enzyme inhibition and reduction of P. falciparum growth suggesting that Fp2 inhibition represents just one of the various mechanisms through which gold compounds effectively antagonize P. falciparum replication.     

Targets of antithrombotic drugs

New antithrombotic agents are being developed not only to improve efficacy, but also to increase safety in comparison with widely used conventional agents such as the oral anticoagulants. New anticoagulant, antiplatelet, and profibrinolytic compounds are currently under study in drug development programs, and most of those in phase II or III of development are derived from the observation of natural phenomena and merely mimic processes developed by mammalians, including humans, to avoid thrombosis, or by blood-sucking insects or animals to prevent coagulation of the blood their are feeding on. By contrast, drug candidates identified by means of rigorous research and designed to target new pathways and achieve direct and specific inhibition of factors that are presumed to play an important role in thrombogenesis have generally failed to show any benefit and sometimes even induce deleterious effects. The clinical development of new drugs, even those mimicking natural phenomena, improves our knowledge of the pathogenesis of thrombosis and sheds light, retrospectively, on previous conceptual errors. The improvement in our basic knowledge and the development of new types of drugs suggest that, in contrast to the current antithrombotic compounds that are used in a broad range of clinical settings, use of new drugs should be restricted to specific situations in which their mechanisms of action are predicted to deliver the highest medical benefit. A major obstacle resides in the fact that current drug development programs are still required to comply with long obsolete guidelines based on the characteristics of first-generation antithrombotic agents, and that do not take into account the specific mechanisms of action of new drugs. This situation should change, however, and new antithrombotic drugs should soon be able to benefit from adapted development programs that will make it possible to determine their optimal risk-benefit ratio.