Antidepressant efficacy of a selective organic cation transporter blocker in a mouse model of depression.

Current antidepressants act principally by blocking monoamine reuptake by high-affinity transporters in the brain. However, these antidepressants show important shortcomings such as slow action onset and limited efficacy in nearly a third of patients with major depression disorder. Here, we report the development of a prodrug targeting organic cation transporters (OCT), atypical monoamine transporters recently implicated in the regulation of mood. Using molecular modeling, we designed a selective OCT2 blocker, which was modified to increase brain penetration. This compound, H2-cyanome, was tested in a rodent model of chronic depression induced by 7-week corticosterone exposure. In male mice, prolonged administration of H2-cyanome induced positive effects on several behaviors mimicking symptoms of depression, including anhedonia, anxiety, social withdrawal, and memory impairment. Importantly, in this validated model, H2-cyanome compared favorably with the classical antidepressant fluoxetine, with a faster action on anhedonia and better anxiolytic effects. Integrated Z-scoring across these depression-like variables revealed a lower depression score for mice treated with H2-cyanome than for mice treated with fluoxetine for 3 weeks. Repeated H2-cyanome administration increased ventral tegmental area dopaminergic neuron firing, which may underlie its rapid action on anhedonia. H2-cyanome, like fluoxetine, also modulated several intracellular signaling pathways previously involved in antidepressant response. Our findings provide proof-of-concept of antidepressant efficacy of an OCT blocker, and a mechanistic framework for the development of new classes of antidepressants and therapeutic alternatives for resistant depression and other psychiatric disturbances such as anxiety.

Targeting Degradation of EGFR through the Allosteric Site Leads to Cancer Cell Detachment-Promoted Death.

Targeting epidermal growth factor receptor (EGFR) with tyrosine kinase inhibitors (TKI) has been widely exploited to disrupt aberrant phosphorylation flux in cancer. However, a bottleneck of potent TKIs is the acquisition of drug resistance mutations, secondary effects, and low ability to attenuate tumor progression. We have developed an alternative means of targeting EGFR that relies on protein degradation through two consecutive routes, ultimately leading to cancer cell detachment-related death. We describe furfuryl derivatives of 4-allyl-5-[2-(4-alkoxyphenyl)-quinolin-4-yl]-4H-1,2,4-triazole-3-thiol that bind to and weakly inhibit EGFR tyrosine phosphorylation and induce strong endocytic degradation of the receptor in cancer cells. The compound-promoted depletion of EGFR resulted in the sequestration of non-phosphorylated Bim, which no longer ensured the integrity of the cytoskeleton machinery, as shown by the detachment of cancer cells from the extracellular matrix (ECM). Of particular note, the longer CH3(CH2)n chains in the terminal moiety of the anti-EGFR molecules confer higher hydrophobicity in the allosteric site located in the immediate vicinity of the catalytic pocket. Small compounds accelerated and enhanced EGFR and associated proteins degradation during EGF and/or glutamine starvation of cultures, thereby demonstrating high potency in killing cancer cells by simultaneously modulating signaling and metabolic pathways. We propose a plausible mechanism of anti-cancer action by small degraders through the allosteric site of EGFR. Our data represent a rational and promising perspective in the treatment of aggressive tumors.

Sulfheme formation during homocysteine S-oxygenation by catalase in cancers and neurodegenerative diseases.

Accumulating evidence suggests that abnormal levels of homocysteine are associated with vascular dysfunctions, cancer cell proliferation and various neurodegenerative diseases. With respect to the latter, a perturbation of transition metal homeostasis and an inhibition of catalase bioactivity have been reported. Herein, we report on some of the molecular bases for the cellular toxicity of homocysteine and demonstrate that it induces the formation of sulfcatalase, an irreversible inactive state of the enzyme, without the intervention of hydrogen sulfide. Initially, homocysteine reacts with native catalase and/or redox-active transition metal ions to generate thiyl radicals that mediate compound II formation, a temporarily inactive state of the enzyme. Then, the ferryl centre of compound II intervenes into the unprecedented S-oxygenation of homocysteine to engender the corresponding sulfenic acid species that further participates into the prosthetic heme modification through the formation of an unusual Fe(II) sulfonium. In addition, our ex cellulo studies performed on cancer cells, models of neurodegenerative diseases and ulcerative colitis suggest the likelihood of this scenario in a subset of cancer cells, as well as in a cellular model of Parkinson's disease. Our findings expand the repertoire of heme modifications promoted by biological compounds and point out another deleterious trait of disturbed homocysteine levels that could participate in the aetiology of these diseases.

Bioactivation of clopidogrel and prasugrel: factors determining the stereochemistry of the thiol metabolite double bond.

The antithrombotics of the tetrahydrothienopyridine series, clopidogrel and prasugrel, are prodrugs that must be metabolized in two steps to become pharmacologically active. The first step is the formation of a thiolactone metabolite. The second step is a further oxidation with the formation of a thiolactone sulfoxide whose hydrolytic opening leads to a sulfenic acid that is eventually reduced into the corresponding active cis thiol. Very few data were available on the formation of the isomer of the active cis thiol having a trans configuration of the double bond, the most striking result in that regard being that both cis and trans thiols were formed upon the metabolism of clopidogrel by human liver microsomes in the presence of glutathione (GSH), whereas only the cis thiol was detected in the sera of patients treated with this drug. This article shows that trans thiols are also formed upon the microsomal metabolism of prasugrel or its thiolactone metabolite in the presence of GSH and that metabolites having the trans configuration of the double bond are only formed when microsomal incubations are done in the presence of thiols, such as GSH, N-acetyl-cysteine, and mercaptoethanol. Intermediate formation of thioesters resulting from the reaction of GSH with the thiolactone sulfoxide metabolite appears to be responsible for trans thiol formation. Addition of human liver cytosol to the microsomal incubations led to a dramatic decrease of the formation of the trans thiol metabolites. These data suggest that cytosolic esterases would accelerate the hydrolytic opening of thiolactone sulfoxide intermediates and disfavor the formation of thioesters resulting from the reaction of these intermediates with GSH that is responsible for trans isomer formation. This would explain why trans thiols have not been detected in the sera of patients treated with clopidogrel.

New peptides with metal binding abilities and their use as drug carriers.

Many new designed molecules that target efficiently in vitro bacterial metalloproteases were completely inactive in cellulo against Gram negative bacteria. Their activities were limited by the severe restriction of the penetration/diffusion rate through the outer membrane barrier. To bypass this limitation, we have assayed the strategy of metallodrugs, to improve the delivery of hydroxamic acid inhibitors to peptide deformylase. In this metal-chaperone, to facilitate bacterial uptake, the ancillary ligand tris(2-pyridylmethyl)amine (TPA) or di(picolyl)amine (DPA) was functionalized by a tetrapeptide analogue of antimicrobial peptide, RWRW(OBn) (AA08 with TPA) and/or an efflux pump modulator PAßN (AA09 with TPA and AA27 with DPA). We prepared Co(III), Zn(II), and Cu(II) metallodrugs. Using a fluorescent hydroxamic acid, we showed that, in contrast to Cu(II) metallodrugs, Co(III) metallodrugs were stable in the Mueller Hinton (MH) broth during the time required for bacterial assays. The antibacterial activities were determined against E. coli strain wild-type (AG100) and E. coli strain deleted from acrAB efflux pump (AG100A). While none of the PDFinhs used in this study (SMP289 with an indole scaffold, AT015 and AT019 built on a 1,2,4-oxadiazole scaffold) displayed activity higher than 128 µM, all the metallodrugs were active with MICs around 8 µM both against AG100 and AG100A. However, compared to the activities of equimolar combinations of PDFinhs and the free chelating peptides (AA08, AA09, or AA27), they showed similar activities. A synergistic association between AT019 and AA08 or AA09 was determined using the fractional inhibitory concentration with AG100 and AG100A. Combinations of peptides lacking the chelating group with PDFinhs were inefficient. LC-MS analyses showed that the chelating peptides bind Zn(II) cation when incubated in MH broth. These results support the in situ formation of a zinc metallodrug, but we failed to detect it by LC-MS in MH. Nevertheless, this chelating peptides metalated with zinc act as permeabilizers which are more efficient than PAßN to facilitate the uptake of PDFinhs by Gram(-) bacteria.

A versatile strategy for appending a single functional group to a multifunctional host through host-guest covalent-capture.

Mono-functionalization of a molecular host is a key step for the development of various efficient systems ranging from supramolecular fluorescent probes to supramolecular catalysts. The presence of several identical reactive groups on the host makes its selective mono-functionalization a challenge. We propose a general two-step strategy to achieve this, based on the receptor properties of the host. A guest bearing two orthogonal functions is first reacted with the host presenting itself a reactive function that is complementary to one of those of the guest. As a result, the host is selectively mono-functionalized by the covalent capture of the guest, which inhibits further reaction of the host. The second function that was present on the guest and which is now covalently linked to the host can be activated in the second step for the grafting of various objects. As a proof of concept, the strategy is described on a calix[6]arene scaffold presenting three identical reactive units. Using Huisgen thermal azide-alkyne cycloaddition for the host-guest covalent-capture step, three examples of post-functionalization are described, allowing cavities bearing a single redox tag, fluorescent probe or polydentate ligand through esterification, Schiff base formation or nucleophilic substitution to be obtained.

Supramolecular control of a mononuclear biomimetic copper(II) center: bowl complexes vs funnel complexes.

Modeling the mononuclear site of copper enzymes is important for a better understanding of the factors controlling the reactivity of the metal center. A major difficulty stems from the difficult control of the nuclearity while maintaining free sites open to coordination of exogenous ligands. A supramolecular approach consists in associating a hydrophobic cavity to a tripodal ligand that will define the coordination spheres as well as access to the metal ion. Here, we describe the synthesis of a bowl Cu(II) complex based on the resorcinarene scaffold. This study supplements a previous work on Cu(I) coordination. It provides a complete picture of the cavity-copper system in its two oxidation states. The first XRD structure of such a bowl complex was obtained, evidencing a 5-coordinate Cu(II) ion with the three imidazole donors bound to the metal (two in the base of the pyramid, one in the apical position) and with an acetate anion, completing the base of the pyramid, and deeply included in the bowl. Solution studies conducted by EPR and UV-vis absorption spectroscopies as well as cyclic voltammetry highlighted interaction with coordinating solvents, various carboxylates that can sit either in the endo or in the exo position depending on their size as well as possible stabilization of hydroxo species in a mononuclear state. A comparison of the binding and redox properties of the bowl complex with funnel complexes based on the calix[6]arene core further highlights the importance of supramolecular features defining the first, second, and third coordination sphere for control of the metal ion.

Thiolactone sulfoxides as new reactive metabolites acting as bis-electrophiles: implication in clopidogrel and prasugrel bioactivation.

The antithrombotics of the tetrahydrothienopyridine series, clopidogrel and prasugrel, are prodrugs that must be metabolized in two steps to become pharmacologically active. The first step is the formation of a thiolactone metabolite. The second step is a cytochrome P450 (P450)-dependent oxidation of this thiolactone resulting in the formation of a sulfenic acid that is eventually reduced into the corresponding active thiol. It has been postulated that the sulfenic acid metabolite resulted from a nucleophilic attack of water on a highly reactive thiolactone sulfoxide derived from P450-dependent oxidation of the thiolactone primary metabolite. The data described in the present article are in complete agreement with this proposition as they show that it was possible to trap these thiolactone sulfoxides by a series of nucleophiles such as amines, thiols, or cyclopentane-1,3-dione (CPDH), an equivalent of dimedone that is used as a sulfenic acid trapping agent. HPLC-MS studies showed that various bis-adducts having incorporated two nucleophile molecules were formed in these reactions. One of them that resulted from the oxidation of 2-oxo-prasugrel by human liver microsomes in the presence of ethanolamine and CPDH was isolated and completely characterized by (1)H and (13)C NMR spectroscopy in addition to MS and MS(2) spectrometry. All metabolites derived from an attack of H2O or an amine at the CO carbon of the intermediate thiolactone sulfoxide existed as a mixture of two diastereomers having a cis configuration of the double bond, whereas those formed in the presence of thiols appeared as a mixture of four diastereomers with a cis or trans configuration of the double bond. This led us to propose tentative mechanisms for the previously reported formation of trans isomers of the active thiol metabolite of clopidogrel upon microsomal metabolism of this antithrombotic in the presence of thiols. The results described in this article showed that thiolactone sulfoxides are formed as reactive metabolites during the metabolism of clopidogrel and prasugrel and are able to react as bis-electrophiles with a variety of nucleophiles. The possible implications of the formation of these reactive metabolites in the pharmacological and/or secondary toxic effects of these drugs remain to be studied.