N-Heterocyclic Carbene-Iridium Complexes as Photosensitizers for In Vitro Photodynamic Therapy to Trigger Non-Apoptotic Cell Death in Cancer Cells.

A series of seven novel iridium complexes were synthetized and characterized as potential photosensitizers for photodynamic therapy (PDT) applications. Among them, four complexes were evaluated in vitro for their anti-proliferative activity with and without irradiation on a panel of five cancer cell lines, namely PC-3 (prostate cancer), T24 (bladder cancer), MCF7 (breast cancer), A549 (lung cancer) and HeLa (cervix cancer), and two non-cancerous cell models (NIH-3T3 fibroblasts and MC3T3 osteoblasts). After irradiation at 458 nm, all tested complexes showed a strong selectivity against cancer cells, with a selectivity index (SI) ranging from 8 to 34 compared with non-cancerous cells. The cytotoxic effect of all these complexes was found to be independent of the anti-apoptotic protein Bcl-xL. The compound exhibiting the best selectivity, complex 4a, was selected for further investigations. Complex 4a was mainly localized in the mitochondria. We found that the loss of cell viability and the decrease in ATP and GSH content induced by complex 4a were independent of both Bcl-xL and caspase activation, leading to a non-apoptotic cell death. By counteracting the intrinsic or acquired resistance to apoptosis associated with cancer, complex 4a could be an interesting therapeutic alternative to be studied in preclinical models.

Novel molecule combinations and corresponding hybrids targeting artemisinin-resistant Plasmodium falciparum parasites.

Malaria is still considered as the major parasitic disease and the development of artemisinin resistance does not improve this alarming situation. Based on the recent identification of relevant malaria targets in the artemisinin resistance context, novel drug combinations were evaluated against artemisinin-sensitive and artemisinin-resistant Plasmodium falciparum parasites. Corresponding hybrid molecules were also synthesized and evaluated for comparison with combinations and individual pharmacophores (e.g. atovaquone, mefloquine or triclosan). Combinations and hybrids showed remarkable antimalarial activity (IC50 = 0.6 to 1.1 nM for the best compounds), strong selectivity, and didn't present any cross-resistance with artemisinin. Moreover, the combination triclosan + atovaquone showed high activity against artemisinin-resistant parasites at the quiescent stage but the corresponding hybrid lost this pharmacological property. This result is essential since only few molecules active against quiescent artemisinin-resistant parasites are reported. Our promising results highlight the potential of these combinations and paves the way for pharmacomodulation work on the best hybrids.

Ethionamide biomimetic activation and an unprecedented mechanism for its conversion into active and non-active metabolites.

Ethionamide (ETH), a second-line anti-tubercular drug that is regaining a lot of interest due to the increasing cases of drug-resistant tuberculosis, is a pro-drug that requires an enzymatic activation step to become active and to exert its therapeutic effect. The enzyme responsible for ETH bioactivation in Mycobacterium tuberculosis is a monooxygenase (EthA) that uses flavin adenine dinucleotide (FAD) as a cofactor and is NADPH- and O2-dependant to exert its catalytic activity. In this work, we investigated the activation of ETH by various oxygen-donor oxidants and the first biomimetic ETH activation methods were developed (KHSO5, H2O2, and m-CPBA). These simple oxidative systems, in the presence of ETH and NAD+, allowed the production of short-lived radical species and the first non-enzymatic formation of active and non-active ETH metabolites. The intermediates and the final compounds of the activation pathway were well characterized. Based on these results, we postulated a consistent mechanism for ETH activation, not involving sulfinic acid as a precursor of the iminoyl radical, as proposed so far, but putting forward a novel reactivity for the S-oxide ethionamide intermediate. We proposed that ETH is first oxidized into S-oxide ethionamide, which then behaves as a "ketene-like" compound via a formal [2 + 2] cycloaddition reaction with peroxide to give a dioxetane intermediate. This unstable 4-membered intermediate in equilibrium with its open tautomeric form decomposes through different pathways, which would explain the formation of the iminoyl radical and also that of different metabolites observed for ETH oxidation, including the ETH-NAD active adduct. The elucidation of this unprecedented ETH activation mechanism was supported by the application of isotopic labelling experiments.

Terpyridine-based heteroditopic ligand for Ru(II)Ln3(III) metallostar architectures (Ln = Gd, Eu, Nd, Yb) with MRI/optical or dual-optical responses.

A new ditopic ligand (L) based on a 2,2':5',4″-terpyridine unit substituted in the 2″,6″ positions with iminodiacetate arms has been designed and synthesized for the construction of Ru(II)L3Ln3(III) supramolecular architectures. The two components of this system, a 2,2'-bipyridine unit for Ru(II) coordination and a pyridine-bis(iminodiacetate) core for Ln(III) coordination, are tightly connected via a covalent Carom(py)-Carom(py) bond. The paramagnetic and photophysical properties of the corresponding tetrametallic Ru(II)L3Gd3(III) complex have been evaluated, highlighting the potential of this metallostar structure to act as a bimodal MRI/optical imaging agent. Variable-temperature (17)O NMR and proton nuclear magnetic relaxation dispersion (NMRD) measurements showed that this complex exhibits (i) a remarkable relaxivity per metallostar molecule, particularly at clinical and high magnetic fields (r1(310K) = 51.0 and 36.0 mM(-1) s(-1) at 20 and 300 MHz, respectively) and (ii) a near-optimal residence lifetime of Gd(III) coordinated water molecule (τM(310K) = 77.5 ns). This is the result of the presence of two inner-sphere water molecules in the Gd(III) components of the metallostar and a slow tumbling rate of the molecule (τR(310K) = 252 ps). Upon excitation in the visible domain (λexc = 472 nm), the Ru(II) component of the complex exhibits a bright-red luminescence centered at 660 nm with a quantum yield of 2.6% in aqueous solutions at pH 7.4. Moreover, this Ru(II)L3Gd3(III) assembly is also characterized by a high kinetic inertness in biological media (PBS and human serum solutions) and a high photostability (photobleaching). Finally, preliminary photophysical studies on RuL3Nd3 and RuL3Yb3 assemblies revealed that the Ru(II) center acts as an effective sensitizer for Ln(III)-based luminescence in the near-IR region. The Nd(III) species was found to be the most effective at quenching the (3)MLCT luminescence of the Ru center.

Biology-oriented synthesis of a natural-product inspired oxepane collection yields a small-molecule activator of the Wnt-pathway.

In Biology Oriented Synthesis the scaffolds of biologically relevant compound classes inspire the synthesis of focused compound collections enriched in bioactivity. This criterion is met by the structurally complex scaffolds of natural products (NPs) selected in evolution. The synthesis of NP-inspired compound collections approaching the complexity of NPs calls for the development of efficient synthetic methods. We have developed a one pot 4-7 step synthesis of mono-, bi-, and tricyclic oxepanes that resemble the core scaffolds of numerous NPs with diverse bioactivities. This sequence entails a ring-closing ene-yne metathesis reaction as key step and makes productive use of polymer-immobilized scavenger reagents. Biological profiling of a corresponding focused compound collection in a reporter gene assay monitoring for Wnt-signaling modulation revealed active Wntepanes. This unique class of small-molecule activators of the Wnt pathway modulates the van-Gogh-like receptor proteins (Vangl), which were previously identified in noncanonical Wnt signaling, and acts in synergy with the canonical activator protein (Wnt-3a).

Psoromic acid is a selective and covalent Rab-prenylation inhibitor targeting autoinhibited RabGGTase.

Post-translational attachment of geranylgeranyl isoprenoids to Rab GTPases, the key organizers of intracellular vesicular transport, is essential for their function. Rab geranylgeranyl transferase (RabGGTase) is responsible for prenylation of Rab proteins. Recently, RabGGTase inhibitors have been proposed to be potential therapeutics for treatment of cancer and osteoporosis. However, the development of RabGGTase selective inhibitors is complicated by its structural and functional similarity to other protein prenyltransferases. Herein we report identification of the natural product psoromic acid (PA) that potently and selectively inhibits RabGGTase with an IC(50) of 1.3 µM. Structure-activity relationship analysis suggested a minimal structure involving the depsidone core with a 3-hydroxyl and 4-aldehyde motif for binding to RabGGTase. Analysis of the crystal structure of the RabGGTase:PA complex revealed that PA forms largely hydrophobic interactions with the isoprenoid binding site of RabGGTase and that it attaches covalently to the N-terminus of the α subunit. We found that in contrast to other protein prenyltransferases, RabGGTase is autoinhibited through N-terminal (α)His2 coordination with the catalytic zinc ion. Mutation of (α)His dramatically enhances the reaction rate, indicating that the activity of RabGGTase is likely regulated in vivo. The covalent binding of PA to the N-terminus of the RabGGTase α subunit seems to potentiate its interaction with the active site and explains the selectivity of PA for RabGGTase. Therefore, psoromic acid provides a new starting point for the development of selective RabGGTase inhibitors.

Pd-catalyzed C-C and C-N cross-coupling reactions in 2-aminothieno[3,2-d]pyrimidin-4(3H)-one series for antiplasmodial pharmacomodulation.

In 2015, we identified gamhepathiopine (M1), a 2-tert-butylaminothieno[3,2-d]pyrimidin-4(3H)-one antiplasmodial hit targeting all development stages of the human malarial parasite P. falciparum. However, this hit compound suffers from sensitivity to hepatic oxidative metabolism. Herein, we describe the synthesis of 33 new compounds in the 2-aminothieno[3,2-d]pyrimidin-4(3H)-one series modulated at position 6 of this scaffold. The modulations were performed using three palladium-catalyzed cross coupling reactions, namely Suzuki-Miyaura, Sonogashira, and Buchwald-Hartwig. For the latter, we developed the reaction conditions. Then, we evaluated the synthesized compounds for their antiplasmodial activity on the K1 P. falciparum strain and their cytotoxicity on the human HepG2 cell line. Although we did not obtain a compound better than M1 in terms of the antiplasmodial activity, we identified compound 1g bearing a piperidine at position 6 of the thieno[3,2-d]pyrimidin-4(3H)-one ring with an improved cytotoxicity and metabolic stability. 1g is an interesting new starting point for further pharmacomodulation studies. This study also provides valuable antiplasmodial SAR data regarding the nature of the ring at position 6, the possible substituent on this ring, and the introduction of a spacer between this ring and the thienopyrimidinone moiety.

Analysis of the eukaryotic prenylome by isoprenoid affinity tagging.

Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl-tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents.

Chemical synthesis, biological evaluation and structure-activity relationship analysis of azaisoindolinones, a novel class of direct enoyl-ACP reductase inhibitors as potential antimycobacterial agents.

The synthesis and biological evaluation of azaisoindolinone compounds embedding a lipophilic chain on the framework were performed. These compounds were designed as InhA inhibitors and as anti-Mycobacterium tuberculosis agents. Structure-activity relationships concerning the length and the location of the lipophilic chain around the azaisoindolinone framework, the suppression of the phenyl group, the bioisosteric substitution of ether link and alkylating of the tertiary hydroxyl and the hemiamidal nitrogen were also investigated, revealing insightful information and thereby enabling further diversification of the azaisoindolinone scaffold for new antitubercular agents.

New 8-Nitroquinolinone Derivative Displaying Submicromolar in Vitro Activities against Both Trypanosoma brucei and cruzi.

An antikinetoplastid pharmacomodulation study was conducted at position 6 of the 8-nitroquinolin-2(1H)-one pharmacophore. Fifteen new derivatives were synthesized and evaluated in vitro against L. infantum, T. brucei brucei, and T. cruzi, in parallel with a cytotoxicity assay on the human HepG2 cell line. A potent and selective 6-bromo-substituted antitrypanosomal derivative 12 was revealed, presenting EC50 values of 12 and 500 nM on T. b. brucei trypomastigotes and T. cruzi amastigotes respectively, in comparison with four reference drugs (30 nM ≤ EC50 ≤ 13 µM). Moreover, compound 12 was not genotoxic in the comet assay and showed high in vitro microsomal stability (half life >40 min) as well as favorable pharmacokinetic behavior in the mouse after oral administration. Finally, molecule 12 (E° = -0.37 V/NHE) was shown to be bioactivated by type 1 nitroreductases, in both Leishmania and Trypanosoma, and appears to be a good candidate to search for novel antitrypanosomal lead compounds.

Synthesis and mechanistic investigation of iron(II) complexes of isoniazid and derivatives as a redox-mediated activation strategy for anti-tuberculosis therapy.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB) represents a major threat to global health. Isoniazid (INH) is a prodrug used in the first-line treatment of tuberculosis. It undergoes oxidation by a catalase-peroxidase KatG, leading to generation of an isonicotinoyl radical that reacts with NAD(H) forming the INH-NADH adduct as the active metabolite. A redox-mediated activation of isoniazid using an iron metal complex was previously proposed as a strategy to overcome isoniazid resistance due to KatG mutations. Here, we have prepared a series of iron metal complexes with isoniazid and analogues, containing alkyl substituents at the hydrazide moiety, and also with pyrazinamide derivatives. These complexes were activated by H2O2 and studied by ESR and LC-MS. For the first time, the formation of the oxidized INH-NAD adduct from the pentacyano(isoniazid)ferrate(II) complex was detected by LC-MS, supporting a redox-mediated activation, for which a mechanistic proposition is reported. ESR data showed all alkylated hydrazides, in contrast to non-substituted hydrazides, only generated alkyl-based radicals. The structural modifications did not improve minimal inhibitory concentration (MIC) against MTB in comparison to isoniazid iron complex, providing support to isonicotinoyl radical formation as a requirement for activity. Nonetheless, the pyrazinoic acid hydrazide iron complex showed redox-mediated activation using H2O2 with generation of a pyrazinoyl radical intermediate and production of pyrazinoic acid, which is in fact the active metabolite of pyrazinamide prodrug. Thereby, this strategy can also unveil new opportunities for activation of this type of drug.

Update of Antitubercular Prodrugs from a Molecular Perspective: Mechanisms of Action, Bioactivation Pathways, and Associated Resistance.

The place of prodrugs in the current antitubercular therapeutic arsenal is preponderant, since two of the four first-line antitubercular agents, isoniazid (INH) and pyrazinamide (PZA), need to be activated by Mycobacterium tuberculosis before exerting their activity. In addition, six other prodrugs can be found in the second- and third-line therapeutic regimens. The emergence of mycobacterial strains resistant to one or several antitubercular agents is one of the main issues of the antitubercular therapy. In the case of prodrugs, the resistance phenomenon is often related to a mutation in the gene encoding for the activation enzymes, resulting thus in a default of these enzymes that are no more able to activate prodrugs. Consequently, identification of the prodrugs targets and a better understanding of their modes of action and also of their activation mechanisms are of crucial importance. Related to their molecular mechanism of activation, these prodrugs may thus be classified in four categories: activation via oxidation (catalase-peroxidase (KatG) or flavin monooxygenase (EthA) enzymes), condensation (FolP1 and FolC), hydrolysis (by the amidase PncA) and reduction (by the nitroreductase DnD) mechanisms. For each prodrug, these mechanisms are described in details, as well as the mechanism of action of its active metabolite. Finally, the reported resistance related to these mechanisms of activation/action are also addressed in a molecular perspective.

Influence of chelators and iron ions on the production and degradation of H2O2 by beta-amyloid-copper complexes.

beta-Amyloid peptide (Abeta) 1-42, involved in the pathogenesis of Alzheimer's disease, binds copper ions to form AbetaxCu(n) complexes that are able to generate H(2)O(2) in the presence of a reductant and O(2). The production of H(2)O(2) can be stopped with chelators. More reactive than H(2)O(2) itself, hydroxyl radicals HO() (generated when a reduced redox active metal complex interacts with H(2)O(2)) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H(2)O(2) associated to a pre-incubation step between ascorbate and AbetaxCu(n) allows to study the formation of H(2)O(2) but also, at the same time, its transformation by the copper complexes. AbetaxCu(n) peptides produce but do not efficiently degrade H(2)O(2). The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H(2)O(2) production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from AbetaxCu(n), whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with AbetaxCu(n). The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.

Preparation and study of new poly-8-hydroxyquinoline chelators for an anti-Alzheimer strategy.

Fourteen different ligands have been synthesized with two covalently linked 8-hydroxyquinoline motifs that favor metal complexation. These bis-chelators include different bridges at the C2 positions and different substituents to modulate their physicochemical properties. They can form metal complexes in a ratio of one ligand per metal ion with Cu II and Zn II, two metal ions involved in the formation of amyloid aggregates of the toxic Abeta-peptides in the Alzheimer disease. The apparent affinity of all bis-8-hydroxyquinoline ligands for Cu II and Zn II are similar with logK Cu II approximately 16 and logK Zn II approximately 13 and are 10,000 times more efficient than for the corresponding 8-hydroxyquinoline monomers. Their strong chelating capacities allow them to inhibit more efficiently than the corresponding monomers the precipitation of Abeta-peptides induced by Cu II and Zn II and also to inhibit the toxic formation of H2O2 due to copper complexes of Abeta. The best results were obtained with a one-atom linker between the two quinoline units. X-ray analyses of single-crystals of Cu II, Zn II or Ni II complexes of 2,2'-(2,2-propanediyl)-bis(8-hydroxyquinoline), including a one-atom linker, showed that all heteroatoms of the bis-8-hydroxyquinoline ligand chelate the same metal ion in a distorted square-planar geometry. The Cu II and Zn II complexes include a fifth axial ligand and are pentacoordinated.

Insights into the cholecystokinin 2 receptor binding site and processes of activation.

The cholecystokinin (CCK) 2 receptor (CCK2R) appears as a pharmacological target for the treatment of many major diseases. To complete the mapping of the CCK2R binding site and its activation processes, we have looked for the receptor residues that interact with Trp6, an essential residue for CCK binding and activity. In our molecular model of the CCK-occupied CCK2R, the indole group of Trp6 stacked with the phenyl ring of Phe120 (ECL1) and interacted with the imidazole group of His381(H7.39) and the phenyl ring of Tyr385(H7.43). Mutagenesis and pharmacological studies validated these interactions. It is noteworthy that the mutation of Phe120 to Trp conferred constitutive activity to the CCK2R. Molecular modeling and experimental works identified the residues involved in the activation cascade initiated by Trp6 and revealed that the constitutively active F120W mutation mimics the conformational changes induced by Trp6 resulting in: 1) the exposure of Glu151(E3.49) of the conserved E/DRY motif 2) the formation of an amphiphatic pocket involving protonated Glu151(E3.49) and Leu330 (ICL3), and 3) the opening of the intracellular loops 2 and 3 and the release of Arg158 (ICL2). The R158A mutation was shown to affect inositol phosphate production, whereas the E151A and L330E mutations induced constitutive inositol phosphate production. Given that a constitutively active variant of the CCK2R has been identified in different cancers and the fact that the E151A mutant has been reported to induce tumors, these studies should help in the development of potent inverse agonists to inhibit the constitutive activation of the CCK2R.