Methylation-dependent T cell immunity to Mycobacterium tuberculosis heparin-binding hemagglutinin.

Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides. Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice. Methylated HBHA provides high levels of protection against M. tuberculosis challenge in mice, whereas nonmethylated HBHA does not. Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guérin, the only available anti-tuberculosis vaccine. Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.

Synthesis and antimalarial activity of carbamate and amide derivatives of 4-anilinoquinoline.

A series of 4-anilinoquinolines bearing an amino side chain linked to the aromatic ring with a carbamate or an amide bond were synthesized and evaluated for their antimalarial activity and their cytotoxicity upon MRC-5 cells. Among the 17 compounds, a majority was found to be active in the low nanomolar range against both chloroquine-sensitive and -resistant strains of Plasmodium falciparum in vitro with relative low cytotoxicity. Two compounds were then tested on mice infected by Plasmodium berghei and were found to exhibit reasonable in vivo activity.

Synthesis and antimalarial activity of new analogues of amodiaquine.

In order to determine the real significance of the 4'-phenolic group in the antimalarial activity and/or cytotoxicity of amodiaquine (AQ), analogues for which this functionality was shifted or modified were synthesized. Good in vitro antimalarial activity was obtained for compounds unable to form intramolecular hydrogen bond. Among the compounds synthesized, new amino derivative 5 displayed the greatest selectivity index towards the most CQ-resistant strain tested and was active in mice infected by Plasmodium berghei.

Biochemical properties and cellular localization of Plasmodium falciparum protein disulfide isomerase.

We have previously reported the isolation of a 52,000 M(r) protein (Pf52) displaying consensus sequences for thiol:disulfide oxidoreductases. Pf52 therefore represents the plasmodial protein disulfide isomerase (PDI). It has been renamed PfPDI and correlates to MAL8P1.17 in the annotated genome of P. falciparum (3D7 strain). Antibodies were raised against recombinant (His)(6)-tagged forms of PfPDI devoid of its signal peptide sequence, demonstrating a major co-localization of PfPDI with endoplasmic reticulum-resident proteins, PfBIP and PfERC, but not with the Golgi marker PfERD2. Recombinant PfPDI displayed typical biochemical functions of PDIs: oxidase/isomerase and reductase activities, as well as a chaperone-like behavior on the denaturated protein rhodanese. These activities were comparable to those measured for the purified native bovine PDI and the human recombinant PDI. The antiplasmodial compound DS61 does inhibit the recombinant PfPDI oxidase/isomerase activity but not that of the human recombinant PDI, suggesting structural differences between both enzymes. However, a discrepancy between the inhibitory activity of DS61 on the recombinant PfPDI (IC(50) of 430 microM) and its in vitro antiplasmodial activity (IC(50) of 0.1 microM) was observed, suggesting that PfPDI is not the only target of DS61. Taking into account its biochemical properties and its intracellular localization, the involvement of PfPDI in the parasite protein folding is discussed, as well as its potential for the development of alternative antimalarial chemotherapy strategies.

Glucose regulates LXRalpha subcellular localization and function in rat pancreatic beta-cells.

Liver X receptors (LXRs) are members of the nuclear receptor superfamily, which have been implicated in lipid homeostasis and more recently in glucose metabolism. Here, we show that glucose does not change LXRalpha protein level, but affects its localization in pancreatic beta-cells. LXRalpha is found in the nucleus at 8 mM glucose and in the cytoplasm at 4.2 mM. Addition of glucose translocates LXRalpha from the cytoplasm into the nucleus. Moreover, after the activation of LXR by its synthetic non-steroidal agonist (T0901317), insulin secretion and glucose uptake are increased at 8 mM and decreased at 4.2 mM glucose in a dose-dependent manner. Furthermore, at low glucose condition, okadaic acid reversed LXRalpha effect on insulin secretion, suggesting the involvement of glucose signaling through a phosphorylation-dependent mechanism.

Chloroquine and chloroquinoline derivatives as models for the design of modulators of amyloid Peptide precursor metabolism.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD). Preventing deregulated APP processing by inhibiting amyloidogenic processing of carboxy-terminal fragments (APP-CTFs), and reducing the toxic effect of amyloid beta (Aß) peptides remain an effective therapeutic strategy. We report the design of piperazine-containing compounds derived from chloroquine structure and evaluation of their effects on APP metabolism and ability to modulate the processing of APP-CTF and the production of Aß peptide. Compounds which retained alkaline properties and high affinity for acidic cell compartments were the most effective. The present study demonstrates that (1) the amino side chain of chloroquine can be efficiently substituted by a bis(alkylamino)piperazine chain, (2) the quinoline nucleus can be replaced by a benzyl or a benzimidazole moiety, and (3) pharmacomodulation of the chemical structure allows the redirection of APP metabolism toward a decrease of Aß peptide release, and increased stability of APP-CTFs and amyloid intracellular fragment. Moreover, the benzimidazole compound 29 increases APP-CTFs in vivo and shows promising activity by the oral route. Together, this family of compounds retains a lysosomotropic activity which inhibits lysosome-related Aß production, and is likely to be beneficial for therapeutic applications in AD.

Synthesis and in vitro and in vivo antimalarial activity of N1-(7-chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine derivatives.

Three series of monoquinolines consisting of a 1,4-bis(3-aminopropyl)piperazine linker and a large variety of terminal groups were synthesized. Our aim was to prove that in related bisquinoline, it is the second quinoline moiety that is responsible for cytotoxicity and that it is not an absolute requirement for overcoming resistance to chloroquine (CQ). Eleven compounds displayed a higher selectivity index (ratio CC50/IC50 activity) than CQ, and one of them cured mice infected by Plasmodium berghei.

Synthesis, antimalarial activity, and molecular modeling of new pyrrolo[1,2-a]quinoxalines, bispyrrolo[1,2-a]quinoxalines, bispyrido[3,2-e]pyrrolo[1,2-a]pyrazines, and bispyrrolo[1,2-a]thieno[3,2-e]pyrazines.

Three pyrrolo[1,2-a]quinoxalines, 15 bispyrrolo[1,2-a]quinoxalines, bispyrido[3,2-e]pyrrolo[1,2-a]pyrazines, and bispyrrolo[1,2-a]thieno[3,2-e]pyrazines were synthesized from various substituted nitroanilines or nitropyridines and tested for their in vitro activity upon the erythrocytic development of Plasmodium falciparum strains with different chloroquine-resistance status. Bispyrrolo[1,2-a]quinoxalines showed superior antimalarial activity with respect to monopyrrolo[1,2-a]quinoxalines. The best activity was observed with bispyrrolo[1,2-a]quinoxalines linked by a bis(3-aminopropyl)piperazine. Moreover, it was observed that the presence of a methoxy group on the pyrrolo[1,2-a]quinoxaline nucleus increased the pharmacological activity. Drug effects upon beta-hematin formation were assayed and showed similar or higher inhibitory activities than CQ. A possible mechanism of interaction implicating binding of pyrroloquinoxalines to beta-hematin was supported by molecular modeling.

Design, synthesis and in vitro antimalarial activity of an acylhydrazone library.

A library of acylhydrazone iron chelators was synthesized and tested for its ability to inhibit the growth of a chloroquine-resistant strain of Plasmodium falciparum. Some of these new compounds are significantly more active than desferrioxamine DFO, the iron chelator in widespread clinical use and also than the most effective chelators.

Synthesis and biological evaluation of benzimidazole derivatives as potent AMP-activated protein kinase activators.

Design, synthesis and structure-activity relationships of benzimidazole derivatives as activators of the AMP-activated protein kinase (AMPK) are presented in this paper. AMPK is the central component of a protein kinase cascade that plays a key role in the regulation of energy balance. Once activated, AMPK initiates a series of responses that are aimed at restoring the energy balance of the cell and recent studies have indicated that AMPK plays an important role in regulation of the whole-body energy metabolism. The following study based on the lead compound S27847 involved modification of three regions of this compound. Preliminary structure-activity relationships are being described.

Conversion of sterically hindered diacylated 1,2-phenylenediamines into 2-substituted benzimidazoles.

A series of bulky 2-substituted benzimidazoles was designed in order to find new leads for several biological targets. Formation by cyclodehydration from their monoacylated counterparts was shown to be strongly dependent upon the nature of the acyl group. In the case of a dicyclohexylmethyl group, cyclization was only observed in a p-toluenesulfonic acid/toluene mixture from the symmetrical diacylated precursor. Analysis of the mechanism was begun starting from mixed diacylated derivatives.

Synthesis and antimalarial evaluation of new N1-(7-chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine derivatives.

Synthesis and evaluation of the activity of new N(1)-(7-chloro-4-quinolyl)-1,4-bis(3-aminopropyl)piperazine derivatives against a chloroquine-resistant strain of Plasmodium falciparum are described. Selectivity indices were improved for two compounds versus the lead 1, the bis-cyclopropylmethyl derivative, thus increasing the therapeutic interest of our family. As our previous studies conducted on the mode of action of our compounds made us hypothesize the existence of original mechanisms and/or original targets, terminal amino derivatives can be considered as promising tools further mechanistical studies, as probes for affinity chromatography.

Synthesis and pharmacological evaluation of Tic-hydantoin derivatives as selective sigma1 ligands. Part 2.

Herein is described a new class of selective sigma1 ligands consisting of tetrahydroisoquinoline-hydantoin (Tic-hydantoin) derivatives. Compound 1a has high affinity (IC50 = 16 nM) for sigma1 receptor and is selective in a large panel of therapeutic targets. This study presents structural changes on the side chain of the Tic-hydantoin core. Analogs of higher affinity could be identified (IC50 approximately 2-3 nM).

Synthesis and pharmacological evaluation of Tic-hydantoin derivatives as selective sigma1 ligands. Part 1.

Herein is described a new class of selective sigma1 ligands consisting of tetrahydroisoquinoline-hydantoin (Tic-hydantoin) derivatives. Compound 3a has high affinity (IC50 = 16 nM) for the sigma1 receptor and is selective in a large panel of therapeutic targets. This first study presents structural changes around the Tic-hydantoin core, leading to a Tic-hydantoin analogue with a higher sigma1 affinity (IC50 approximately 1 nM).

Novel N-(4-Piperidinyl)benzamide antimalarials with mammalian protein farnesyltransferase inhibitory activity.

Protein farnesyltransferase of Plasmodium falciparum is a potential target in the treatment of malaria for which increased drug resistance is observed. The design, synthesis and evaluation of a series of N-(4-piperidinyl)benzamides is reported. The most potent compounds showed in vitro activity against the parasite at submicromolar concentrations.

Design, synthesis and antimalarial activity of novel, quinoline-based, zinc metallo-aminopeptidase inhibitors.

PfA-M1, a neutral zinc aminopeptidase of Plasmodium falciparum, is a new potential target for the discovery of antimalarials. The design and synthesis of a library of 45 quinoline-based inhibitors of PfA-M1 is reported. The best inhibitor displays an IC(50) of 854 nM. The antimalarial activity on a CQ-resistant strain and the specificity towards mammalian aminopeptidase N are also discussed.

Antitrypanosomal activities and cytotoxicity of 5-nitro-2-furancarbohydrazides.

A series of 5-nitro-2-furancarbohydrazides were synthesized. In vitro antitrypanosomal activities of these compounds were determined against the closely related protozoa Trypanosoma cruzi Trypanosoma brucei and discussed in relation to potential targets.

Characterization of boar sperm cytoskeletal cylicin II as an actin-binding protein.

The presence of actin-binding proteins in the perinuclear theca of boar spermatozoa has been investigated, using stepwise extractions of proteins from sperm heads. Proteins extracted with the alkaline buffer 1M Na(2)CO(3), pH 11, were found to contain a 66kDa protein that binds F-actin in actin pelleting assays. Sequence studies and immunological characterization with antibodies specific for human cylicin II identified the 66kDa protein as the homologue of bovine and human cylicin II. Immunocytochemical studies showed the presence of porcine cylicin II in the acrosomal region of round spermatids and in the postacrosomal region of late spermatids and spermatozoa, in agreement with the previously described localization of cylicins. Taken together, the results suggest that cylicin II, a protein of the sperm perinuclear cytoskeleton, is a novel actin-binding protein, which probably plays a role in the actin-related events that occur during spermiogenesis and the early events of fertilization.

Characterization of aggregates of hepatitis C virus glycoproteins.

Hepatitis C virus (HCV) encodes two glycoproteins, E1 and E2, which assemble in oligomeric structures. Studies of HCV glycoprotein assembly using heterologous expression systems have shown that these glycoproteins can follow two pathways: a productive pathway leading to the formation of a non-covalent heterodimer; and a non-productive pathway leading to the formation of large disulfide-linked aggregates. The non-covalent HCV glycoprotein complex is probably the functional complex which plays an active role in the entry process in host cells. The aggregates are believed to be waste products; however, one can imagine that, in infected cells, they could provide HCV glycoproteins with additional functions. To further understand the potential role played by HCV glycoprotein aggregates in HCV infection, a MAb (H14) was produced which specifically recognizes these aggregates but not the non-covalent E1E2 heterodimer. The H14 epitope was shown to be present on both HCV glycoproteins and was sensitive to deglycosylation. An additional characterization of HCV glycoprotein aggregates, with the help of MAb H14, indicates that they share an epitope with a cellular protein called Mac-2 binding protein. The presence of such an epitope on HCV glycoprotein aggregates could potentially lead to the production of autoantibodies recognizing Mac-2 binding protein in HCV-infected patients.

Synthesis and antimalarial evaluation of new 1,4-bis(3-aminopropyl)piperazine derivatives.

Synthesis and evaluation of the activity of a new family of 1,4-bis(3-aminopropyl)piperazine derivatives against a chloroquine-resistant strain of Plasmodium falciparum, and as inhibitors of beta-hematin formation, are described. The highest antimalarial activities were obtained for compounds displaying the highest predicted vacuolar accumulation ratios and the best potencies as inhibitors of beta-hematin formation. The most potent compound displayed an activity 3-fold better than chloroquine for a comparable selectivity index upon MRC-5 cells. Therefore, in this series, the replacement of the 7-chloroquinoline group can constitute a strong rationale for further investigation.