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A highly efficient and facile protocol for the selective reduction of carboxylic acid of Fenofibric acid to corresponding alcohol was developed. The selective reduction was carried out by activation of carboxylic acid by mixed anhydride followed by the reaction of sodium borohydride in presence of methanol. This is the first example of chemoselective reduction of carboxylic acid to alcohol in presence of a ketone without any external catalyst or ligand in a single step. The reaction offers wide applicability for the selective carboxylic group reduction methodology. The chemoselective reduction was demonstrated by the reduction of Fenofibric acid, an active metabolite of the drug Fenofibrate, to corresponding alcohol in excellent selectivity, yield, and purity.

The correlation of mycobactericidal property of macrophages with its potential to deliver bacteria to hydrolytic lysosomes, augmented with ubiquitin-derived peptides (Ub2), activates the process of autophagy. This leads to the formation of phagolysosomes supported by factor involving increased cationic charges which regulate the acidic pH causing elimination of Mycobacterium. To better understand this interaction of cationic-rich ubiquitin-derived peptides with mycobacteria and to identify putative mycobacterial intrinsic resistance mechanisms for phagolysosome formation, we have synthesized a new series of Ub2 peptides, wherein the Gly residues are replaced with azaGly with the aim to improve metabolic stability. In addition to that a new methodology is reported for the synthesis of heteroaryl tethered peptides using azaGly as a linker. We have demonstrated that positive puncta (directly proportional to the acidification of lysosome) in cytosol was significantly increased after 6 hours on the treatment of macrophage with Ub2 peptide derivatives (1, 6, 10, and 11) causing the higher intensity of lysosome observed through LysoTracker Red Dye. The circular dichroism spectral studies are carried out in water and water:TFE mixture and demonstrated that the Ub2 peptides have helix-forming tendency in the presence of TFE. The recognizable intracellular killing of Mycobacterium tuberculosis by Ub2 peptides provides a new approach for host-directed therapy.

An approach for the synthesis of a variety of new β-aryl-β-amino acids has been developed via a palladium-catalyzed auxiliary-directed regioselective Csp3-H arylation of the unactivated β-methylene bond of β-alanine. The use of 8-aminoquinoline amide as an auxiliary efficiently directs the desired regioselective β-Csp3-H functionalization. The developed protocol enables the easy and straightforward access to several high-value β-aryl-β-amino acids useful for peptide engineering, starting from inexpensive and readily available β-alanine precursors in moderate to excellent yields.

An expeditious method for the synthesis of homo and heterochiral dipeptides containing l-alanine and d/l 2-methyl allo-hydroxyl prolines was developed using direct aminolysis of bicyclic lactones derived from d/l alanine. The impact of C-2 methylation and its spatial orientation on the pyrrolidine ring puckering and prolyl amide bond configuration was ascertained by solution NMR studies. The present studies reveal that C-2 methylation causes the prolyl amide bond to exist exclusively in the trans geometry in both homo- and heterochiral dipeptides. However, the spatial orientation of the C-2 methyl group and its i + 2 position in appropriately capped model dipeptides may nucleate into a turn like structure.

A simple and straightforward process for the synthesis of rapamycin peptide conjugates in a regio and chemoselective manner was developed. The methodology comprises the tagging of chemoselective functionalities to rapamycin and peptides which enables the conjugation of free peptides, without protecting the functionality of the side chain amino acids, in high yield and purity. From this methodology, we successfully conjugate free peptides containing up to 15 amino acids. Rapamycin is also conjugated to the peptides known for inhibiting the kinase activity of Akt protein. These conjugates act as dual target inhibitors and inhibit the kinase activity of both mTOR and Akt.

Kalaimathi Murugesan,1 Padmapriya Srinivasan,1,* Raghunandan Mahadeva,1,* Chhitar M Gupta,1 Wahajul Haq2 1Institute of Bioinformatics and Applied Biotechnology (IBAB), Bangalore, India; 2Central Drug Research Institute (CDRI), Medicinal and Process Chemistry Division, Lucknow, India*These authors contributed equally to this workCorrespondence: Kalaimathi MurugesanInstitute of Bioinformatics and Applied Biotechnology (IBAB), Biotech Park, Electronic City Phase I, Bangalore 560100, IndiaTel +919585986415Email mathi.biotech@gmail.comBackground: Targeted multidrug-loaded delivery systems have emerged as an advanced strategy for cancer treatment. In this context, antibodies, hormones, and small peptides have been coupled to the surface of drug carriers, such as liposomes, polymeric and metallic nanoparticles loaded with drugs, as tumor-specific ligands. In the present study, we have grafted a natural macrophage stimulating peptide, tuftsin, on the surface of the liposomes (LPs) that were loaded with doxorubicin (DOX) and/or curcumin (CUR), by attaching to its C-terminus a palmitoyl residue (Thr-Lys-Pro-Arg-CO-NH-(CH2)2-NH-COC15H31, P.Tuft) to enable its grafting within the liposome’s bilayer.Methods: The prepared drug-loaded liposomes (DOX LPs, CUR LPs, DOX-CUR LPs, P.Tuft-LPs, P.Tuft-DOX LPs, P.Tuft-CUR LPs, P.Tuft-DOX-CUR LPs) were thoroughly characterised in terms of particle size, drug content, encapsulation efficiency and structural properties using UV–visible spectroscopy, dynamic light scattering (DLS) and Fourier transform infrared spectroscopy (FTIR). The anti-cancer activity and drug toxicity of the liposomal formulations were examined on Ehrlich ascites carcinoma (EAC) tumor-induced mice model.Results: A significant reduction in the tumor weight and volume was observed upon treating the tumor-bearing mice with palmitoyl tuftsin-grafted dual drug-loaded liposomes (P.Tuft-DOX-CUR LPs), as compared to the single drug/peptide-loaded formulation (DOX LPs, CUR LPs, DOX-CUR LPs, P.Tuft- LPs, P.Tuft-DOX LPs, P.Tuft-CUR LPs). Western blot analysis revealed that the tumor inhibition was associated with p53-mediated apoptotic pathway. Further, the biochemical and histological analysis revealed that the various liposomal preparation used in this study were non-toxic to the animals at the specified dose (10mg/kg).Conclusion: In conclusion, we have developed a targeted liposomal formulation of P.Tuftsin-bearing liposomes co-encapsulated with effective anti-cancer drugs such as doxorubicin and curcumin. In experimental animals, tumor inhibition by P.Tuft-DOX-CUR LPs indicates the synergistic therapeutic effect of the peptide and the dual drug.Keywords: palmitoyl-tuftsin, antitumor, doxorubicin, curcumin

A series of novel 4-aminoquinoline analogues bearing a methyl group at 4-aminoquinoline moiety were synthesized via a new and robust synthetic route comprising in situ tert-butoxycarbonyl (Boc) deprotection-methylation cascade resulting in the corresponding N-methylated secondary amine using Red-Al and an efficient microwave-assisted strategy for the fusion of N-methylated secondary amine with 4-chloroquinoline nucleus to access the series of novel 4-N-methylaminoquinoline analogues. The new series of compounds were evaluated for their antimalarial activity in in vitro and in vivo models. Among 21 tested compounds, 9a-i have shown a half-maximal inhibitory concentration (IC50) value less than 0.5 μM (i.e.

Synthetic routes for the preparation of (i) isopropyl 2-(4-(4-chlorobenzyl)phenoxy)-2-methyl propanoate (Reduced Fenofibrate, Fibrane) (2) from isopropyl 2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanoate (Fenofibrate) (1) in a single step is established in good yield and purity under mild conditions. The newly synthesized derivative of Fenofibrate has been characterized by NMR and IR spectroscopy techniques. Selective conversion of biphenyl ketone moiety, in the presence of ester carbonyl group in Fenofibrate to its corresponding alkane can be performed by Pd catalyst reduction with hydrogen transfer agent ammonium formate.

Background: Smac mimetics (also known as IAP antagonist) are a new class of targeted drugs having a goal to suppress the IAPs, reestablishing the apoptotic pathways and inducing cancer cell death. Therefore, development of Smac mimetics was considered an attractive strategy for the development of new anticancer drugs. Lots of reviews have come in yesteryears which mainly discussed the biology of IAPs and their role in cancer development. None of these reviews focused on the chemical synthesis of Smac mimetics. Methods: Literature study was done by using standard bibliographic search engines like scifinder, pubmed etc. The characteristic features of screened articles were described in the review. Results: The review gives an introduction of IAP proteins and Smac mimetics. Readers will gain an overview of the development of Smac mimetics with representative examples of both monovalent and bivalent Smac mimetics as anticancer agents and an understanding of their structure-activity relationships. Chemical synthesis of biologically important Smac mimetics was discussed briefly in this review. Conclusion: Small molecules that mimic Smac are continuously progressing towards clinical development. Smac mimetics are generally well tolerated and have demonstrated rapid suppression of their target (the IAPs), activation of apoptosis and anti-tumor activity. Continuous research has been done to generate even more insight into the function of IAP proteins to significantly enhance the therapeutical potential of Smac mimetics.

Practical synthetic route for the formation of enantiomeric mixture of Isopropyl 2-(4-((4-chlorophenyl)(hydroxyl)methyl)phenoxy)-2-methylpropanoate (Fibratol 2a/b) from isopropyl 2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanoate (Fenofibrate 1) has been developed. Method has also been established for the chiral separation of enantiomers of Fibratol 2a/b that is synthesized using the route mentioned above. The optical activity determined for enantiomerically separated Fibratol (2a) and Fibratol (2b) are −5.2° and 8.0° which reflect their ability to rotate plane polarized light counterclockwise (levo) and clockwise (dextro), respectively.

We report the synthesis and biological and physiochemical properties of a series of azaGly-appended peptidomimetics. We have developed a simple and facile synthesis for azapeptides on solid support without any side reaction. The azaGly is inserted by in situ reaction of disuccinimidyl carbonate with free amine followed by treatment of hydrazine hydrate at room temperature. The new series of peptidomimetics was prepared by azaGly scanning of heptapeptide Arg-Pro-Arg-Nle-Tyr-Dap-Nle (Akt-01), a GSK-3β-derived Akt inhibitor. The azaGly-appended peptides showed significant improvement in biological activity and serum stability, with retention of conformation as evidenced by NMR and CD studies. The results clearly demonstrate that azaGly-appended peptides are new peptidomimetics. Their synthesis makes this approach highly useful for the development of novel peptidomimetics of therapeutic potential.

Background The mechanistic (or mammalian) target of rapamycin (mTOR), a Ser/Thr kinase, associates with different subunits forming two functionally distinct complexes, mTORC1 and mTORC2, regulating a diverse set of cellular functions in response to growth factors, cellular energy levels, and nutrients. The mechanisms regulating mTORC1 activity are well characterized; regulation of mTORC2 activity, however, remains obscure. While studies conducted in Dictyostelium suggest a possible role of Ras protein as a potential upstream regulator of mTORC2, definitive studies delineating the underlying molecular mechanisms, particularly in mammalian cells, are still lacking. Methods Protein levels were measured by Western blotting and kinase activity of mTORC2 was analyzed by in vitro kinase assay. In situ Proximity ligation assay (PLA) and co-immunoprecipitation assay was performed to detect protein-protein interaction. Protein localization was investigated by immunofluorescence and subcellular fractionation while cellular function of mTORC2 was assessed by assaying extent of cell migration and invasion. Results Here, we present experimental evidence in support of the role of Ras activation as an upstream regulatory switch governing mTORC2 signaling in mammalian cancer cells. We report that active Ras through its interaction with mSIN1 accounts for mTORC2 activation, while disruption of this interaction by genetic means or via peptide-based competitive hindrance, impedes mTORC2 signaling. Conclusions Our study defines the regulatory role played by Ras during mTORC2 signaling in mammalian cells and highlights the importance of Ras-mSIN1 interaction in the assembly of functionally intact mTORC2.

Development of a suitable vaccine against visceral leishmaniasis (VL), a fatal parasitic disease, is considered to be vital for maintaining the success of kala-azar control programs. The fact that Leishmania-infected individuals generate life-long immunity offers a viable proposition in this direction. Our prior studies demonstrated that T-helper1 (Th1) type of cellular response was generated by six potential recombinant proteins viz. elongation factor-2 (elF-2), enolase, aldolase, triose phosphate isomerase (TPI), protein disulfide isomerase (PDI) and p45, derived from a soluble antigenic fraction (89.9–97.1 kDa) of Leishmania (Leishmania) donovani promastigote, in treated Leishmania patients and golden hamsters and showed significant prophylactic potential against experimental VL. Moreover, since, it is well-known that our immune system, in general, triggers production of specific protective immunity in response to a small number of amino acids (peptide), this led to the identification of antigenic epitopes of the above-stated proteins utilizing immunoinformatics. Out of thirty-six, three peptides-P-10 (enolase), P-14, and P-15 (TPI) elicited common significant lymphoproliferative as well as Th1-biased cytokine responses both in golden hamsters and human subjects. Further, immunization with these peptides plus BCG offered 75% prophylactic efficacy with boosted cellular immune response in golden hamsters against Leishmania challenge which is indicative of their candidature as potential vaccine candidates.

Additional file 1 : Figure S1. Superoxide anion generation in Pyrogallol treated cells. (A) MDA-MB-231 cells were treated with Pyrogallol (10, 20, 50 and 100 μM) for 24 h followed by 30 mins incubation with 10 μM DHE and analyzed for superoxide anion detection using a fluorescence microscope (above). Phase-contrast images of cells after indicated concentration of Pyrogallol treatment (below). Scale bars, 50 μm. (B) MDA-MB-231 cells were treated with 20 μM Pyrogallol for time point as indicated in the figure, and Western blotting was done for mTORC2 specific markers. All data are representative of three independent experiments. Figure S2. Pyrogallol prevents Ras localization to the plasma membrane. MDA-MB-231 cells pre-treated with FTI (Lonafarnib 1 μM) for 4 h followed by stimulation with Pyrogallol (20 μM) for another 24 h. Cells were analyzed for Ras localization by immunofluorescence microscopy. Data are representative of three independent experiments. Figure S3. Evaluation of sequences of peptides. Evaluation of sequences of synthetic peptides identical to the Ras-binding domain (RBD) of mSIN1. Figure S4. Peptide penetration and mutation analysis. (A) Immunofluorescence images of MDA-MB-231 cells treated with FITC-conjugated P4 (50 μg/ml) for 24 h. (B) RBD sequences of wild-type and mutant. Two of the amino acid residues Tyr-323(Y323) and Leu-325(L325) within the region corresponding to P4 in the wild-type RBD, were conserved across the species. The conserved amino acids tyrosine and leucine were mutated with alanine substitutions. Figure S5. Quantification of data of Fig. 1b Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data (represented in Fig. 1b). **P ≤ 0.01, ***P ≤ 0.001. Figure S6. Quantification of data of Fig. 2c, d, and e. (A) Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data (represented in Fig. 2c). (B) Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data (represented in Fig. 2d). (C) Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data (represented in Fig. 2e). Pyr (Pyrogallol) ns (not significant). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Figure S7. Quantification of data of Fig. 3a and f. (A) Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data represented in Fig. 3a. (B) Densitometric quantification of Western blot data represented in Fig. 3f. VC (Vehicle control), Pyr (Pyrogallol), and ns, not significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Figure S8. Quantification of data of Fig. 4b and c. Densitometric quantification of protein phosphorylation of mTORC2 specific markers by Western blot data (represented in Fig. 4b). ns, not significant. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

4-Hydroxy-2-methylproline diastereomers are successfully prepared without the use of an external chiral auxiliary. Dihydroxylation of the key intermediate 2 resulted in lactone 4 as a mixture of diastereomers in good yield. Mesylation, hydrogenation and concomitant intramolecular cyclization of 4 led to the formation of both (2 R ,4 R )- and (2 R ,4 S )-4-hydroxy-2-methylprolines as a mixture of diastereomers. Appropriate protection followed by chromatographic separation resulted in isolation of both cis - and trans -diastereomers in enantiomerically pure form and in equal quantity. In subsequent experiments, the synthesis of the more challenging diastereomers (2 R ,4 R )- and (2 S ,4 S )-4-hydroxy-2-methylproline was achieved by diastereoselective iodolactonization of ( R )- or ( S )-allylalanine obtained after hydrolysis of intermediate 2 , followed by pyrrolidine ring closer under mild alkaline conditions. After selective protection and deprotection, Fmoc-(2 R ,4 R )-α-Me-Hyp( t Bu)-OH 14 , a building block suitable for solid phase peptide synthesis was obtained.

Antimicrobial peptides (AMPs) are the promising future therapeutic candidates because of their multifunctional roles and unique mode of action against microbes. Despite several advantages, developing AMPs into therapeutic antibiotics is often associated with limitations, such as thermal and enzymatic stability, moderate antimicrobial activity and higher toxicity. We here report the synthesis of 2-fluoro- and 2,6-difluorophenyalanine, their introduction into naturally occurring antimicrobial peptide Temporin L (TL). We also report the antimicrobial and hemolytic activity of parent TL as well as the fluorinated variant in plasma and buffer conditions. Circular dichroism studies clearly show that fluorination reduces the helical propensity, thus accounting for lower activity. We further demonstrated that fluorinated TL can act as antimicrobial agents in creams and gels used for treating skin infections.

A new series of side chain-modified 4-aminoquinolines were synthesized and screened for in vitro antiplasmodial activity against both chloroquine-sensitive (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum. Among the series, compounds 30 and 31 showed significant inhibition of parasite growth against K1 strain of P. falciparum with IC50 values 0.28 and 0.31 µM, respectively, whereas compounds 34, 35, and 38 exhibited superior activity against K1 strain with IC50 values 0.18, 0.22, and 0.17 µM, respectively, as compared to 0.255 µM for chloroquine (CQ). All the compounds displayed good resistance factor between 1.54 and >34.48 as against 51.0 for CQ. All these analogues were found to form strong complex with hematin and inhibited the β-hematin formation in vitro, suggesting that this class of compounds act on a heme polymerization target. Overall results suggest that present series of compounds appear to be promising for further lead optimization to obtain compounds active against drug-resistant parasites.

A simple and stereoselective synthesis of 3-methylthalidomide, a configurationally stable thalidomide analog, is presented. Herein we describe the synthesis of (R)-3-methylthalidomide starting from (S)-alanine by piperidin-2-one ring assembly approach in high yield and enantiomeric purity without using a chiral auxiliary or reagent. Starting from (R)-alanine, the corresponding (S)-3-methylthalidomide can be prepared using the same methodology. Chirality 27:619–624, 2015. © 2015 Wiley Periodicals, Inc.

The synthesis of sitagliptin phosphate 1 , a novel DPP-IV inhibitor for the treatment of type 2 diabetes mellitus has been accomplished starting from the chiral synthon (1,4-bis[( R )-1-phenylethyl]piperazine-2,5-dione) 2 , involving highly stereocontrolled (>98%) alkylation as a key step, in a good overall yield of 50% over six steps.

A number of thiazolidin-4-one and thiazinan-4-one derivatives were prepared by three component condensation in one pot reaction method. These compounds were evaluated for anti-hyperglycemic activity by in vitro and in vivo assay systems. The compounds with thiazolidin-4-one and thiazinan-4-one moieties exhibited significant anti-hyperglycemic activity. A few compounds (3a, 3b, 4a and 4b) have exhibited both anti-hyperglycemic and anti-dyslipidemic activities. Among them the thiazinan-4-one derivative 4a showed maximal (45%) improvement in oral glucose tolerance test in db/db mice at 30 mg/kg oral dose.

A versatile diastereoselective Friedel-Crafts alkylation reaction of heteroaryl systems with a cyclic enecarbamate for the preparation of 5-heteroaryl-substituted proline analogues in good yields has been developed. These heterocyclic tethered cyclic amino acid building blocks constitute important structural motifs in many biologically active molecules. The impact of the substitution on proline cis/trans isomerization was explored by carrying out solution conformational studies by NMR on 5-furanyl-substituted proline-containing peptides. Conformational analysis revealed that the peptide bond is constrained in an exclusively trans conformation.

A novel 4-aminoquinoline derivative [( S )-7-chloro- N -(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-quinolin-4-amine triphosphate] exhibiting curative activity against chloroquine-resistant malaria parasites has been identified for preclinical development as a blood schizonticidal agent. The lead molecule selected after detailed structure-activity relationship (SAR) studies has good solid-state properties and promising activity against in vitro and in vivo experimental malaria models. The in vitro absorption, distribution, metabolism, and excretion (ADME) parameters indicate a favorable drug-like profile.

A versatile diastereoselective Friedel-Crafts alkylation reaction of heteroaryl systems with a cyclic enecarbamate for the preparation of 5-heteroaryl-substituted proline analogues in good yields has been developed. These heterocyclic tethered cyclic amino acid building blocks constitute important structural motifs in many biologically active molecules. The impact of the substitution on proline cis/trans isomerization was explored by carrying out solution conformational studies by NMR on 5-furanyl-substituted proline-containing peptides. Conformational analysis revealed that the peptide bond is constrained in an exclusively trans conformation.

Development of efficient and safe nucleic acid carriers (vectors) is one of the essential requirements for the success of gene therapy. Here, we have evaluated the gene transfer capability of chitosan-PEI (CP) conjugates prepared by conjugating low molecular weight branched polyethylenimine (LMWP) with depolymerized chitosans (7 and 10 kDa) via their terminal aldehyde/keto groups. The CP conjugates interacted efficiently with nucleic acids and also showed higher cellular uptake. These conjugates on complexation with DNA yielded nanoparticles in the size range of 100–130 nm (in case of C7P) and 115–160 nm (in case of C10P), which exhibited significantly higher transfection efficiency (∼2–42 folds) in vitro compared to chitosans (high and low mol. wt.) and the commercially available transfection reagents retaining cell viability almost comparable to the native chitosan. Of the two CP conjugates, chitosan 7 kDa-LMWP (C7P) displayed higher gene transfer ability in the presence and absence of serum. Luciferase reporter gene analysis in male Balb/c mice receiving intravenous administration of C7P3/DNA polyplex showed the maximum expression in their spleen. Further, tuftsin, a known macrophage targeting molecule, was tethered to C7P3 and the resulting complex, i.e., C7P3-T/DNA, exhibited significantly higher gene expression in cultured mouse peritoneal macrophages as compared to unmodified C7P3/DNA complex without any cytotoxicity demonstrating the suitability of the conjugate for targeted applications. Conclusively, the study demonstrates the potential of the projected conjugates for gene delivery for wider biomedical applications.

The present study describes the synthesis of a series of new 4-aminoquinoline-derived thiazolidines and evaluation of their antimalarial activity against a NF-54 strain of Plasmodium falciparum in vitro and N-67 strain of Plasmodium yoelii in vivo. Among the series, two compounds, 2-(4-chloro-phenyl)-thiazolidine-4-carboxylic acid [2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide hydrochloride (14) and 2-(2,6-dichloro-phenyl)-thiazolidine-4-carboxylic acid [2-(7-chloro-quinolin-4-ylamino)-ethyl]-amide hydrochloride (22) exhibited significant suppression of parasitaemia in the in vivo assay. All the analogues were found to form strong complex with haematin and inhibited the β-haematin formation in vitro. These results suggest that these compounds act on heme polymerization target.

It is widely appreciated that the vascular endothelium is capable of modulating vascular smooth muscle tone suiting it well for its role as an important regulator of a number of diverse biological processes. Endothelial dysfunction is an early manifestation of atherothrombosis and a consequence of the established disease. Although several arginine derivatives alkylated at one of the guanidino nitrogen were found to inhibit vasorelaxation induced by acetylcholine, activity of the corresponding arginine esters is not reported. The present work was therefore designed to synthesize and evaluate series of novel arginine derivatives to obtain further insight into structure-activity relationship in this series of compounds. Present study involves assessment of activity of these novel compounds on the vascular tone of rat thoracic aorta in comparison with l-arginine analog, that is, l-nitro-arginine methyl ester (l-NAME). Results from the present study showed that full reversal of phenylephrine-mediated contraction was achieved by cumulative applications of acetylcholine (3nm-300μm), which were abolished when the aortic rings were pretreated with l-NAME (300μm). Results from the present study demonstrated that these novel arginine derivatives cause significant yet reversible reduction in acetylcholine-mediated relaxation, similar to that of L-NAME.

In view of the severe immunosuppression in visceral leishmaniasis (VL), a rational approach to effectively combat the parasitic scourge would be to enhance the immune status of the host. Use of CpG oligodeoxynucleotide (CpG-ODN) against leishmaniasis has previously been reported, especially as an immunomodulator and adjuvant with various immunogens. In the present study, experiments were carried out with BALB/c mice and hamsters infected with Leishmania donovani . Immunostimulating class B bacterial CpG-ODN namely, ODN-2006, was administered at various doses by the intraperitoneal (i.p.) route. The dose of CpG-ODN-2006 (1 nM/single dose) showing the most antileishmanial activity was given as free and liposomal forms with different doses of miltefosine, namely, 5 and 10 mg/kg of body weight, for 5 days in mice and hamsters, respectively. Among the various groups, mice coadministered liposomal CpG-ODN and miltefosine (5 mg/kg) showed the best inhibitory effect (97% parasite inhibition) compared with free CpG-ODN plus miltefosine and miltefosine, free CpG-ODN, and liposomal CpG-ODN given separately. Similar responses were observed in the case of hamsters, where the combination of liposomal CpG-ODN with miltefosine (10 mg/kg) gave 96% parasite inhibition. Promising antileishmanial efficacy was observed in animals treated with liposomal CpG-ODN and miltefosine.

Objectives To evaluate the combination of CpG oligodeoxynucleotide (CpG ODN) and miltefosine for the treatment of experimental visceral leishmaniasis (VL). Methods The experiments were carried out using BALB/c mice and hamsters, infected with Leishmania donovani. CpG ODN was administered at various doses by the intraperitoneal (ip) route. The dose of CpG ODN (1 nM/single dose) showing best antileishmanial activity was given as free and liposomal forms with a subcurative dose of miltefosine, namely 2.5 and 5 mg/kg x 5 days in mice and hamsters, respectively. Results Among the various groups of mice, co-administered liposomal CpG ODN and miltefosine showed the best inhibitory effect (85% inhibition) compared with free CpG ODN and miltefosine, and miltefosine, free CpG ODN and liposomal CpG ODN separately. Production of Th1 cytokines, nitric oxide (NO), reactive oxygen species (ROS) and H(2)O(2) was enhanced. A remarkable increase in the phagocytosis index was also observed, indicating overall immunological support to antileishmanial activity of miltefosine by CpG ODN. Similar responses were observed in hamsters. Conclusions Promising antileishmanial efficacy was observed in animals treated with liposomal CpG ODN and miltefosine, strongly supported by enhancement of Th1 cytokines as well as NO, ROS and H(2)O(2) levels. The correlation of experimental findings in both the models (mouse and hamster) strengthens the potential of CpG ODN as an immunomodulator in combination with miltefosine against VL.

Human β-casein fragment (54–59) having the amino acid sequence Val–Glu–Pro–Ile–Pro–Tyr, has shown potent immunostimulant activity. Several analogs of this hexapeptide have been synthesized with modification at the N-terminal region and two analogs, viz. peptide I and peptide II have shown significant immunosuppressant activity in-vivo mouse model. Effect on cell mediated immunity (CMI) and humoral immunity was studied in mouse/SRBC model. Both the peptides failed to stimulate immune response in vivo and showed inhibition of CMI and humoral response to sheep red blood cells (SRBC). Peptides showed inhibition in alloantigen induced lymphocyte proliferation, i.e., mixed lymphocyte reaction (MLR) in vitro . Treatment with peptides inhibited the production of interferon-γ (IFN-γ), and increased the production of interleukin-4 (IL-4) as well as improved the skin graft survival. Cyclosporine a known immunosuppressant showed similar effect on mouse model. Present study thus provides a lead for the development of safe and effective immunosuppressant.

AKR1B10 is an aldose reductase (AR) homologue overexpressed in liver cancer and various forms of that enzyme in carcinomas catalyze the reduction of anticancer drugs, potential cytostatic drug, and dl-glyceraldehyde but do not catalyze the reduction of glucose. Kinetic parameters for wild-type and C299S mutant AKR1B10 indicate that substitution of serine for cysteine at position 299 reduces the affinity of this protein for dl-glyceraldehyde and enhances its catalytic activity. Fibrates suppress peroxisome proliferation and the development of liver cancer in human. Here we report the potency of fibrate-mediated inhibition of the carbonyl reduction catalyzed by wild-type and C299S mutant AKR1B10 and compare it with known AR inhibitors. Wild-type AKR1B10-catalyzed carbonyl reduction follows pure non-competitive inhibition kinetics using zopolrestat, EBPC or sorbinil, whereas fenofibrate, Wy 14,643, ciprofibrate and fenofibric acid follow mixed non-competitive inhibition kinetics. In contrast, catalysis of reaction by the C299S AKR1B10 mutant is not inhibited by sorbinil and EBPC. Despite these differences, the C299S AKR1B10 mutant still manifests kinetics similar to the wild-type protein with other fibrates including zopolrestat, fenofibrate, Wy 14,346, gemfibrozil and ciprofibrate that show mixed non-competitive inhibition kinetics. The reaction of the mutant AKR1B10 is inhibited by fenofibric acid, but manifests pure non-competitive inhibition kinetics that are different from those demonstrated for the wild-type enzyme.

The cellular regulatory protein p53 is overexpressed by almost 50% of all malignancies making it an attractive target for a vaccine approach to cancer. A number of immunotherapy approaches targeting p53 have been evaluated successfully in murine models, but translation of these preclinical findings to the clinic has been unsuccessful. Prior studies in our laboratory employing murine models demonstrated that a modified vaccinia virus Ankara (MVA) vaccine expressing murine p53 could stimulate p53 specific immunity. Systemic administration of the MVA vaccine was able to effect the rejection of established tumors. To better understand the immunologic mechanisms that underlie the vaccine function of human p53, we utilized a murine model in which the murine germ line copy of p53 was replaced with a modified human one. These mice, referred to as Hupki, were evaluated as a tolerant model to explore the capacity of MVA expressing human p53 to overcome tolerance and reject human p53-expressing tumors.MVAp53 immunization of Hupki mice resulted in the generation of p53-specific CD8(+) T cells and the rejection of a highly aggressive murine mammary carcinoma cell line 4T1(H-2d) transfected with human p53 (4T1p53). An immunologic correlate of tumor protection was evaluated utilizing an overlapping peptide library spanning the full length of human p53. This reagent was also used in combination with MVAp53 to stimulate p53-specific CD8(+) T cell responses in cancer patients.These studies demonstrate the potential of MVAp53 to overcome tolerance to p53 for cancer immunotherapy.

Two HLA-A*02-restricted epitopes have been identified within the VP1 polypeptide of a human polyomavirus, BK virus, which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Immunization of transgenic mice with recombinant modified vaccinia Ankara expressing BKV VP1 (rMVA-BKV VP1) elicited functional CTL populations recognizing the sequences LLMWEAVTV (amino acids residues 108–116, BKV VP1p108) and AITEVECFL (residues 44–52, BKV VP1p44) and cross-reactive to the previously described JC virus VP1 homologs. Flow-based analyses of PBMC from a panel of thirty healthy HLA-A*02 human volunteers indicated that the majority of these subjects harbored functional CTL populations recognizing the BKV epitopes and cross-reactive with the JCV homologs. CTL recognizing the JCV VP1p100 and JCV VP1p36 epitopes have previously been associated with prolonged survival in progressive multifocal leukoencephalopathy patients. These findings suggest that infection with BKV or JCV could potentially induce cross-protective T-cell immunity against diseases associated with these viruses.

A transgenic mouse model was used to identify an HLA-A*02-restricted epitope within the VP1 polypeptide of a human polyomavirus, BK virus (BKV), which is associated with polyomavirus-associated nephropathy in kidney transplant patients. Peptide stimulation of splenocytes from mice immunized with recombinant modified vaccinia virus Ankara expressing BKV VP1 resulted in expansion of cytotoxic T lymphocytes (CTLs) recognizing the sequence LLMWEAVTV corresponding to amino acid residues 108 to 116 (BKV VP1p108). These effector T-cell populations represented functional CTLs as assessed by cytotoxicity and cytokine production and were cross-reactive against antigen-presenting cells pulsed with a peptide corresponding to the previously described JC virus (JCV) VP1 homolog sequence ILMWEAVTL (JCV VP1p100) (I. J. Koralnik et al., J. Immunol. 168: 499-504, 2002). A panel of 10 healthy HLA-A*02 human volunteers and two kidney transplant recipients were screened for T-cell immunity to this BK virus VP1 epitope by in vitro stimulation of their peripheral blood mononuclear cells (PBMC) with the BKV VP1p108 peptide, followed by tetramer labeling combined with simultaneous assays to detect intracellular cytokine production and degranulation. PBMC from 4/10 subjects harbored CTL populations that recognized both the BKV VP1p108 and the JCV VP1p100 peptides with comparable efficiencies as measured by tetramer binding, gamma interferon production, and degranulation. CTL responses to the JCV VP1p100 epitope have been associated with prolonged survival in progressive multifocal leukoencephalopathy patients (R. A. Du Pasquier et al., Brain 127: 1970-1978, 2004; I. J. Koralnik et al., J. Immunol. 168: 499-504, 2002). Given that both human polyomaviruses are resident in a high proportion of healthy individuals and that coinfection occurs (W. A. Knowles et al., J. Med. Virol. 71: 115-123, 2003), our findings suggest a reinterpretation of this protective T-cell immunity, suggesting that the same VP1 epitope is recognized in HLA-A*02 persons in response to either BK or JC virus infection.

In search of a potent immunomodulator to be used as an immunoprophylactic agent and as adjunct to chemotherapy against Leishmania infection, two analogs of muramyl dipeptide, viz. N.Ac-norMur-MeVal-D-isoGln (86/448) and N.AcMur-Acc-D-isoGln (89/729) were evaluated for desired activity. Effect of these peptides on cell mediated and humoral immunity was studied by immunizing the peptide treated mouse with sheep red blood cells (SRBC) and determining HA-titer, plaque forming cells assay and delayed type of hypersensitivity (DTH) response after 4-5 days. Both the peptides stimulated cell mediated immunity (CMI), humoral response as well as macrophage function in terms of super oxide anion (O2-) and nitric oxide (NO) generation. Mitogen induced lymphocyte proliferation and production of IL-2 and INF-gamma increased while that of IL-4 and IL-10 decreased by both the peptides showing a typical Th1 type response. After establishing the immunostimulatory activity, these peptides were evaluated for immunoprophylactic efficacy as well as for use as adjunct to chemotherapy with stibanate (SSG) against Leishmania donovani infection in golden hamster. These peptides were found quite effective in both the modes. In adjunct use the treatment may require lower dose of SSG and thereby reduce the chances of drug toxicity.

Vaccination strategies remain elusive that are effective against viral disease pathogens yet remain gentle enough for widespread human use. We developed a model system that relies on the recognition of specific T-cell epitopes from immunodominant antigens of HIV to explore single-stranded CpG-oligodeoxynucleotides (ODN) (CpG) as an adjuvant. We improved upon current strategies of utilizing CpG in combination with peptide vaccines by covalently modifying epitope fusion peptides with CpG motifs. Characterization of the immune recognition of DNA-peptide conjugates was carried out in a murine model of human HLA A2. Immunogenicity of DNA-peptide conjugates was superior in sensitivity to non-covalently linked mixtures of the same functional molecules as measured by peptide-mediated cytotoxicity and IFN-gamma release, as well as protection against viral infection. Enhancement of sensitivity of immune recognition by covalent attachment of DNA to epitope peptides should be further evaluated as a novel prophylactic vaccine strategy for HIV infection and other infectious diseases.

In a one pot procedure, amines 1a-c, cyclic ketones 3a-f and mercapto acids 2a-c were converted into 1-thia-4-azaspiro(4.n)alkan-3-ones (n = 4-7) 4a-e, 8a-e, 9a and 1-thia-4,8-diaza- spiro(4.5)decan-3-one 9b. The 4-thiazolidinone moiety of 4b and the piperidine ring of 9b were subsequently derivatized furnishing products 5-7 and 10, 12a-d, respectively. The products were evaluated as potential antimycobacterial agents, ten compounds were found active at 25 µg/mL concentration; with the most active compound 12a showing more than 90% inhibition.

A panel of 7 human cytomegalovirus (CMV) epitope peptides and corresponding major histocompatibility class 1 tetramers was used to evaluate cellular immunity in healthy seropositive donors and in hematopoietic stem-cell transplant recipients. Broad CMV-specific T cell responses to epitopes were found within several CMV polypeptides and were restricted by multiple human leukocyte antigen alleles. Their cytotoxic functionality was evaluated by use of an assay that measures transient surface levels of lysosomal membrane proteins LAMP-1 (CD107a) and LAMP-2 (CD107b) after peptide stimulation. This assay can be combined with tetramer staining of antigen-specific CD8(+) T lymphocytes and has potential as a surrogate marker for cytotoxic function. CD8(+) T lymphocytes specific for epitopes within the pp65 or pp50 gene products exhibited significantly higher functionality, compared with populations recognizing CMV major immediate early-1 epitopes. These functional differences between T lymphocyte populations within the same individual may have implications for protection against CMV.

In continuation to our earlier studies with peptidomimetic opioid compounds, we have further investigated immunosuppressive properties of one of our peptidomimetic compound (Tyr-NH-CH2-CH2-O-Phe-NH2) using peripheral blood mononuclear cells (PBMCs) of healthy volunteers. Peptidomimetic compound was evaluated for its effect on anti-CD3 and recombinant human interleukin-2 (rhIL-2) stimulated lymphocyte proliferation in vitro and lipopolysaccharide (LPS) induced activation of mitogen activated protein kinase (MAPK, pp42/44) in mouse macrophage cells (RAW 264.7). Our results show the immunosuppressive potential of synthetic peptidomimetic compound. This compound significantly inhibited anti-CD3 and rhIL-2 stimulated lymphocyte proliferation in vitro. However, this peptidomimetic compound did not show any effect on LPS induced MAPK activation. These observations suggest that above peptidomimetic compound has potential to inhibit immune responses mediated by lymphocytes.

In the present study, 4-thiazolidinones have been assembled by DCC mediated three-component reaction of amine, aldehyde and mercaptoacetic acid. The final compounds are obtained in quantitative yields within one hour.

A simple and stereoselective synthesis of 3-methylthalidomide, a configurationally stable thalidomide analog, is presented. Herein we describe the synthesis of (R)-3-methylthalidomide starting from (S)-alanine by piperidin-2-one ring assembly approach in high yield and enantiomeric purity without using a chiral auxiliary or reagent. Starting from (R)-alanine, the corresponding (S)-3-methylthalidomide can be prepared using the same methodology.