Identification and quantification of phytochemicals of Chamaecostus cuspidatus (Nees & Mart.) C.D.Specht & D.W.Stev and Cheilocostus speciosus (J. Koenig) C.D.Specht by LC-MS techniques and their in-vitro anti-adipogenic screening.

Chamaecostus cuspidatus (Nees & Mart.) C.D.Specht & D.W.Stev and Cheilocostus speciosus (J.Koenig) C.D.Specht contain bioactive compounds that possess many pharmacological activities including antidiabetic and hypolipidemic. These plants are used to treat diabetes by herbal healers. Considering the traditional use of C. cuspidatus and C. speciosus, the present study is designed to perform qualitative and quantitative analysis as well as in-vitro anti-adipogenesis against 3T3-L1 cells to ensure efficacy. A total of thirty-eight compounds were identified using HPLC-QTOF-MS/MS. Quantification of ten bioactive compounds among identified compounds was performed by UPLC-QqQLIT-MS/MS. The quantification method was validated according to ICH guidelines (International conference on harmonization guidelines). Quantification of bioactive compounds of different organs of C. cuspidatus and C. speciosus showed remarkable differences in the content. Microscopic and ORO absorbance confirmed the antiadipogenic potential of leaves (L-02), roots (R-02) of C. cuspidatus and leaves of C. speciosus (L-01) in 3T3-L1 cells.

6,7-Dihydroxycoumarin ameliorates crystal-induced necroptosis during crystal nephropathies by inhibiting MLKL phosphorylation.

AIMS: Mineralization of crystalline particles and the formation of renal calculi contribute to the pathogenesis of crystal nephropathies. Several recent studies on the biology of crystal handling implicated intrarenal crystal deposition-induced necroinflammation in their pathogenesis. We hypothesized that 6,7-dihydroxycoumarin (DHC) inhibit intrarenal crystal cytotoxicity and necroinflammation, and ameliorate crystal-induced chronic kidney disease (CKD). MAIN METHODS: An unbiased high content screening coupled with fluorescence microscopy was used to identify compounds that inhibit CaOx crystal cytotoxicity. The ligand-protein interactions were identified using computational models e.g. molecular docking and molecular dynamics simulations. Furthermore, mice and rat models of oxalate-induced CKD were used for in-vivo studies. Renal injury, crystal deposition, and fibrosis were assessed by histology analysis. Western blots were used to quantify the protein expression. Data were expressed as boxplots and analyzed using one way ANOVA. KEY FINDINGS: An unbiased high-content screening in-vitro identified 6,7-DHC as a promising candidate. Further, 6,7-DHC protected human and mouse cells from calcium oxalate (CaOx) crystal-induced necroptosis in-vitro as well as mice and rats from oxalate-induced CKD in either preventive or therapeutic manner. Computational modeling demonstrated that 6,7-DHC interact with MLKL, the key protein in the necroptosis machinery, and inhibit its phosphorylation by ATP, which was evident in both in-vitro and in-vivo analyses. SIGNIFICANCE: Together, our results indicate that 6,7-DHC possesses a novel pharmacological property as a MLKL inhibitor and could serve as a lead molecule for further development of coumarin-based novel MLKL inhibitors. Furthermore, our data identify 6,7-DHC as a novel therapeutic strategy to combat crystal nephropathies.

Determination of bioactive compounds of Artemisia Spp. plant extracts by LC-MS/MS technique and their in-vitro anti-adipogenic activity screening.

Ultra Performance Liquid Chromatography coupled with hybrid triple quadrupole linear ion trap tandem mass spectrometry (UPLC-ESI-QqQLIT-MS/MS) method in multiple reaction monitoring (MRM) acquisition mode was developed and validated for identification and simultaneous determination of potential anti-diabetic and anti-malarial compounds in ethanolic extracts of different Artemisia species. The chromatographic separation was carried out on an Acquity BEH™ C18 column (1.7 µm, 2.1 × 50 mm) with 0.1 % (v/v) formic acid in water and acetonitrile as mobile phase under gradient condition in 6 min. The developed method was validated in terms of linearity, LOD, LOQ, precision, stability and recovery according to international conference on harmonization guidelines. The correlation coefficients of all the calibration curves were ≥0.9902 and recoveries ranged from 98.22 to 104.49% (RSD ≤2.18 %). Relative standard deviations of intra-day, inter-day precisions and stability were ≤ 1.04, 1.09 and 2.80 %, respectively. The quantitative results showed remarkable differences in the content of all the compounds in different Artemisia species. The quantitative values of each peak were summarized as mean ± SD. The statistical analysis for comparison of observed quantitative differences of each compound was done to show that they are statistically significant. In-vitro assessment of extracts of selected Artemisia species inhibited adipocyte differentiation in 3T3-L1 cells, hence it may have certain phytochemicals which are responsible for reducing obesity and related metabolic disorders.

Temporal immmunometabolic profiling of adipose tissue in HFD-induced obesity: manifestations of mast cells in fibrosis and senescence.

BACKGROUND/OBJECTIVES: Chronic low-grade inflammation/meta-inflammation in adipose tissue leads to obesity-associated metabolic complications. Despite growing understanding, the roles of immune cell subsets, their interrelationship, and chronological events leading to progression of obesity-associated insulin resistance (IR) remains unclear. METHODS: We carried out temporal immunometabolic profiling of adipose tissue from C57BL/6 mice fed a high-fat diet (HFD) for 4, 8, 12, 16, and 20 weeks. We used clodronate sodium liposomes (CLODs) to deplete macrophages and disodium cromoglycate sodium liposomes (DSCGs) to stabilize mast cells. RESULTS: In the temporal HFD settings, mice showed progressive glucose intolerance, insulin resistance, and adipose tissue senescence. Histochemistry analysis of epididymal white adipose tissue (eWAT) using picro-sirius red and Masson's trichrome staining showed extensive collagen deposition in the 16th and 20th weeks. Flow cytometry analysis of the stromal vascular fraction (SVF) from eWAT revealed T-cell subsets as early-phase components and pro-inflammatory macrophages, as well as mast cells as the later phase components during obesity progression. In our therapeutic strategies, macrophage depletion by CLOD and mast stabilization by DSCG attenuated obesity, adipose tissue fibrosis, and improved whole-body glucose homeostasis. In addition, mast cell stabilization also attenuated senescence (p53 and X-gal staining) in eWAT, signifying the role of mast cells over macrophages during obesity. CONCLUSION: New-generation mast cell stabilizers can be exploited for the treatment of obesity-associated metabolic complications.

Chronic hyperinsulinemia promotes meta-inflammation and extracellular matrix deposition in adipose tissue: Implications of nitric oxide.

Various imperative studies support the notion that hyperinsulinemia (HI) itself serves as the common link between adipose tissue inflammation (ATI) and metabolic syndrome. However, the contribution of HI mediated ATI and its metabolic consequences are yet to be explored. We induced chronic HI per se in mice by administration of exogenous insulin for 8 weeks through mini-osmotic pumps. For the reduction of circulating insulin in response to excess calorie intake, we have partially ablated ß-cells by using streptozotocin (STZ) in the diet-induced obesity (DIO) and genetic mice models (db/db). Flow cytometry analysis was performed for the quantification of immune cells in stromal vascular fraction (SVF) isolated from epididymal white adipose tissue (eWAT). Our studies demonstrated that chronic HI augmented ATI in terms of elevated pro-inflammatory cells (M1 macrophages and NK-cells) and suppressed anti-inflammatory cells (M2 macrophages, eosinophils and regulatory T-cells). These results were correlated with altered obesity-associated metabolic phenotype. Partial reduction of circulating insulin level attenuated excess calorie-induced ATI and improved insulin sensitivity. Mechanistically, an imbalance in M1 and M2 macrophage proportions in eWAT promoted iNOS (inducible nitric oxide synthase): arginase-1 imbalance that resulted into extracellular matrix (ECM) deposition and insulin resistance (IR) development. However, iNOS-/- mice were protected from HI-induced M1:M2 macrophage imbalance, ECM deposition and IR in adipose tissue. Overall, we conclude that chronic HI per se contributed in ATI and iNOS corroborated ECM deposition.

Aegeline inspired synthesis of novel ß3-AR agonist improves insulin sensitivity in vitro and in vivo models of insulin resistance.

BACKGROUND AND PURPOSE: In our drug discovery program of natural product, earlier we have reported Aegeline that is N-acylated-1-amino-2- alcohol, which was isolated from the leaves of Aeglemarmelos showed anti-hyperlipidemic activity for which the QSAR studies predicted the compound to be the ß3-AR agonist, but the mechanism of its action was not elucidated. In our present study, we have evaluated the ß3-AR activity of novel N-acyl-1-amino-3-arylopropanol synthetic mimics of aegeline and its beneficial effect in insulin resistance. In this study, we have proposed the novel pharmacophore model using reported molecules for antihyperlipidemic activity. The reported pharmacophore features were also compared with the newly developed pharmacophore model for the observed biological activity. EXPERIMENTAL APPROACH: Based on 3D pharmacophore modeling of known ß3AR agonist, we screened 20 synthetic derivatives of Aegeline from the literature. From these, the top scoring compound 10C was used for further studies. The in-slico result was further validated in HEK293T cells co-trransfected with human ß3-AR and CRE-Luciferase reporter plasmid for ß3-AR activity.The most active compound was selected and ß3-AR activity was further validated in white and brown adipocytes differentiated from human mesenchymal stem cells (hMSCs). Insulin resistance model developed in hMSC derived adipocytes was used to study the insulin sensitizing property. 8 week HFD fed C57BL6 mice was given 50 mg/Kg of the selected compound and metabolic phenotyping was done to evaluate its anti-diabetic effect. RESULTS: As predicted by in-silico 3D pharmacophore modeling, the compound 10C was found to be the most active and specific ß3-AR agonist with EC50 value of 447 nM. The compound 10C activated ß3AR pathway, induced lipolysis, fatty acid oxidation and increased oxygen consumption rate (OCR) in human adipocytes. Compound 10C induced expression of brown adipocytes specific markers and reverted chronic insulin induced insulin resistance in white adipocytes. The compound 10C also improved insulin sensitivity and glucose tolerance in 8 week HFD fed C57BL6 mice. CONCLUSION: This study enlightens the use of in vitro insulin resistance model close to human physiology to elucidates the insulin sensitizing activity of the compound 10C and edifies the use of ß3AR agonist as therapeutic interventions for insulin resistance and type 2 diabetes.

Saroglitazar reduces obesity and associated inflammatory consequences in murine adipose tissue.

Prevailing knowledge links chronic low-grade inflammation in the adipose tissue to obesity and its associated metabolic complications. In this study, we evaluated immunometabolic effects of a recently launched dual peroxisome proliferator-activated receptor (PPAR) α & γ agonist 'Saroglitazar' in a mouse model of diet-induced obesity (DIO). Body composition analysis revealed that saroglitazar treatment promoted hepatic weight gain, while attenuated epididymal white adipose tissue (eWAT) mass in DIO. In the eWAT of saroglitazar treated mice, histological analysis showed reduced adipocyte hypertrophy and matrix deposition (picrosirius red staining). Immunological profiling of stromal vascular fraction isolated from eWAT showed decreased pro-inflammatory cells (M1 macrophages, CD4 and CD8 T-cells) and increased anti-inflammatory M2 macrophages. Gene expression and western blot analysis suggested that saroglitazar promoted energy expenditure machinery and attenuated inflammatory as well as fibrotic markers in eWAT during DIO. In conclusion, for the first time we are reporting immunometabolic effects of dual PPARα & γ agonist saroglitazar in DIO and insulin resistance (IR). Saroglitazar exerted its beneficial effects on adipose tissue by limiting, diet-induced adipose tissue dysfunction, adipocyte hypertrophy, adipocyte cell damage and extracellular matrix deposition in obesity.

Novel indole and triazole based hybrid molecules exhibit potent anti-adipogenic and antidyslipidemic activity by activating Wnt3a/ß-catenin pathway.

Obesity and dyslipidemia is the two facet of metabolic syndrome, which needs further attention. Recent studies indicate triazole and indole derivatives have remarkable anti-obesity/antidyslipidemic activity. To harness the above-mentioned potential, a series of novel triazole clubbed indole derivatives were prepared using click chemistry and evaluated for anti-adipogenic activity. Based on the structure-activity relationship, essential functional groups which potentiate anti-adipogenic activity were identified. The lead compound 13m exhibited potent anti-adipogenic activity compared to its parent compounds with the IC-50 value of 1.67 µM. Further evaluation of anti-adipogenic activity was conducted in different cell lines such as C3H10T1/2 and hMSC with positive result. The anti-adipogenic effect of compound 13m was most prominent in the early phase of adipogenesis, which is driven by the G1 to S phase cell cycle arrest during mitotic clonal expansion. The mechanistic study suggests that compound 13m exhibit anti-adipogenic property by activating Wnt3a/ß-catenin pathway, a known suppressor of key adipogenic genes PPARγ and C/EBPα. It is noteworthy that the compound 13m also reduced serum triglyceride, LDL and total cholesterol in Syrian Golden hamster model of dyslipidemia. The anti-adipogenic activity of compound 13m can also be correlated with decreased expression of PPARγ and increased expression of ß-catenin in epididymal white adipose tissue (eWAT) in vivo. The compound 13m also increased the expression of genes involved in reverse cholesterol transport (RCT) such as PPARα and LXR1α indicating another mechanism by which compound 13m ameliorates dyslipidemia in Syrian Golden hamster model. Overall this study provides a unique perspective into the anti-adipogenic/antidyslipidemic property of triazole and indole hybrids molecules with further scope to increase the anti-adipogenic potency for therapeutic intervention of obesity and metabolic syndrome.

PPP2R5B, a regulatory subunit of PP2A, contributes to adipocyte insulin resistance.

Insulin resistance is associated with deregulation of insulin signaling owing to the chronic exposure of insulin (hyperinsulinemia) to the tissues. Phosphorylation and dephosphorylation events in insulin signaling pathway play an essential role in signal transduction and glucose uptake. Amongst all, Akt protein is considered to be central to the overall insulin signaling proteins. In glucose responsive tissues like adipose and muscles, activation of Akt is responsible for triggering GLUT4 translocation and glucose transport. Several phosphatases such as PTEN, PP2A have been reported to be involved in dephosphorylation and inactivation of Akt protein. We have identified increased PP2A activity during state of chronic hyperinsulinemia exposure along-with development of adipocyte insulin resistance. This increased phosphatase activity leads activation of cAMP/PKA axis, which in turn increased cAMP levels in insulin resistant (IR) adipocytes. Okadaic acid, an inhibitor of PP2A restored and increased insulin stimulated glucose uptake in insulin resistant (IR) and insulin sensitive (IS) adipocytes respectively. In IS adipocyte, chemical activation of PP2A through MG132 and FTY720 showed decreased insulin sensitivity corroborated with decreased Akt phosphorylation and glucose uptake. We also observed an increased expression of PP2A-B (regulatory) subunit in IR adipocytes. We found PPP2R5B, a regulatory subunit of PP2A is responsible for the dephosphorylation and inactivation of Akt protein. Increased expression of PPP2R5B was also confirmed in white adipose tissue of high fat diet induced IR mice model. Overexpression and suppression strategies confirmed the role of PPP2R5B in regulating insulin signaling. Thus, we conclude that PPP2R5B, a B subunit of PP2A is a negative regulator of Akt phosphorylation contributing partly to the chronic hyperinsulinemia induced insulin resistance in adipocytes.

Chronic hyperinsulinemia reduces insulin sensitivity and metabolic functions of brown adipocyte.

The growing pandemics of diabetes have become a real threat to world economy. Hyperinsulinemia and insulin resistance are closely associated with the pathophysiology of type 2 diabetes. In pretext of brown adipocytes being considered as the therapeutic strategy for the treatment of obesity and insulin resistance, we have tried to understand the effect of hyperinsulinemia on brown adipocyte function. We here with for the first time report that hyperinsulinemia-induced insulin resistance in brown adipocyte is also accompanied with reduced insulin sensitivity and brown adipocyte characteristics. CI treatment decreased expression of brown adipocyte-specific markers (such as PRDM16, PGC1α, and UCP1) and mitochondrial content as well as activity. CI-treated brown adipocytes showed drastic decrease in oxygen consumption rate (OCR) and spare respiratory capacity. Morphological study indicates increased accumulation of lipid droplets in CI-treated brown adipocytes. We have further validated these findings in vivo in C57BL/6 mice implanted with mini-osmotic insulin pump for 8weeks. CI treatment in mice leads to increased body weight gain, fat mass and impaired glucose intolerance with reduced energy expenditure and insulin sensitivity. CI-treated mice showed decreased BAT characteristics and function. We also observed increased inflammation and ER stress markers in BAT of CI-treated animals. The above results conclude that hyperinsulinemia has deleterious effect on brown adipocyte function, making it susceptible to insulin resistance. Thus, the above findings have greater implication in designing approaches for the treatment of insulin resistance and diabetes via recruitment of brown adipocytes.

Stable Tricyclic Antitubercular Ozonides Derived from Artemisinin.

New, highly stable tricyclic antitubercular ozonides 9 and 10 derived from artemisinin are reported in 39 and 9% yields, respectively. The ozonide groups of 9 and 10 were found to be stable under strong basic and acidic conditions. The absolute configuration of ozonides 9 was confirmed by X-ray crystallography. Ozonide 10 shows promising antitubercular activity against M. tuberculosis H37Ra and M. tuberculosis H37Rv with MIC values of 0.39 and 3.12 µg/mL, respectively.

Cucumis melo ssp. Agrestis var. Agrestis Ameliorates High Fat Diet Induced Dyslipidemia in Syrian Golden Hamsters and Inhibits Adipogenesis in 3T3-L1 Adipocytes.

BACKGROUND: Cucumis melo ssp. agrestis var. agrestis (CMA) is a wild variety of C. melo. This study aimed to explore anti-dyslipidemic and anti-adipogenic potential of CMA. MATERIALS AND METHODS: For initial anti-dyslipidemic and antihyperglycemic potential of CMA fruit extract (CMFE), male Syrian golden hamsters were fed a chow or high-fat diet with or without CMFE (100 mg/kg). Further, we did fractionation of this CMFE into two fractions namely; CMA water fraction (CMWF) and CMA hexane fraction (CMHF). Phytochemical screening was done with liquid chromatography-mass spectrometry LC- (MS)/MS and direct analysis in real time-MS to detect active compounds in the fractions. Further, high-fat diet fed dyslipidemic hamsters were treated with CMWF and CMHF at 50 mg/kg for 7 days. RESULTS: Oral administration of CMFE and both fractions (CMWF and CMHF) reduced the total cholesterol, triglycerides, low-density lipoprotein cholesterol, and very low-density lipoprotein-cholesterol levels in high fat diet-fed dyslipidemic hamsters. CMHF also modulated expression of genes involved in lipogenesis, lipid metabolism, and reverse cholesterol transport. Standard biochemical diagnostic tests suggested that neither of fractions causes any toxicity to hamster liver or kidneys. CMFE and CMHF also decreased oil-red-O accumulation in 3T3-L1 adipocytes. CONCLUSION: Based on these results, it is concluded that CMA possesses anti-dyslipidemic and anti-hyperglycemic activity along with the anti-adipogenic activity. SUMMARY: The oral administration of Cucumis melo agrestis fruit extract (CMFE) and its fractions (CMWF and CMHF) improved serum lipid profile in HFD fed dyslipidemic hamsters.CMFE, CMWF and CMHF significantly attenuated body weight gain and eWAT hypertrophy.The CMHF decreased lipogenesis in both liver and adipose tissue.CMFE and CMHF also inhibited adipogenesis in 3T3-L1 adipocytes. Abbreviation used: CMA: Cucumis melo ssp. agrestis var. agrestis, CMFE: CMA fruit extract, CMWF: CMA water fraction, CMHF: CMA hexane fraction, FAS: Fatty acid synthase, SREBP1c: Sterol regulatory element binding protein 1c, ACC: Acetyl CoA carboxylase, LXR α: Liver X receptor α.

Poliothrysoside and its derivatives as novel insulin sensitizers potentially driving AMPK activation and inhibiting adipogenesis.

In our efforts to develop safe and active chemical entities from nature, we first identified poliothrysoside (1), a phytoconstituent isolated from Flacourtia indica, possessing antidiabetic potential. Subsequently, fifteen derivatives (2-16) were synthesized to assess the activity profile of this class. All the compounds were analyzed for their glucose uptake potency in chronic insulin-induced insulin resistant 3T3-L1 adipocytes. Interestingly, compound 2 exhibited strong ability to increase the insulin sensitivity, primarily activating the AMPK signaling pathway and also inhibited the adipogenesis in 3T3-L1 adipocytes, in concentration dependent manner. Overall, these studies suggest the potential of poliothrysoside and its derivatives as promising leads for non-insulin dependent type 2 diabetes (T2D).

Mycobacterium tuberculosis persistence in various adipose depots of infected mice and the effect of anti-tubercular therapy.

The adipocytes are one of the non-professional phagocytes postulated to be a haven for Mycobacterium tuberculosis during persistence in the human host. The adipocyte - M. tuberculosis interaction data available to date are ex vivo. The present study was primarily aimed to investigate M. tuberculosis infection of adipocytes in course of infection of mouse model. Using primary murine adipocytes, the study first confirmed the infection and immunomodulation of natural adipocytes by M. tuberculosis. The bacilli could be isolated form visceral, subcutaneous, peri renal and mesenteric adipose depots of immunocompetent mice infected with M. tuberculosis intravenously. The bacilli could be isolated from adipocytes and the stromal vascular fraction, even though the numbers were significantly higher in the latter. The bacterial burden in the adipose depots was comparable to those in lungs in the early phase of infection. But with time, the burden in the adipose depots was either decreased or kept under control, despite the increasing burden in the lungs. Infected mice treated with standard anti tubercular drugs, despite effective elimination of bacterial loads in the lungs, continued to harbour M. tuberculosis in adipose depots at loads similar to untreated mice in the late infection phase.

High-throughput screening assays for cyclooxygenase-2 and 5-lipoxygenase, the targets for inflammatory disorders.

High-throughput screening (HTS) involves testing of compound libraries against validated drug targets using quantitative bioassays to identify 'hit' molecules that modulate the activity of target, which forms the starting point of a drug discovery effort. Eicosanoids formed via cyclooxygenase (COX) and lipoxygenase (LOX) pathways are major players in various inflammatory disorders. As the conventional non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit both the constitutive (COX-1) and the inducible (COX-2) isoforms have gastric and renal side effects and the recently developed COX-2 selective anti-inflammatory drugs (COXIBs) have cardiac side effects, efforts are being made to develop more potent and safer antiinflammatory drugs. Current assay methods for these enzymes, such as oxygraphic, radioisotopic, spectrophotometric etc. are not compatible for screening of large number of compounds as in drug discovery programs. In the present study, HTS-compatible assays for COX-1, COX-2 and 5-LOX were developed for screening of compound libraries with the view to identify potential anti-inflammatory drug candidates. A spectrophotometric assay involving co-oxidation of tetramethyl-p-phenylene diamine (TMPD) during the reduction of prostaglandin G2 (PGG2) to PGH2 was adopted and standardized for screening of compounds against COX-1 and COX-2. Similarly, the HTS-compatible FOX (ferrous oxidation-xylenol orange) based spectrophotometric assay involving the formation of Fe3+/xylenol orange complex showing absorption in the visible range was developed for screening of compounds against 5-LOX.

A facile synthesis of alpha,alpha'-(EE)-bis(benzylidene)-cycloalkanones and their antitubercular evaluations.

An economical and facile synthesis of alpha,alpha'-(EE)-bis(benzylidene)-cycloalkanones was achieved by the reaction of cycloalkanones with different aromatic aldehydes using ethanolic KOH in good yields. Few of the selected compounds were reduced with NaBH(4) to the respective alpha,alpha'-(EE)-bis(benzylidene)-cycloalkanols. All these compounds and our earlier synthesized cyclohexyl phenyl methanols were evaluated for their antitubercular, antifungal and antibacterial activities. Several compounds displayed moderate antitubercular activity with MIC=12.5-1.56 microg/mL. However, none of the compounds displayed any significant antifungal activity.

C-3 alkyl/arylalkyl-2,3-dideoxy hex-2-enopyranosides as antitubercular agents: synthesis, biological evaluation, and QSAR study.

A series of C-3 alkyl and arylalkyl 2,3-dideoxy hex-2-enopyranoside derivatives were synthesized by Morita-Baylis-Hillman reaction using enulosides 4, 5, and 6 and various aliphatic and aromatic aldehydes. The compounds were evaluated in vitro for the complete inhibition of growth of Mycobacterium tuberculosis H37Rv. They exhibited moderate to good activity in the range of 25-1.56 mug/mL. Among these, 4d, 4h, 5c, and 4hr showed activity at minimum inhibitory concentrations, 3.12, 6.25, 1.56, and 1.56 mug/mL, respectively. These compounds were safe against cytotoxicity in VERO cell line and mouse macrophage cell line J 744A.1. A QSAR analysis by CP-MLR with alignment-free 3D-descriptors indicated the relevance of structure space comparable to the minimum energy conformation (from conformational analysis) of 5c to the activity. The study indicates that the compounds attaining the conformational space of 5c and reflecting some symmetry, minimum eccentricity, and closely placed geometric and electronegativity centers therein are favorable for activity.

Effect of substituents on diarylmethanes for antitubercular activity.

Aminoalkyl derivatives of diarylmethanes were prepared using Grignard, Friedel-Crafts arylation and aminohydrochloride chain formation reactions. These series of compounds were evaluated against Mycobacterium tuberculosis H(37)R(v) and showed the activity in the range of 6.25-25 microg/mL. Effect of heteroaryl, anthracenyl and phenanthrene groups on diarylmethane pharmacophores for antitubercular activity is described.

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses.

Baylis-Hillman reaction: convenient ascending syntheses and biological evaluation of acyclic deoxy monosaccharides as potential antimycobacterial agents.

A series of acyclic deoxy carbohydrate derivatives from easily available carbohydrate enals 1, 2, 3 or 5 were prepared involving the Baylis-Hillman reaction. These newly formed carbohydrate based Baylis-Hillman adducts and their amino derivatives were evaluated for their antimycobacterial activity against Mycobacterium tuberculosis H(37)R(v). Among the compounds evaluated for their antimycobacterial activity, compound (10) showed the desired activity in the range of 3.125 microg/mL.