Discovery of tetrahydrocarbazoles as dual pERK and pRb inhibitors.

The extracellular signal-regulated kinase (ERK) is one of the most important molecular targets for cancer that controls diverse cellular processes such as proliferation, survival, differentiation and motility. Similarly, the Rb (retinoblastoma protein) is a tumor suppressor protein and its function is to prevent excessive cell growth by inhibiting cell cycle progression. When the cell is ready to divide, pRb is phosphorylated, becomes inactive and allows cell cycle progression. Herein, we discovered a new series of tetrahydrocarbazoles as dual inhibitors of pERK and pRb phosphorylation. The in-house small molecule library was screened for inhibition of pERK and pRb phosphorylation, which led to the discovery of tetrahydrocarbazole series of compounds as potential leads. N-(3-methylcyclopentyl)-6-nitro-2,3,4,4a,9,9a-hexahydro-1H-carbazol-2-amine (1) is the dual inhibitor lead identified through screening, displaying inhibition of pERK and pRb phosphorylation with IC50 values of 5.5 and 4.8 µM, respectively. A short structure-activity relationship (SAR) study has been performed, which identified another dual inhibitor 9-methyl-N-(4-methylbenzyl)-2,3,4,4a,9,9a-hexahydro-1H-carbazol-2-amine (16) with IC50 values 4.4 and 3.5 µM for inhibition of pERK and pRb phosphorylation, respectively. This compound has a potential for further lead optimization to discover promising molecularly-targeted anticancer agents.

(E)-9-oxooctadec-10-en-12-ynoic acid from Ixora brachiata Roxb. increases glucose uptake in L6 myotubes by activating the PI3K pathway.

In an attempt to discover new scaffolds for anti-diabetic activity from plants, we screened extracts from Ixora brachiata Roxb. for their effect on glucose uptake in L6 myotubes. The petroleum (PE) extract of the plant showed a significant increase in insulin stimulated glucose uptake by L6 myotubes. The bioactivity guided fractionation of the crude extract yielded a compound (E)-9-oxooctadec-10-en-12-ynoic acid (OEA). The compound induced a dose dependent increase in insulin stimulated glucose uptake in L6 myotubes with an EC50 of 22.96µM. OEA also increased the phosphorylation of IRS-1, Akt and AS160 leading to increased GLUT4 translocation to the plasma membrane indicating that it promotes insulin stimulated glucose uptake in L6 myotubes by activating the PI3K pathway.

A semi-synthetic natural product blocks collagen induced arthritis by preferentially suppressing the production of IL-6.

Rheumatoid arthritis (RA), an autoimmune-inflammatory disease is characterized by dysregulation of signal transduction pathways, increased production of pro-inflammatory cytokines, enhanced leukocyte infiltration into synovial microvascular endothelium, extensive formation of hyper proliferative pannus, degradation of cartilage and bone erosion. Several compounds that abrogate cytokine production demonstrate a therapeutic effect in experimental models of arthritis. In this study, we report that a novel semi-synthetic natural product (Compound A) being a preferential IL-6 inhibitor, is efficacious in a murine model of arthritis. In vitro evaluations of pro-inflammatory cytokine production reveal that Compound A preferentially inhibits induced production of IL-6 and not TNF-α from THP-1 cells and isolated human monocytes. Furthermore, Compound A robustly inhibits the spontaneous production of IL-6 from pathologically relevant synovial tissue cells isolated from patients with active RA. In a physiologically relevant assay, Compound A selectively inhibits the activated T cell contact-mediated production of IL-6 from human monocytes. Compound A, at pharmacologically efficacious concentrations, does not significantly curtail the LPS-induced activation of p38 MAPKs. In the collagen-induced arthritis (CIA) mouse model (i) macroscopic observations demonstrate that Compound A, administered subcutaneously in a therapeutic regimen, significantly and dose-dependently inhibits disease associated increases in articular index and paw thickness; (ii) histological analyses of paw tissues reveal that Compound A prominently diminishes joint destruction, hyperproliferative pannus formation and infiltration of inflammatory cells. Collectively, these results provide direct evidence that Compound A, a novel preferential IL-6 inhibitor, suppresses collagen-induced arthritis, and may be a potential therapeutic for treating patients with active RA.

Anti-inflammatory properties of mutolide isolated from the fungus Lepidosphaeria species (PM0651419).

Mutolide an anti-inflammatory compound was isolated from the coprophilous fungus Lepidosphaeria sp. (PM0651419). The compound mitigated LPS-induced secretion of pro-inflammatory cytokines TNF-α and IL-6 from THP-1 cells as well as human peripheral blood mononuclear cells (hPBMCs). Mutolide also inhibited secretion of another pro-inflammatory cytokine IL-17 from anti-hCD3/anti-hCD28 stimulated hPBMCs. NF-κB is the major transcription factor involved in the secretion of pro-inflammatory cytokines including IL-17. Mechanistic evaluations revealed that mutolide inhibited induced NF-κB activation and translocation from cytoplasm into the nucleus. However, mutolide did not significantly affect activity of p38 MAPK enzyme, a serine/threonine kinase involved in cell cycle proliferation and cytokine secretion. These results indicate that mutolide may exert its anti-inflammatory effect via NF-κB inhibition. Oral administration of mutolide at 100 mg/kg showed significant inhibition of LPS-induced release of TNF-α from Balb/c mice in an acute model of inflammation. Our results highlight the anti-inflammatory properties of mutolide and suggest that further evaluation in a chronic model of inflammation is required to confirm the potential of mutolide as a druggable candidate for the treatment of inflammatory diseases.

NFAT-133 increases glucose uptake in L6 myotubes by activating AMPK pathway.

NFAT-133 is an aromatic compound with cinammyl alcohol moiety, isolated from streptomycetes strain PM0324667. We have earlier reported that NFAT-133 increases insulin stimulated glucose uptake in L6 myotubes using a PPARγ independent mechanism and reduces plasma or blood glucose levels in diabetic mice. Here we investigated the effects of NFAT-133 on cellular signaling pathways leading to glucose uptake in L6 myotubes. Our studies demonstrate that NFAT-133 increases glucose uptake in a dose- and time-dependent manner independent of the effects of insulin. Treatment with Akti-1/2, wortmannin and increasing concentrations of insulin had no effect on NFAT-133 mediated glucose uptake. NFAT-133 induced glucose uptake is completely mitigated by Compound C, an AMPK inhibitor. Further, the kinases upstream of AMPK activation namely; LKB-1 and CAMKKß are not involved in NFAT-133 mediated AMPK activation nor does the compound NFAT-133 have any effect on AMPK enzyme activity. Further analysis confirmed that NFAT-133 indirectly activates AMPK by reducing the mitochondrial membrane potential and increasing the ratio of AMP:ATP.

Screening of Microbial Extracts for Anticancer Compounds Using Streptomyces Kinase Inhibitor Assay.

Eukaryotic kinases are known to play an important role in signal transduction pathways by phosphorylating their respective substrates. Abnormal phosphorylations by these kinases have resulted in diseases. Hence inhibitors of kinases are of considerable pharmaceutical interest for a wide variety of disease targets, especially cancers. A number of reports have been published which indicate that eukaryotic-like kinases may complement two-component kinase systems in several bacteria. In Streptomyces sp. such kinases have been found to have a role in formation of aerial hyphae, spores, pigmentation & even in antibiotic production in some strains. Eukaryotic kinase inhibitors are seen to inhibit formation of aerial mycelia in Streptomyces without inhibiting vegetative mycelia. This property has been used to design an assay to screen for eukaryotic kinase inhibitors. The assay involves testing of compounds against Streptomyces 85E ATCC 55824 using agar well diffusion method. Inhibitors of kinases give rise to "bald" colonies where aerial mycelia and sporulation inhibition is seen. The assay has been standardized using known eukaryotic protein kinase inhibiting anticancer agents like AG-490, AG-1295, AG-1478, Flavopiridol and Imatinib as positive controls, at a concentration ranging from 10 µg/well to 100 µg/well. Anti-infective compounds which are not reported to inhibit eukaryotic protein kinases were used as negative controls. A number of microbial cultures have been screened for novel eukaryotic protein kinase inhibitors. Further these microbial extracts were tested in various cancer cell lines like Panel, HCT116, Calul, ACHN and H460 at a concentration of 10 µg/mL/ well. The anticancer data was seen correlating well with the Streptomyces kinase assay thus validating the assay.

Discovery of thiazolyl-phthalazinone acetamides as potent glucose uptake activators via high-throughput screening.

With the aim to discover orally active small molecules that stimulate glucose uptake, high throughput screening of a library of 5000 drug-like compounds was conducted in differentiated skeletal muscle cells in presence of insulin. N-Substituted phthalazinone acetamide was identified as a potential glucose uptake modulator. Several novel derivatives were synthesized to establish structure activity relationships. Identified lead thiazolyl-phthalazinone acetamide (7114863) increased glucose uptake (EC50 of 0.07±0.02 µM) in differentiated skeletal muscle cells in presence of insulin. Furthermore, 7114863 was superior to rosiglitazone under similar experimental conditions without inducing PPAR-γ agonist activity thus making it a very interesting scaffold.

Lead optimization of isocytosine-derived xanthine oxidase inhibitors.

We report our attempts at improving the oral efficacy of low-nanomolar inhibitors of xanthine oxidase from isocytosine series through chemical modifications. Our lead compound had earlier shown good in vivo efficacy when administered intraperitoneally but not orally. Several modifications are reported here which achieved more than twofold improvement in exposure. A compound with significant improvement in oral efficacy was also obtained.

Isocytosine-based inhibitors of xanthine oxidase: design, synthesis, SAR, PK and in vivo efficacy in rat model of hyperuricemia.

Structure-activity relationship studies were carried out for lead generation following structure-guided design approach from an isocytosine scaffold identified earlier for xanthine oxidase inhibition. A 470-fold improvement in in vitro IC(50) was obtained in the process. Five most potent compounds with nanomolar IC(50) values were selected for pharmacokinetics and in vivo experiments. The best compound showed good in vivo activity when administered intraperitoneally but was not active by oral route. The results suggest that improvement in oral exposure could improve the in vivo efficacy of this series.

NF-κB dependent anti-inflammatory activity of chlorojanerin isolated from Saussurea heteromalla.

Medicinal plants have shown great promise as a source of novel drug compounds for the treatment of inflammatory disorders. In our search for new entities with anti-inflammatory potential, the extracts of the whole plant of Saussurea heteromalla (family-Asteraceae), collected from Himalayas, were evaluated in the high throughput screen for TNF-α and IL-6 inhibitors. The extract blocked TNF-α and IL-6 production in LPS stimulated THP-1 cells (human acute monocyte leukemia cell line) completely at 10 and 30 µg/ml. The plant has been found as a new source of chlorojanerin, a guaianolide type of sesquiterpene lactone. Chlorojanerin was shown to be significantly effective in inhibiting TNF-α and IL-6 production in LPS-stimulated THP-1 cells (IC(50)=2.3±0.2 µM and 1.8±0.7 µM respectively). The compound also blocked TNF-α and IL-6 production from LPS-stimulated human monocytes (IC(50)=1.5±0.4 and 0.7±0.2 µM respectively) and synovial cells from a patient with rheumatoid arthritis (IC(50)<0.03 and 0.5 µM respectively). Transcriptional profiling of the LPS stimulated THP-1 cells revealed that chlorojanerin exerted its anti-inflammatory effect by inhibiting the expression of 8 genes involved in activating the transcription factor - NF-κB. Real time analysis of these genes validated the effect of chlorojanerin on the classical downstream targets of NF-κB. Thus, this study clearly delineated 8 genes which were specifically mitigated due to the effect of chlorojanerin on NF-κB induced signaling at the mRNA level. Further, chlorojanerin at 5 µM also inhibited the binding of NF-κB in a GFP reporter assay system by 55.5% thus validating the microarray gene expression data. This work is a step towards the isolation and characterization of lead anti-inflammatory agents from the extract of Saussurea heteromalla, which can be developed into better therapeutic molecules targeted towards some specific inflammatory diseases.

Identification of novel isocytosine derivatives as xanthine oxidase inhibitors from a set of virtual screening hits.

In recent years, xanthine oxidase has emerged as an important target not only for gout but also for cardiovascular and metabolic disorders involving hyperuricemia. Contrary to popular belief, recent clinical trials with uricosurics have demonstrated that enhanced excretion of uric acid is, by itself, not adequate to treat hyperuricemia; simultaneous inhibition of production of uric acid by inhibition of xanthine oxidase is also important. Virtual screening of in-house synthetic library followed by in vitro and in vivo testing led to the identification of a novel scaffold for xanthine oxidase inhibition. In vitro activity results corroborated the results from molecular docking studies of the virtual screening hits. The isocytosine scaffold maintains key hydrogen bonding and pi-stacking interactions in the deep end of the xanthine-binding pocket, which anchors it in an appropriate pose to inhibit binding of xanthine and shows promise for further lead optimization using structure-based drug design approach.

Vitamin E is a MIF Inhibitor.

Macrophage migration inhibitory factor (MIF) is known to contribute to the pathogenesis of inflammatory hyperalgesia and neuropathic pain. Prior studies have shown that Vitamin E treatment is associated with attenuated hyperalgesia and reduced neuropathic pain in rodents. Given these observations, we investigated the possibility that Vitamin E is a MIF inhibitor. Dopachrome tautomerase assays revealed that Vitamin E inhibits the enzymatic activity of purified human recombinant MIF (rhMIF) in a dose-dependent manner (45%, 74%, 92% and 100% inhibition at 3, 10, 30 and 100µM, respectively). Cell-free ELISA based assays showed that Vitamin E binds onto rhMIF thereby blocking its recognition (48% inhibition at 100µM). Circular dichroism studies indicated the Vitamin E has a strong affinity to bind to rhMIF (binding constant 19.52±1.4µM). In silico studies demonstrated that Vitamin E docks well in the active site of MIF with the long aliphatic chain of Vitamin E exhibiting strong van der Waals interactions with MIF. Most importantly, human cell-based assays revealed that Vitamin E significantly inhibits rhMIF-induced production of pro-inflammatory cytokines in a dose-dependent manner (77%, 80%, and 96% inhibition of IL-6 production, respectively, at 10, 30 and 100µM). Taken together, these results demonstrate that Vitamin E inhibits not only the enzymatic activity of MIF but more importantly the biological function of MIF. Our findings suggest that Vitamin E may be attenuating hyperalgesia and reducing neuropathic pain at least in part by inhibiting MIF activity.

Fermentation, Isolation, Structure, and antidiabetic activity of NFAT-133 produced by Streptomyces strain PM0324667.

Type-2 diabetes is mediated by defects in either insulin secretion or insulin action. In an effort to identify extracts that may stimulate glucose uptake, similar to insulin, a high throughput-screening assay for measuring glucose uptake in skeletal muscle cells was established. During the screening studies to discover novel antidiabetic compounds from microbial resources a Streptomyces strain PM0324667 (MTCC 5543, the Strain accession number at Institute of Microbial Technology, Chandigarh, India), an isolate from arid soil was identified which expressed a secondary metabolite that induced glucose uptake in L6 skeletal muscle cells. By employing bioactivity guided fractionation techniques, a tri-substituted simple aromatic compound with anti-diabetic potential was isolated. It was characterized based on MS and 2D NMR spectral data and identified as NFAT-133 which is a known immunosuppressive agent that inhibits NFAT-dependent transcription in vitro. Our investigations revealed the antidiabetic potential of NFAT-133. The compound induced glucose uptake in differentiated L6 myotubes with an EC50 of 6.3 ± 1.8 µM without activating the peroxisome proliferator-activated receptor-γ. Further, NFAT-133 was also efficacious in vivo in diabetic animals and reduced systemic glucose levels. Thus it is a potential lead compound which can be considered for development as a therapeutic for the treatment of type-2 diabetes. We have reported herewith the isolation of the producer microbe, fermentation, purification, in vitro, and in vivo antidiabetic activity of the compound.

A preferential p110alpha/gamma PI3K inhibitor attenuates experimental inflammation by suppressing the production of proinflammatory mediators in a NF-kappaB-dependent manner.

A promising therapeutic approach to diminish pathological inflammation is to inhibit the increased production and/or biological activity of proinflammatory cytokines (e.g., TNF-alpha, IL-6). The production of proinflammatory cytokines is controlled at the gene level by the activity of transcription factors, such as NF-kappaB. Phosphatidylinositol 3-kinase (PI3K), a lipid kinase, is known to induce the activation of NF-kappaB. Given this, we hypothesized that inhibitors of PI3K activation would demonstrate anti-inflammatory potential. Accordingly, we studied the effects of a preferential p110alpha/gamma PI3K inhibitor (compound 8C; PIK-75) in inflammation-based assays. Mechanism-based assays utilizing human cells revealed that PIK-75-mediated inhibition of PI3K activation is associated with dramatic suppression of downstream signaling events, including AKT phosphorylation, IKK activation, and NF-kappaB transcription. Cell-based assays revealed that PIK-75 potently and dose dependently inhibits in vitro and in vivo production of TNF-alpha and IL-6, diminishes the induced expression of human endothelial cell adhesion molecules (E-selectin, ICAM-1, and VCAM-1), and blocks human monocyte-endothelial cell adhesion. Most importantly, PIK-75, when administered orally in a therapeutic regimen, significantly suppresses the macroscopic and histological abnormalities associated with dextran sulfate sodium-induced murine colitis. The efficacy of PIK-75 in attenuating experimental inflammation is mediated, at least in part, due to the downregulation of pertinent inflammatory mediators in the colon. Collectively, these results provide first evidence that PIK-75 possesses anti-inflammatory potential. Given that PIK-75 is known to exhibit anti-cancer activity, the findings from this study thus reinforce the cross-therapeutic functionality of potential drugs.

Anti-inflammatory and anticancer activity of ergoflavin isolated from an endophytic fungus.

Biodiversity is a major resource for identification of new molecules with specific therapeutic activities. To identify such an active resource, high throughput screening (HTS) of the extracts prepared from such diversity are examined on specific functional assays. Based on such HTS studies and bioactivity-based fractionation, we have isolated ergoflavin, a pigment from an endophytic fungus, growing on the leaves of an Indian medicinal plant Mimosops elengi (bakul). We report here the isolation, structure elucidation, and biological properties of this compound, which showed good anti-inflammatory and anticancer activities.

Discovery of diacylphloroglucinols as a new class of GPR40 (FFAR1) agonists.

In this letter, we report discovery of diacylphloroglucinol compounds as a new class of GPR40 (FFAR1) agonists. Several diacylphloroglucinols with varying length of acyl functionality and substitution on aromatic hydroxyls were synthesized and evaluated for GPR40 agonism using functional calcium-flux assay. Out of 17 compounds evaluated, 14, 17, 19 and 25 exhibited good GPR40 agonistic activity with EC(50) values ranging from 0.07 to 8 microM (pEC(50) 7.12-5.09), respectively, with maximal agonistic response of 84-102%.

Synthesis and evaluation of pyrazolo[3,4-b]pyridines and its structural analogues as TNF-alpha and IL-6 inhibitors.

In the present article, we have synthesized three different series of pyrazolo[3,4-b]pyridines and their structural analogues using novel synthetic strategy involving one-pot condensation of 5,6-dihydro-4H-pyran-3-carbaldehyde/2-formyl-3,4,6-tri-O-methyl-D-glucal/chromone-3-carbaldehyde with heteroaromatic amines. All synthesized compounds were evaluated for their anti-inflammatory activity against TNF-alpha and IL-6. Out of 28 compounds screened, 40, 51, 52 and 56 exhibited promising activity against IL-6 with 60-65% inhibition at 10 microM concentration. Amongst these, 51, 52 and 56 showed potent IL-6 inhibitory activity with IC(50)'s of 0.2, 0.3 and 0.16 microM, respectively. Compound 56 was not cytotoxic in CCK-8 cells up to the concentration of >100 microM.

Novel leads from Heliotropium ovalifolium, 4,7,8-trimethoxy-naphthalene-2-carboxylic acid and 6-hydroxy-5,7-dimethoxy-naphthalene-2-carbaldehyde show specific IL-6 inhibitory activity in THP-1 cells and primary human monocytes.

From our screening program, we identified the anti-inflammatory effects of the extracts of Heliotropium ovalifolium in its ability to inhibit specific cytokines. The H. ovalifolium extract was found to be moderately active with an IC(50) equaling 10 microg/ml for inhibition of interleukin-6 (IL-6) in a human monocytic cell line. Interleukin-6 is a pleiotropic cytokine with implications in the regulation of the immune response, inflammation and hematopoiesis. This prompted us to examine and identify the active molecules that are responsible for the bioactivity in THP-1 cells. Bioassay guided fractionation identified two compounds 4,7,8-trimethoxy-naphthalene-2-carboxylic acid and 6-hydroxy-5,7-dimethoxy-naphthalene-2-carbaldehyde with an IC(50) of 2.4 and 2.0 microM for IL-6 inhibition and an IC(50) of 15.6 and 7.0 microM for tumor necrosis factor-alpha (TNF-alpha) inhibition in THP-1 cells. The protein expression data were supported by the inhibitory effect on mRNA gene expression. The compounds isolated from H. ovalifolium were also non-toxic in human peripheral blood monocytes from normal donors and the activity profile was similar to that obtained on THP-1 cells. Thus, we believe that these scaffolds may be of interest to develop leads for treating rheumatoid arthritis, psoriasis, ulcerative colitis, Crohn's disease and other inflammatory disorders. However, more detailed investigations need to be carried out to explain the efficacy of these compounds as drugs.