The Development of LAT1 Efflux Agonists as Mechanistic Probes of Cellular Amino Acid Stress.

Amino acid restriction induces cellular stress and cells often respond via the induction of autophagy. Autophagy or 'self-eating' enables the recycling of proteins and provides the essential amino acids needed for cell survival. Of the naturally occurring amino acids, methionine restriction has pleiotropic effects on cells because methionine also contributes to the intracellular methyl pools required for epigenetic controls as well as polyamine biosynthesis. In this report, we describe the chemical synthesis of four diastereomers of a methionine depletion agent and demonstrate how controlled methionine efflux from cells significantly reduces intracellular methionine, S-adenosylmethionine (SAM), S-adenosyl homocysteine (SAH), and polyamine levels. We also demonstrate that human pancreatic cancer cells respond via a lipid signaling pathway to induce autophagy. The methionine depletion agent causes the large amino acid transporter 1 (LAT1) to preferentially work in reverse and export the cell's methionine (and leucine) stores. The four diastereomers of the lead methionine/leucine depletion agent were synthesized and evaluated for their ability to (a) efflux 3H-leucine from cells, (b) dock to LAT1 in silico, (c) modulate intracellular SAM, SAH, and phosphatidylethanolamine (PE) pools, and (d) induce the formation of the autophagy-associated LC3-II marker. The ability to modulate the intracellular concentration of methionine regardless of exogenous methionine supply provides new molecular tools to better understand cancer response pathways. This information can then be used to design improved therapeutics that target downstream methionine-dependent processes like polyamines.

Discovery of Anthranilic Acid Derivatives as Difluoromethylornithine Adjunct Agents That Inhibit Far Upstream Element Binding Protein 1 (FUBP1) Function.

Polyamine biosynthesis is regulated by ornithine decarboxylase (ODC), which is transcriptionally activated by c-Myc. A large library was screened to find molecules that potentiate the ODC inhibitor, difluoromethylornithine (DFMO). Anthranilic acid derivatives were identified as DFMO adjunct agents. Further studies identified the far upstream binding protein 1 (FUBP1) as the target of lead compound 9. FUBP1 is a single-stranded DNA/RNA binding protein and a master controller of specific genes including c-Myc and p21. We showed that 9 does not inhibit 3H-spermidine uptake yet works synergistically with DFMO to limit cell growth in the presence of exogenous spermidine. Compound 9 was also shown to inhibit the KH4 FUBP1-FUSE interaction in a gel shift assay, bind to FUBP1 in a ChIP assay, reduce both c-Myc mRNA and protein expression, increase p21 mRNA and protein expression, and deplete intracellular polyamines. This promising hit opens the door to new FUBP1 inhibitors with increased potency.

Bis-Cyclic Guanidine Heterocyclic Peptidomimetics as Opioid Ligands with Mixed µ-, κ- and δ-Opioid Receptor Interactions: A Potential Approach to Novel Analgesics.

The design and development of analgesics with mixed-opioid receptor interactions has been reported to decrease side effects, minimizing respiratory depression and reinforcing properties to generate safer analgesic therapeutics. We synthesized bis-cyclic guanidine heterocyclic peptidomimetics from reduced tripeptides. In vitro screening with radioligand competition binding assays demonstrated variable affinity for the mu-opioid receptor (MOR), delta-opioid receptor (DOR), and kappa-opioid receptor (KOR) across the series, with compound 1968-22 displaying good affinity for all three receptors. Central intracerebroventricular (i.c.v.) administration of 1968-22 produced dose-dependent, opioid receptor-mediated antinociception in the mouse 55 °C warm-water tail-withdrawal assay, and 1968-22 also produced significant antinociception up to 80 min after oral administration (10 mg/kg, p.o.). Compound 1968-22 was detected in the brain 5 min after intravenous administration and was shown to be stable in the blood for at least 30 min. Central administration of 1968-22 did not produce significant respiratory depression, locomotor effects or conditioned place preference or aversion. The data suggest these bis-cyclic guanidine heterocyclic peptidomimetics with multifunctional opioid receptor activity may hold potential as new analgesics with fewer liabilities of use.

Development of a Redox-Sensitive Spermine Prodrug for the Potential Treatment of Snyder Robinson Syndrome.

Snyder Robinson Syndrome (SRS) is a rare disease associated with a defective spermine synthase gene and low intracellular spermine levels. In this study, a spermine replacement therapy was developed using a spermine prodrug that enters cells via the polyamine transport system. The prodrug was comprised of three components: a redox-sensitive quinone "trigger", a "trimethyl lock (TML)" aryl "release mechanism", and spermine. The presence of spermine in the design facilitated uptake by the polyamine transport system. The quinone-TML motifs provided a redox-sensitive agent, which upon intracellular reduction generated a hydroquinone, which underwent intramolecular cyclization to release free spermine and a lactone byproduct. Rewardingly, most SRS fibroblasts treated with the prodrug revealed a significant increase in intracellular spermine. Administering the spermine prodrug through feeding in a Drosophila model of SRS showed significant beneficial effects. In summary, a spermine prodrug is developed and provides a lead compound for future spermine replacement therapy experiments.

Indolyl-α-keto-1,3,4-oxadiazoles: Synthesis, anti-cell proliferation activity, and inhibition of tubulin polymerization.

A series of novel indolyl-α-keto-1,3,4-oxadiazole derivatives have been synthesized by employing molecular iodine-mediated oxidative cyclization of acylhydrazones. In vitro anti cell proliferation activity of these derivatives against various cancer cells lines such as human lymphoblast (U937), leukemia (Jurkat & SB) and human breast (BT474) was investigated. Among the synthesized indolyl-α-keto-1,3,4-oxadiazoles 19a-p, only one compound (19e) exhibited significant antiproliferative activity against a panel of cell lines. The compound 19e with 3,4,5-trimethoxyphenyl motif, endowed strong cytotoxicity against U937, Jurkat, BT474 and SB cancer cells with IC50 values of 7.1, 3.1, 4.1, and 0.8 µM, respectively. Molecular docking studies suggested a potential binding mode for 19e in the colchicine binding site of tubulin. When tested for in vitro tubulin polymerizaton, 19e inhibited tubulin polymezations (IC50 = 10.66 µM) and induced apoptosis through caspase 3/7 activation. Further, the derivative 19e did not cause necrosis when measured using lactate dehydrogenase assay.

NMK-BH2, a novel microtubule-depolymerising bis (indolyl)-hydrazide-hydrazone, induces apoptotic and autophagic cell death in cervical cancer cells by binding to tubulin at colchicine - site.

BACKGROUND: Microtubules, the key components of the eukaryotic cytoskeleton and mitotic spindle, are one of the most sought-after targets for cancer chemotherapy, especially due to their indispensible role in mitosis. Cervical cancer is a prevalent malignancy among women of developing countries including India. In spite of the remarkable therapeutic advancement, the non-specificity of chemotherapeutic drugs adversely affect the patients' survival and well-being, thus, necessitating the quest for novel indole-based anti-microtubule agent against cervical cancer, with high degree of potency and selectivity. METHODS: For in vitro studies, we used MTT assay, confocal microscopy, fluorescence microscopy, flow cytometry and Western blot analysis. Study in cell free system was accomplished by spectrophotometry, fluorescence spectroscopy and TEM and computational analysis was done by AutodockTools 1.5.6. RESULTS: NMK-BH2 exhibited significant and selective anti-proliferative activity against cervical cancer HeLa cells (IC50 = 1.5 µM) over normal cells. It perturbed the cytoskeletal and spindle microtubules of HeLa cells leading to mitotic block and cell death by apoptosis and autophagy. Furthermore, NMK-BH2 targeted the tubulin-microtubule system through fast and strong binding to the αß-tubulin heterodimers at colchicine-site. CONCLUSION: This study identifies and characterises NMK-BH2 as a novel anti-microtubule agent and provides insights into its key anti-cancer mechanism through two different cell death pathways: apoptosis and autophagy, which are mutually independent. GENERAL SIGNIFICANCE: It navigates the potential of the novel bis (indolyl)-hydrazide-hydrazone, NMK-BH2, to serve as lead for development of new generation microtubule-disrupting chemotherapeutic with improved efficacy and remarkable selectivity towards better cure of cervical cancer.

Metal-free regioselective formation of C-N and C-O bonds with the utilization of diaryliodonium salts in water: facile synthesis of N-arylquinolones and aryloxyquinolines.

Regioselective construction of crucial C-N and C-O bonds leading to N-arylquinolones and aryloxyquinolines has been accomplished by employing easily accessible diaryliodonium salts and quinolones in water under metal- and ligand-free conditions. This operationally simple strategy is significant due to mild reaction conditions, high product yields, recyclability of released iodoarenes and scalability to the gram level. The practical utility of the developed protocol was proved by the arylation of medicinally important heterocycles like acridin-9(10H)-one, 3-methylquinoxalin-2(1H)-one and 1H-benzo[d]imidazol-2(3H)-one.

Design and synthesis of bis(indolyl)ketohydrazide-hydrazones: Identification of potent and selective novel tubulin inhibitors.

A novel series of ketohydrazide-hydrazones as analogues of naturally occurring coscinamides has been synthesized and evaluated for their anticancer activity against five cancer cell lines. Of the twenty-synthesized ketohydrazide-hydrazones, compounds, 21c, 21f, 21g, 21k and 21o showed cytotoxic effects (less than 50% cell survival) against multiple cancer cell lines when tested at a final concentration of 10 µM. IC50 of three compounds 21f, 21k and 21o was determined to be less than 5 µM for all tested cancer cell lines. Compound 21k exhibited significant anticancer activity against MCF-7, MDA-MB-231, HCT-116 and JURKAT cancer cell lines with IC50 values of 0.8 µM, 0.50 µM, 0.15 µM, and 0.22 µM, respectively. Also, 21k was found to be more selectively cytotoxic against tumor cells when compared to normal cells. Preliminary mechanism of action studies indicated that the most active compound 21k induced caspase-dependent apoptosis in cells. 21k arrests cell cycle in G2/M phase by inhibiting of tubulin polymerization (IC50 = 0.6 µM).

Design, synthesis and in vitro cytotoxicity studies of novel ß-carbolinium bromides.

A series of novel ß-carbolinium bromides has been synthesized from easily accessible ß-carbolines and 1-aryl-2-bromoethanones. The newly synthesized compounds were evaluated for their in vitro anticancer activity. Among the synthesized derivatives, compounds 16l, 16o and 16s exhibited potent anticancer activity with IC50 values of <10µM against tested cancer cell lines. The most potent analogue 16l was broadly active against all the tested cancer cell lines (IC50=3.16-7.93µM). In order to test the mechanism of cell death, we exposed castration resistant prostate cancer cell line (C4-2) to compounds 16l and 16s, which resulted in increased levels of cleaved PARP1 and AO/EB staining, indicating that ß-carbolinium salts induce apoptosis in these cells. Additionally, the most potent ß-carbolines 16l and 16s were found to inhibit tubulin polymerization.

A Facile and Microwave-assisted Rapid Synthesis of 2-Arylamino-4-(3'-indolyl)- thiazoles as Apoptosis Inducing Cytotoxic Agents.

BACKGROUND AND OBJECTIVE: The clinical success of the chemotherapeutic drugs is restricted by the nonspecific toxicity-related adverse side effects. The diverse implication of indoles and thiazoles in medicinal chemistry prompted us to develop a new series of novel 2-aryl-amino-4-(3'-indolyl)thiazoles as more effective and less toxic anti-cancer compounds. METHOD AND RESULTS: One-pot microwave-assisted rapid and high yielding synthesis of 2-arylamino-4-(3'- indolyl)thiazoles involved the reaction of easily available α-tosyloxy-ketones with N-arylthioureas in polyethylene glycol-400 (PEG-400). In vitro cytotoxicity study of 2-arylamino-4-(3'-indolyl)thiazoles against a panel of human cancer cell lines by MTT assay revealed IC50 values in the low micromolar range. Of the fifteen synthesized arylaminothiazoles, compounds 17b, 17d, 17g and 17il showed significant anti-proliferative activity against the selected cancer cell lines with IC50 < 10 µM. The compound 17b was identified as the most potent ligand of the series, which exhibited good cytotoxic activity against MCF-7 breast cancer cells with an IC50 value of 1.86 µM but minimal toxicity on normal human cells. Investigation of the underlying mechanism by flow cytometry indicated that 17b induced ROS-mediated apoptosis in MCF-7 cells in a dose-dependent manner as supported by upregulation of Bax and caspase-3 and down-regulation of Bcl-2 (by Western blot). CONCLUSION: Developed an efficient and eco-friendly synthesis for 2-arylamino-4-(3'-indolyl)thiazoles, and their in vitro cytotoxicity studies demonstrate that compound 17b exhibits significant anti-proliferative activity against MCF-7 (breast cancer) cells by activating ROS-mediated apoptosis through the mitochondrial apoptosis pathway.

Synthesis, evaluation and molecular modelling studies of 2-(carbazol-3-yl)-2-oxoacetamide analogues as a new class of potential pancreatic lipase inhibitors.

A series of twenty four 2-(carbazol-3-yl)-2-oxoacetamide analogues were synthesized, characterized and evaluated for their pancreatic lipase (PL) inhibitory activity. Porcine PL was used against 4-nitrophenyl butyrate (method A) and tributyrin (methods B and C) as substrates during the PL inhibition assay. Compounds 7e, 7f and 7p exhibited potential PL inhibitory activity (IC50 values of 6.31, 8.72 and 9.58µM, respectively in method A; and Xi50 of 21.85, 21.94 and 26.2, respectively in method B). Further, inhibition kinetics of 7e, 7f and 7p against PL, using method A, revealed their competitive nature of inhibition. A comparison of the inhibition profiles of the top three compounds in methods B and C, provided a preliminary idea of covalent bonding of the compounds with Ser 152 of PL. Molecular docking studies of the compounds 7a-x into the active site of human PL (PDB ID: 1LPB) was in agreement with the in vitro results, and highlighted probable covalent bond formation with Ser 152 apart from hydrophobic interactions with the lid domain. Molecular dynamics simulation of 7e complexed with PL, further confirmed the role of aromatic groups in stabilising the ligand (RMSD ⩽4Å). The present study led to the identification of 2-(carbazol-3-yl)-2-oxoacetamide analogues 7a-x as a new class of potential PL inhibitors.

Sequential one-pot synthesis of bis(indolyl)glyoxylamides: Evaluation of antibacterial and anticancer activities.

A series of bis(indolyl)glyoxylamides 10a-n has been designed and synthesized. In situ generated indole-3-glyoxalylchloride from the reaction of readily available indole 9 with oxalyl chloride was treated with tryptamine to produce bis(indolyl)glyoxylamides 10a-n in 82-93% yields. All the synthesized bis(indolyl)glyoxylamides were well characterized and tested for their antibacterial activity against Gram-positive and Gram-negative bacterial strains. Compounds 10d, 10g and 10i were found to display potent antibacterial activity against Gram-negative strain. Further, the cytotoxicity of bis(indolyl)glyoxylamides 10a-n were evaluated against a panel of human cancer cell lines. Of the screened analogues, compound 10f (IC50=22.34µM; HeLa, 24.05µM; PC-3, 21.13µM; MDA-MB-231 and 29.94µM; BxPC-3) was identified as the most potent analogue of the series. Exposure of PC-3 cells to either 10a or 10f resulted in increased levels of cleaved PARP1, indicating that bis(indolyl)glyoxylamides induce apoptosis in PC-3 cells. Most importantly, compounds 10d, 10g and 10i were completely ineffective in mammalian cells, suggesting that they target bacterial-specific targets and thus will not display any toxicity in host cells.

Development of Novel Bis(indolyl)-hydrazide-Hydrazone Derivatives as Potent Microtubule-Targeting Cytotoxic Agents against A549 Lung Cancer Cells.

The biological significance of microtubules makes them a validated target of cancer therapy. In this study, we have utilized indole, an important pharmacological scaffold, to synthesize novel bis(indolyl)-hydrazide-hydrazone derivatives (NMK-BH compounds) and recognized NMK-BH3 as the most effective one in inhibiting A549 cell proliferation and assembly of tissue-purified tubulin. Cell viability experiments showed that NMK-BH3 inhibited proliferation of human lung adenocarcinoma (A549) cells, normal human lung fibroblasts (WI38) and peripheral blood mononuclear cells (PBMC) with IC50 values of ∼2, 48.5, and 62 µM, respectively. Thus, the relatively high cytotoxicity of NMK-BH3 toward lung carcinoma (A549) cells over normal lung fibroblasts (WI38) and PBMC confers a therapeutic advantage of reduced host toxicity. Flow cytometry, Western blot, and immunofluorescence studies in the A549 cell line revealed that NMK-BH3 induced G2/M arrest, mitochondrial depolarization, and apoptosis by depolymerizing the cellular interphase and spindle microtubules. Consistent with these observations, study in cell free system revealed that NMK-BH3 inhibited the microtubule assembly with an IC50 value of ∼7.5 µM. The tubulin-ligand interaction study using fluorescence spectroscopy indicated that NMK-BH3 exhibited strong and specific tubulin binding with a dissociation constant of ∼1.4 µM at a single site, very close to colchicine site, on ß-tubulin. Collectively, these findings explore the cytotoxic potential of NMK-BH3 by targeting the microtubules and inspire its development as a potential candidate for lung cancer chemotherapy.

One-pot synthesis and in-vitro anticancer evaluation of 5-(2'-indolyl)thiazoles.

A series of 5-(2'-indolyl)thiazoles were synthesized and evaluated for their cytotoxicity against selected human cancer cell lines. The reaction of thioamides 3 with 3-tosyloxypentane-2,4-dione 4 led to in situ formation of 5-acetylthiazole 5 which upon treatment with arylhydrazines 6 in polyphosphoric acid resulted in the formation of 5-(2'-indolyl)thiazoles 2. Among the synthesized 5-(2'-indolyl)thiazoles, compounds 2d-f, and 2h exhibited encouraging anticancer activity and also selectivity towards particular cell lines (IC50 = 10-30 µM). Further studies on the SAR of compound 2e may result in good anticancer activity.

2-(3'-Indolyl)-N-arylthiazole-4-carboxamides: Synthesis and evaluation of antibacterial and anticancer activities.

A new series of 2-(3'-indolyl)-N-arylthiazole-4-carboxamides 17a-p has been designed and synthesized. Initial reaction of readily available thioamides 15 with bromopyruvic acid under refluxing conditions produced different thiazole carboxylic acids 16 which upon coupling with arylamines by using EDCI·HCl and HOBt afforded diverse arylthiazole-4-carboxamides 17a-p in 78-87% yields. Antibacterial activity evaluation against Gram-positive and Gram-negative bacterial strains led to compounds 17i-k and 17o as potent and selectively (Gram-negative) antibacterial agents. The cytotoxicity of thiazole carboxamides 17a-p was also evaluated on a panel of human cancer cell lines. Among the tested derivatives, compounds 17i (IC50=8.64µM; HEK293T) and 17l (IC50=3.41µM; HeLa) were identified as the most potent analogues of the series. Preliminary mechanism of action studies of thiazole carboxamide 17i suggested that its cytotoxicity against HeLa cells involves the induction of cell death by apoptosis.

Synthesis and identification of α-cyano bis(indolyl)chalcones as novel anticancer agents.

Microwave-assisted synthesis of 23 α-cyano bis(indolyl)chalcones (6a-w) and their in vitro anticancer activity against three human cancer cell lines have been discussed. Among the synthesized chalcones, compound 6n was found to be the most potent and selective against A549 lung cancer cell line (IC50 = 0.8 µM). In a preliminary mechanism of action studies some α-cyano bis(indolyl)chalcones were found to enhance tubulin polymerization suggesting these compounds could act as microtubule stabilizing agents.

NMK-TD-100, a novel microtubule modulating agent, blocks mitosis and induces apoptosis in HeLa cells by binding to tubulin.

Thiadiazoles are one of the most widely utilized agents in medicinal chemistry, having a wide range of pharmacologic activity. Microtubules (MTs) have always remained a sought-after target in rapidly proliferating cancer cells. We screened for the growth inhibitory effect of synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles on cancer cells and identified NMK-TD-100, as the most potent agent. Cell viability experiments using human cervical carcinoma cell line (HeLa cells) indicated that the IC50 value was 1.42±0.11 µM for NMK-TD-100 for 48 h treatment. In further study, we examined the mode of interaction of NMK-TD-100 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. NMK-TD-100 induced arrest in mitotic phase of cell cycle, caused decline in mitochondrial membrane potential and induced apoptosis in HeLa cells. Immunofluorescence studies using an anti-α-tubulin antibody showed a significant depolymerization of the interphase microtubule network and spindle microtubule in HeLa cells in a concentration-dependent manner. However, the cytotoxicity of NMK-TD-100 towards human peripheral blood mononuclear cells (PBMC) was lower compared to that in cancer cells. Polymerization of tissue purified tubulin into microtubules was inhibited by NMK-TD-100 with an IC50 value of 17.5±0.35 µM. The binding of NMK-TD-100 with tubulin was studied using NMK-TD-100 fluorescence enhancement and intrinsic tryptophan fluorescence of tubulin. The stoichiometry of NMK-TD-100 binding to tubulin is 1:1 (molar ratio) with a dissociation constant of ~1 µM. Fluorescence spectroscopic and molecular modeling data showed that NMK-TD-100 binds to tubulin at a site which is very near to the colchicine binding site. The binding of NMK-TD-100 to tubulin was estimated to be ~10 times faster than that of colchicine. The results indicated that NMK-TD-100 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided insights into its potential of being a chemotherapeutic agent.