European journal of medicinal chemistry, Mar 1, 2019
, a quinazolinone-based dual inhibitor of Cdk4 and tubulin polymerization, identified via ligand-... more , a quinazolinone-based dual inhibitor of Cdk4 and tubulin polymerization, identified via ligand-based virtual screening, for efficient anticancer therapy,
Cytochrome P450 family 1 (CYP1) enzymes catalyze the metabolic activation of environmental procar... more Cytochrome P450 family 1 (CYP1) enzymes catalyze the metabolic activation of environmental procarcinogens such as benzo[a]pyrene, B[a]P, into carcinogens, which initiate the process of carcinogenesis. Thus, stopping the metabolic activation of procarcinogens can possibly prevent the onset of cancer. Several natural products have been reported to show unique ability in inhibiting CYP1 enzymes. We found that khellin, a naturally occurring furanochromone from Ammi visnaga, inhibits CYP1A1 enzyme with an IC50 value of 4.02 µM in CYP1A1-overexpressing human HEK293 suspension cells. To further explore this natural product for discovery of more potent and selective CYP1A1 inhibitors, two sets of semisynthetic derivatives were prepared. Treatment of khellin with alkali results in opening of a pyrone ring, yielding khellinone (2). Claisen-Schmidt condensation of khellinone (2) with various aldehydes in presence of potassium hydroxide, at room temperature, provide a series of furanochalcones 3a-v (khellinochalcones). Treatment of khellinone (2) with aryl aldehydes in presence of piperidine, under reflux, affords the flavanone series of compounds 4a-p (khellinoflavanones). The khellinoflavanone 4l potently inhibited CYP1A1 with IC50 value of 140 nM in live cells, with 170-fold selectivity over CYP1B1 (IC50 for CYP1B1 = 23.8 µM). Compound 4l at 3 x IC50 concentration for inhibition of CYP1A1, completely protected HEK293 cells from CYP1A1mediated B[a]P toxicity. Lung cancer cells, A549 (p53 +) and Calu-1 (p53-null), blocked in growth at the S-phase by B[a]P were restored into the cell cycle by compound 4l. The results presented herein strongly indicate the potential of these khellin derivatives for further development as cancer chemopreventive agents.
Journal of Chemical Information and Modeling, May 22, 2017
Target structure-guided virtual screening (VS) is a versatile, powerful and inexpensive alternati... more Target structure-guided virtual screening (VS) is a versatile, powerful and inexpensive alternative to experimental high-throughput screening (HTS). In order to discover potent CYP1A1 enzyme inhibitors for cancer chemoprevention, a commercially library of 50,000 small molecules was utilized for VS guided by both ligand and structure-based strategies. For experimental validation, 300 ligands were proposed based on combined analysis of fitness scores from ligand based e-pharmacophore screening and docking score, prime MMGB/SA binding affinity and interaction pattern analysis from structurebased VS. These 300 compounds were screened, at 10 µM concentration, for in-vitro inhibition of CYP1A1-Sacchrosomes (yeast-derived microsomal enzyme) in the ethoxyresorufin-O-deethylase assay. Thirty-two compounds displayed >50% inhibition of CYP1A1 enzyme activity at 10 µM. 2-Phenylimidazo-[1,2-a]quinoline (5121780, 119) was found to be the most potent with 97% inhibition. It also inhibited ~95% activity of CYP1B1 and CYP1A2, the other two CYP1 enzymes. The compound 5121780 (119) showed high selectivity towards inhibition of CYP1 enzymes with respect to CYP2 and CYP3 enzymes (i.e. there was no detectable inhibition of CYP2D6/ CYP2C9/ CYP2C19 and CYP3A4 at 10 µM). It was further investigated in live CYP-expressing human cell system which confirmed that compound 5121780 (119) potently inhibited CYP1A1, CYP1A2, CYP1B1 enzymes with IC 50 values of 269, 30 and 56 nM, respectively. Like in Sacchrosomes, inhibition of CYP2D6/ CYP2C9/ CYP2C19 and CYP3A4 enzymes, expressed within live human cells, could hardly be detected at 10 µM. The compound 119 rescued CYP1A1 over-expressing HEK293 cells from CYP1A1 mediated B[a]P toxicity and also overcame cisplatin resistance in CYP1B1 over-expressing HEK293 cells. Molecular dynamics simulations of 5121780 (119) with CYP1 enzymes was performed to understand the interaction pattern to CYP isoforms. Results indicate that VS can successfully be used to identify promising CYP1A1 inhibitors, which may have potential in the development of novel cancer chemo-preventive agents.
Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is indu... more Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is induced (a) by polycyclic aromatic hydrocarbons (PAHs)which can be metabolized by CYP1A1 to carcinogens, and (b) in majority of breast cancers. Hence, phytochemicals or dietary flavonoids, if identified as CYP1A1 inhibitors, may help in preventing PAH-mediated carcinogenesis and breast cancer. Herein, we have investigated the cancer chemopreventive potential of a flavonoid-rich Indian medicinal plant, Pongamia pinnata (L.) Pierre. Methanolic extract of its seeds inhibits CYP1A1 in CYP1A1-overexpressing normal human HEK293 cells, with IC 50 of 0.6 µg/mL. Its secondary metabolites, the furanoflavonoids pongapin/ lanceolatin B, inhibit CYP1A1 with IC 50 of 20 nM. Although the furanochalcone pongamol inhibits CYP1A1 with IC 50 of only 4.4 µM, a semisynthetic pyrazole-derivative P5b, has ~10-fold improved potency (IC 50 , 0.49 M). Pongapin/ lanceolatin B and the methanolic extract of P. pinnata seeds protect CYP1A1-overexpressing HEK293 cells from B[a]P-mediated toxicity. Remarkably, they also block the cell cycle of CYP1A1-overexpressing MCF-7 breast cancer cells, at the G 0-G 1 phase, repress cyclin D1 levels and induce cellular-senescence. Molecular modeling studies demonstrate the interaction pattern of pongapin/lanceolatin B with CYP1A1. The results strongly indicate the potential of methanolic seed-extract and pongapin/lanceolatin B for further development as cancer chemopreventive agents.
CYP1A1 is thought to mediate carcinogenesis in oral, lung and epithelial cancers. In order to ide... more CYP1A1 is thought to mediate carcinogenesis in oral, lung and epithelial cancers. In order to identify a CYP1A1 inhibitor from an edible plant, 394 natural products in the IIIM's natural product repository were screened, at 10 μM concentration, using CYP1A1-Sacchrosomes™ (i.e. microsomal enzyme isolated from recombinant baker's yeast). Twenty-seven natural products were identified that inhibited 40-97% of CYP1A1's 7-ethoxyresorufin-O-deethylase activity. The IC 50 values of the 'hits', belonging to different chemical scaffolds, were determined. Their selectivity was studied against a panel of 8 CYP-Sacchrosomes™. In order to assess cellular efficacy, the 'hits' were screened for their capability to inhibit CYP enzymes expressed within live recombinant human embryonic kidney (HEK293) cells from plasmids encoding specific CYP genes (1A2, 1B1, 2C9, 2C19, 2D6, 3A4). Isopimpinellin (IN-475; IC 50 , 20 nM) and karanjin (IN-195; IC 50 , 30 nM) showed the most potent inhibition of CYP1A1 in human cells. Isopimpinellin is found in celery, parsnip, fruits and in the rind and pulp of limes whereas different parts of the Indian beech tree, which contain karanjin, have been used in traditional medicine. Both isopimpinellin and karanjin negate the cellular toxicity of CYP1A1-mediated benzo[a]pyrene. Molecular docking and molecular dynamic simulations with CYP isoforms rationalize the observed trends in the potency and selectivity of isopimpinellin and karanjin.
Renal cell carcinoma (RCC) is the most difficult-to-treat form of kidney cancer with a median 5-y... more Renal cell carcinoma (RCC) is the most difficult-to-treat form of kidney cancer with a median 5-year survival of 10% under metastatic setting. In RCC, although cytoreductive nephrectomy is common, approximately 20-30% of patients will develop recurrent cancer after surgery, which highlights the need for an effective therapy. Rho-GTPases viz, Rac-1 and Cdc42 are the central regulators of cancer cell migration and invasion and thus metastasis in multiple cancer types. Hence, we elucidated the role of Ketorolac, a modulator Rho-GTPases against RCC through potentiation of tumor suppressor Par-4. The effect of Ketorolac alone and in combination on proliferation, apoptosis, cellcycle progression, migration, tumor inhibition and their related markers were studied. Moreover, Ketorolac's impact on metastasis by influencing Rac-1/HIF-1α/DDX3/β-catenin signalling was studied with respect to its ability to modulate the expression of tumor suppressor Par-4, and this mechanism was confirmed by siRNA knockdown studies. Ketorolac induced cytotoxicity in a panel of renal cells including patient derived tumor cells with IC 50 2.8 to 9.02 mM and 0.28 to 3.8 mM in monolayer and anchorage independent clonogenic assays respectively. Ketorolac caused significant down regulation of proliferation (Ki-67, Cyclin D1, pRB and DDX3), migration/invasion (Rac-1, Cdc42, and Tiam1), and angiogenesis (HIF-1α and VEGF) markers as studied by gene and protein expression. Moreover, it caused a significant upregulation of tumor suppressor Par-4 known to be downregulated in RCC. This mechanism was further confirmed by using siRNA knockdown studies where we could demonstrate a negative relation between the expression of Par-4 and Rac-1/Cdc42. Importantly, Ketorolac alone and in combination with Sunitinib showed tumor growth inhibition (TGI) of 73% and 86% respectively in xenograft model. This anti-tumor activity was further corroborated by down regulation of Rac-1/ Cdc42/HIF-1α/DDX3/β-catenin signalling. This is the first report which implicates the role of Ketorolac against RCC by acting as a small molecule secretagogue causing upregulation of Par-4 in autocrine and paracrine manner. Consequently, these findings suggest that Par-4 can serve as a valuable therapeutic target and a prognostic marker for the treatment of RCC.
Glioblastoma (GBM) is an aggressive form of brain tumor with a median survival of approximately 1... more Glioblastoma (GBM) is an aggressive form of brain tumor with a median survival of approximately 12 months. With no new drugs in the last few decades and limited success in clinics for known therapies, drug repurposing is an attractive choice for its treatment. Here, we examined the efficacy of pyronaridine (PYR), an anti-malarial drug in GBM cells. PYR induced anti-proliferative activity in GBM cells with IC50 ranging from 1.16 to 6.82 µM. Synergistic activity was observed when PYR was combined with Doxorubicin and Ritonavir. Mechanistically, PYR triggered mitochondrial membrane depolarization and enhanced the ROS levels causing caspase-3 mediated apoptosis. PYR significantly decreased markers associated with proliferation, EMT, hypoxia, and stemness and upregulated the expression of E-cadherin. Interestingly, PYR induced the expression of intracellular as well as secretory Par-4, a tumor suppressor in GBM cells, which was confirmed using siRNA. Notably, Par-4 levels in plasma sampl...
A novel inhibitor of hypoxia-inducible factor-1a P3155 also modulates PI3K pathway and inhibits g... more A novel inhibitor of hypoxia-inducible factor-1a P3155 also modulates PI3K pathway and inhibits growth of prostate cancer cells
This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted P... more This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. P7170, a novel inhibitor of mTORC1/mTORC2 and activin receptor-like Kinase 1
La presente invention concerne une combinaison pharmaceutique comprenant deux agents antineoplasi... more La presente invention concerne une combinaison pharmaceutique comprenant deux agents antineoplasiques cytotoxiques, la gemcitabine et le carboplatine, et au moins un inhibiteur de kinase dependante de cycline (CDK); ladite combinaison presentant des effets synergiques lorsqu'elle est utilisee dans le traitement du cancer du sein, en particulier du cancer du sein triple negatif. L'invention concerne egalement une methode de traitement du cancer du sein a l'aide d'une quantite therapeutiquement efficace de ladite combinaison.
La presente invention concerne une combinaison pharmaceutique comprenant un inhibiteur de PI3K/mT... more La presente invention concerne une combinaison pharmaceutique comprenant un inhibiteur de PI3K/mTOR selectionne parmi des composes de formule (I) (tels que decrits dans la demande) ou des sels pharmaceutiquement acceptables ou des solvants de ceux-ci ; et au moins un agent anti-proliferatif; des compositions pharmaceutiques contenant lesdits composes de formule (I) et au moins un agent anti-proliferatif; et l'utilisation de ladite combinaison dans le traitement de maladies ou troubles anti-proliferatifs.
Glioblastoma multiforme (GBM) is a commonly diagnosed primary brain tumor with median survival of... more Glioblastoma multiforme (GBM) is a commonly diagnosed primary brain tumor with median survival of about one year. GBM is highly aggressive malignancy with limited treatment options. The standard of care for GBM is Temozolomide (TMZ), given as first-line therapy, however, 90% of recurrent gliomas acquire TMZ resistance. Treatment to GBM remains greatest challenge in the management of cancer patients worldwide due to inherent tumor heterogeneity and drug resistant nature leading to high unmet medical need. New drug development is challenging and takes an enormous amount of time, money and effort. Drug repurposing, the application of an existing therapeutic to a new disease indication holds the promise of rapid clinical impact at a lower cost than de novo drug development. This study reports the anti-cancer efficacy of Pyronaridine (PYR), a benzonaphthyridine derivative, used as an agent for the treatment of malaria. Anti-malarial action of PYR therapy is known to target haematin formation and inhibit activity of P. falciparum DNA topoisomerase II. PYR was earlier reported to show in vitro anticancer activity in combination with other drugs in breast and haematological cancer. However, in this study we report its anticancer activity in GBM with novel mechanism of action selective against GBM resistant cells. In our studies, anticancer activity of PYR was confirmed in panel of cancer cell lines comprising of kidney, pancreatic, ovarian, glioma and bladder cancer (IC50: 0.907 - 11 µM). Interestingly, PYR demonstrated dose dependant anti-proliferative and pro-apoptotic activity in both TMZ sensitive and TP53 and PTEN mutated resistant GBM cells (IC50: 1.82-4.2 µM). The 3D culture multicellular spheroid model (MCS) is important and relevant as it is predictive of in vivo antitumor efficacy, PYR inhibited MCS developed from TMZ resistant cells. Additionally, PYR also showed potent activity against glioma stem cell line and primary culture derived from high grade glioma. Cell cycle analysis indicated G1 and G2/M phase arrest and increased sub G0 population when GBM cells were treated with PYR. While deciphering the mechanism of action of PYR, we astoundingly invented a novel role for PYR as an inducer of Prostate Apoptosis Response-4 (PAR-4), a pro-apoptotic tumor suppressor protein. The expression of PAR-4 is known to be downregulated in most tumors including GBM. Here, we demonstrate that PYR modulates microenvironment by inducing PAR-4 in normal fibroblast cells in a dose dependent manner. The conditioned medium from these cells induced profound cytotoxicity against cancer cells. PYR is relatively safe to use in humans and our data clearly indicates its anti-cancer potential and hence is a promising therapeutic candidate. The detailed studies focusing on its novel mechanism of action alone and in combination in drug resistant models will be discussed. Citation Format: Jeevan D. Ghosalkar, Vinay R. Sonawane, Siddhika R. Raut, Mariamma Thekkumpurath, Radha Pujari, Padma Shastri, Geena Malhotra, Kalpana S. Joshi. Anti-malarial drug Pyronaridine modulates microenvironment leading to potent anti-cancer activity against GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 251.
Cytochrome P450 family 1 (CYP1) enzymes catalyze the metabolic activation of environmental procar... more Cytochrome P450 family 1 (CYP1) enzymes catalyze the metabolic activation of environmental procarcinogens such as benzo[a]pyrene, B[a]P, into carcinogens, which initiate the process of carcinogenesis. Thus, stopping the metabolic activation of procarcinogens can possibly prevent the onset of cancer. Several natural products have been reported to show unique ability in inhibiting CYP1 enzymes. We found that khellin, a naturally occurring furanochromone from Ammi visnaga, inhibits CYP1A1 enzyme with an IC50 value of 4.02 µM in CYP1A1-overexpressing human HEK293 suspension cells. To further explore this natural product for discovery of more potent and selective CYP1A1 inhibitors, two sets of semisynthetic derivatives were prepared. Treatment of khellin with alkali results in opening of a pyrone ring, yielding khellinone (2). Claisen-Schmidt condensation of khellinone (2) with various aldehydes in presence of potassium hydroxide, at room temperature, provide a series of furanochalcones 3a-v (khellinochalcones). Treatment of khellinone (2) with aryl aldehydes in presence of piperidine, under reflux, affords the flavanone series of compounds 4a-p (khellinoflavanones). The khellinoflavanone 4l potently inhibited CYP1A1 with IC50 value of 140 nM in live cells, with 170-fold selectivity over CYP1B1 (IC50 for CYP1B1 = 23.8 µM). Compound 4l at 3 x IC50 concentration for inhibition of CYP1A1, completely protected HEK293 cells from CYP1A1mediated B[a]P toxicity. Lung cancer cells, A549 (p53 +) and Calu-1 (p53-null), blocked in growth at the S-phase by B[a]P were restored into the cell cycle by compound 4l. The results presented herein strongly indicate the potential of these khellin derivatives for further development as cancer chemopreventive agents.
&NA; Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yea... more &NA; Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yeast cells, are extensively used for drug metabolism studies. However, they may not always portray how a developmental drug would behave in human cells with intact intracellular transport mechanisms. This study emphasizes the usefulness of human HEK293 kidney cells, grown in ‘suspension’ for expression of CYPs, in finding potent CYP1A1/CYP1B1 inhibitors, as possible anticancer agents. With live cell‐based assays, quinazolinones 9i/9b were found to be selective CYP1A1/CYP1B1 inhibitors with IC50 values of 30/21 nM, and > 150‐fold selectivity over CYP2/3 enzymes, whereas they were far less active using commercially‐available CYP1A1/CYP1B1 microsomal enzymes (IC50, >10/1.3–1.7 &mgr;M). Compound 9i prevented CYP1A1‐mediated benzo[a]pyrene‐toxicity in normal fibroblasts whereas 9b completely reversed cisplatin resistance in PC‐3/prostate, COR‐L23/lung, MIAPaCa‐2/pancreatic and LS174T/colon cancer cells, underlining the human‐cell‐assays' potential. Our results indicate that the most potent CYP1A1/CYP1B1 inhibitors would not have been identified if one had relied merely on microsomal enzymes.
, a quinazolinone-based dual inhibitor of Cdk4 and tubulin polymerization, identified via ligand-... more , a quinazolinone-based dual inhibitor of Cdk4 and tubulin polymerization, identified via ligand-based virtual screening, for efficient anticancer therapy,
Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is indu... more Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is induced (a) by polycyclic aromatic hydrocarbons (PAHs)which can be metabolized by CYP1A1 to carcinogens, and (b) in majority of breast cancers. Hence, phytochemicals or dietary flavonoids, if identified as CYP1A1 inhibitors, may help in preventing PAH-mediated carcinogenesis and breast cancer. Herein, we have investigated the cancer chemopreventive potential of a flavonoid-rich Indian medicinal plant, Pongamia pinnata (L.) Pierre. Methanolic extract of its seeds inhibits CYP1A1 in CYP1A1-overexpressing normal human HEK293 cells, with IC 50 of 0.6 µg/mL. Its secondary metabolites, the furanoflavonoids pongapin/ lanceolatin B, inhibit CYP1A1 with IC 50 of 20 nM. Although the furanochalcone pongamol inhibits CYP1A1 with IC 50 of only 4.4 µM, a semisynthetic pyrazole-derivative P5b, has ~10-fold improved potency (IC 50 , 0.49 M). Pongapin/ lanceolatin B and the methanolic extract of P. pinnata seeds protect CYP1A1-overexpressing HEK293 cells from B[a]P-mediated toxicity. Remarkably, they also block the cell cycle of CYP1A1-overexpressing MCF-7 breast cancer cells, at the G 0-G 1 phase, repress cyclin D1 levels and induce cellular-senescence. Molecular modeling studies demonstrate the interaction pattern of pongapin/lanceolatin B with CYP1A1. The results strongly indicate the potential of methanolic seed-extract and pongapin/lanceolatin B for further development as cancer chemopreventive agents.
S2 CONTENTS S1. Experimental procedures S2. CYP1A1 screening results in Sacchrosomes TM S3. HPLC ... more S2 CONTENTS S1. Experimental procedures S2. CYP1A1 screening results in Sacchrosomes TM S3. HPLC purity of selected in-house natural product library hits. S4. Karanjin is a competitive inhibitor of CYP1A1 S5. MD simulation and interactions of isopimpinellin (IN-475) with CYP1A1 S6. Interaction of isopimpinellin (IN-475) with CYP1, CYP2 and CYP3 isoforms S7. Interactions of karanjin (IN-195) with CYP2 and CYP3 isoforms S8. NMR, HPLC and HRMS data scans of IN-195 and IN-475
European journal of medicinal chemistry, Mar 1, 2019
, a quinazolinone-based dual inhibitor of Cdk4 and tubulin polymerization, identified via ligand-... more , a quinazolinone-based dual inhibitor of Cdk4 and tubulin polymerization, identified via ligand-based virtual screening, for efficient anticancer therapy,
Cytochrome P450 family 1 (CYP1) enzymes catalyze the metabolic activation of environmental procar... more Cytochrome P450 family 1 (CYP1) enzymes catalyze the metabolic activation of environmental procarcinogens such as benzo[a]pyrene, B[a]P, into carcinogens, which initiate the process of carcinogenesis. Thus, stopping the metabolic activation of procarcinogens can possibly prevent the onset of cancer. Several natural products have been reported to show unique ability in inhibiting CYP1 enzymes. We found that khellin, a naturally occurring furanochromone from Ammi visnaga, inhibits CYP1A1 enzyme with an IC50 value of 4.02 µM in CYP1A1-overexpressing human HEK293 suspension cells. To further explore this natural product for discovery of more potent and selective CYP1A1 inhibitors, two sets of semisynthetic derivatives were prepared. Treatment of khellin with alkali results in opening of a pyrone ring, yielding khellinone (2). Claisen-Schmidt condensation of khellinone (2) with various aldehydes in presence of potassium hydroxide, at room temperature, provide a series of furanochalcones 3a-v (khellinochalcones). Treatment of khellinone (2) with aryl aldehydes in presence of piperidine, under reflux, affords the flavanone series of compounds 4a-p (khellinoflavanones). The khellinoflavanone 4l potently inhibited CYP1A1 with IC50 value of 140 nM in live cells, with 170-fold selectivity over CYP1B1 (IC50 for CYP1B1 = 23.8 µM). Compound 4l at 3 x IC50 concentration for inhibition of CYP1A1, completely protected HEK293 cells from CYP1A1mediated B[a]P toxicity. Lung cancer cells, A549 (p53 +) and Calu-1 (p53-null), blocked in growth at the S-phase by B[a]P were restored into the cell cycle by compound 4l. The results presented herein strongly indicate the potential of these khellin derivatives for further development as cancer chemopreventive agents.
Journal of Chemical Information and Modeling, May 22, 2017
Target structure-guided virtual screening (VS) is a versatile, powerful and inexpensive alternati... more Target structure-guided virtual screening (VS) is a versatile, powerful and inexpensive alternative to experimental high-throughput screening (HTS). In order to discover potent CYP1A1 enzyme inhibitors for cancer chemoprevention, a commercially library of 50,000 small molecules was utilized for VS guided by both ligand and structure-based strategies. For experimental validation, 300 ligands were proposed based on combined analysis of fitness scores from ligand based e-pharmacophore screening and docking score, prime MMGB/SA binding affinity and interaction pattern analysis from structurebased VS. These 300 compounds were screened, at 10 µM concentration, for in-vitro inhibition of CYP1A1-Sacchrosomes (yeast-derived microsomal enzyme) in the ethoxyresorufin-O-deethylase assay. Thirty-two compounds displayed >50% inhibition of CYP1A1 enzyme activity at 10 µM. 2-Phenylimidazo-[1,2-a]quinoline (5121780, 119) was found to be the most potent with 97% inhibition. It also inhibited ~95% activity of CYP1B1 and CYP1A2, the other two CYP1 enzymes. The compound 5121780 (119) showed high selectivity towards inhibition of CYP1 enzymes with respect to CYP2 and CYP3 enzymes (i.e. there was no detectable inhibition of CYP2D6/ CYP2C9/ CYP2C19 and CYP3A4 at 10 µM). It was further investigated in live CYP-expressing human cell system which confirmed that compound 5121780 (119) potently inhibited CYP1A1, CYP1A2, CYP1B1 enzymes with IC 50 values of 269, 30 and 56 nM, respectively. Like in Sacchrosomes, inhibition of CYP2D6/ CYP2C9/ CYP2C19 and CYP3A4 enzymes, expressed within live human cells, could hardly be detected at 10 µM. The compound 119 rescued CYP1A1 over-expressing HEK293 cells from CYP1A1 mediated B[a]P toxicity and also overcame cisplatin resistance in CYP1B1 over-expressing HEK293 cells. Molecular dynamics simulations of 5121780 (119) with CYP1 enzymes was performed to understand the interaction pattern to CYP isoforms. Results indicate that VS can successfully be used to identify promising CYP1A1 inhibitors, which may have potential in the development of novel cancer chemo-preventive agents.
Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is indu... more Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is induced (a) by polycyclic aromatic hydrocarbons (PAHs)which can be metabolized by CYP1A1 to carcinogens, and (b) in majority of breast cancers. Hence, phytochemicals or dietary flavonoids, if identified as CYP1A1 inhibitors, may help in preventing PAH-mediated carcinogenesis and breast cancer. Herein, we have investigated the cancer chemopreventive potential of a flavonoid-rich Indian medicinal plant, Pongamia pinnata (L.) Pierre. Methanolic extract of its seeds inhibits CYP1A1 in CYP1A1-overexpressing normal human HEK293 cells, with IC 50 of 0.6 µg/mL. Its secondary metabolites, the furanoflavonoids pongapin/ lanceolatin B, inhibit CYP1A1 with IC 50 of 20 nM. Although the furanochalcone pongamol inhibits CYP1A1 with IC 50 of only 4.4 µM, a semisynthetic pyrazole-derivative P5b, has ~10-fold improved potency (IC 50 , 0.49 M). Pongapin/ lanceolatin B and the methanolic extract of P. pinnata seeds protect CYP1A1-overexpressing HEK293 cells from B[a]P-mediated toxicity. Remarkably, they also block the cell cycle of CYP1A1-overexpressing MCF-7 breast cancer cells, at the G 0-G 1 phase, repress cyclin D1 levels and induce cellular-senescence. Molecular modeling studies demonstrate the interaction pattern of pongapin/lanceolatin B with CYP1A1. The results strongly indicate the potential of methanolic seed-extract and pongapin/lanceolatin B for further development as cancer chemopreventive agents.
CYP1A1 is thought to mediate carcinogenesis in oral, lung and epithelial cancers. In order to ide... more CYP1A1 is thought to mediate carcinogenesis in oral, lung and epithelial cancers. In order to identify a CYP1A1 inhibitor from an edible plant, 394 natural products in the IIIM's natural product repository were screened, at 10 μM concentration, using CYP1A1-Sacchrosomes™ (i.e. microsomal enzyme isolated from recombinant baker's yeast). Twenty-seven natural products were identified that inhibited 40-97% of CYP1A1's 7-ethoxyresorufin-O-deethylase activity. The IC 50 values of the 'hits', belonging to different chemical scaffolds, were determined. Their selectivity was studied against a panel of 8 CYP-Sacchrosomes™. In order to assess cellular efficacy, the 'hits' were screened for their capability to inhibit CYP enzymes expressed within live recombinant human embryonic kidney (HEK293) cells from plasmids encoding specific CYP genes (1A2, 1B1, 2C9, 2C19, 2D6, 3A4). Isopimpinellin (IN-475; IC 50 , 20 nM) and karanjin (IN-195; IC 50 , 30 nM) showed the most potent inhibition of CYP1A1 in human cells. Isopimpinellin is found in celery, parsnip, fruits and in the rind and pulp of limes whereas different parts of the Indian beech tree, which contain karanjin, have been used in traditional medicine. Both isopimpinellin and karanjin negate the cellular toxicity of CYP1A1-mediated benzo[a]pyrene. Molecular docking and molecular dynamic simulations with CYP isoforms rationalize the observed trends in the potency and selectivity of isopimpinellin and karanjin.
Renal cell carcinoma (RCC) is the most difficult-to-treat form of kidney cancer with a median 5-y... more Renal cell carcinoma (RCC) is the most difficult-to-treat form of kidney cancer with a median 5-year survival of 10% under metastatic setting. In RCC, although cytoreductive nephrectomy is common, approximately 20-30% of patients will develop recurrent cancer after surgery, which highlights the need for an effective therapy. Rho-GTPases viz, Rac-1 and Cdc42 are the central regulators of cancer cell migration and invasion and thus metastasis in multiple cancer types. Hence, we elucidated the role of Ketorolac, a modulator Rho-GTPases against RCC through potentiation of tumor suppressor Par-4. The effect of Ketorolac alone and in combination on proliferation, apoptosis, cellcycle progression, migration, tumor inhibition and their related markers were studied. Moreover, Ketorolac's impact on metastasis by influencing Rac-1/HIF-1α/DDX3/β-catenin signalling was studied with respect to its ability to modulate the expression of tumor suppressor Par-4, and this mechanism was confirmed by siRNA knockdown studies. Ketorolac induced cytotoxicity in a panel of renal cells including patient derived tumor cells with IC 50 2.8 to 9.02 mM and 0.28 to 3.8 mM in monolayer and anchorage independent clonogenic assays respectively. Ketorolac caused significant down regulation of proliferation (Ki-67, Cyclin D1, pRB and DDX3), migration/invasion (Rac-1, Cdc42, and Tiam1), and angiogenesis (HIF-1α and VEGF) markers as studied by gene and protein expression. Moreover, it caused a significant upregulation of tumor suppressor Par-4 known to be downregulated in RCC. This mechanism was further confirmed by using siRNA knockdown studies where we could demonstrate a negative relation between the expression of Par-4 and Rac-1/Cdc42. Importantly, Ketorolac alone and in combination with Sunitinib showed tumor growth inhibition (TGI) of 73% and 86% respectively in xenograft model. This anti-tumor activity was further corroborated by down regulation of Rac-1/ Cdc42/HIF-1α/DDX3/β-catenin signalling. This is the first report which implicates the role of Ketorolac against RCC by acting as a small molecule secretagogue causing upregulation of Par-4 in autocrine and paracrine manner. Consequently, these findings suggest that Par-4 can serve as a valuable therapeutic target and a prognostic marker for the treatment of RCC.
Glioblastoma (GBM) is an aggressive form of brain tumor with a median survival of approximately 1... more Glioblastoma (GBM) is an aggressive form of brain tumor with a median survival of approximately 12 months. With no new drugs in the last few decades and limited success in clinics for known therapies, drug repurposing is an attractive choice for its treatment. Here, we examined the efficacy of pyronaridine (PYR), an anti-malarial drug in GBM cells. PYR induced anti-proliferative activity in GBM cells with IC50 ranging from 1.16 to 6.82 µM. Synergistic activity was observed when PYR was combined with Doxorubicin and Ritonavir. Mechanistically, PYR triggered mitochondrial membrane depolarization and enhanced the ROS levels causing caspase-3 mediated apoptosis. PYR significantly decreased markers associated with proliferation, EMT, hypoxia, and stemness and upregulated the expression of E-cadherin. Interestingly, PYR induced the expression of intracellular as well as secretory Par-4, a tumor suppressor in GBM cells, which was confirmed using siRNA. Notably, Par-4 levels in plasma sampl...
A novel inhibitor of hypoxia-inducible factor-1a P3155 also modulates PI3K pathway and inhibits g... more A novel inhibitor of hypoxia-inducible factor-1a P3155 also modulates PI3K pathway and inhibits growth of prostate cancer cells
This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted P... more This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. P7170, a novel inhibitor of mTORC1/mTORC2 and activin receptor-like Kinase 1
La presente invention concerne une combinaison pharmaceutique comprenant deux agents antineoplasi... more La presente invention concerne une combinaison pharmaceutique comprenant deux agents antineoplasiques cytotoxiques, la gemcitabine et le carboplatine, et au moins un inhibiteur de kinase dependante de cycline (CDK); ladite combinaison presentant des effets synergiques lorsqu'elle est utilisee dans le traitement du cancer du sein, en particulier du cancer du sein triple negatif. L'invention concerne egalement une methode de traitement du cancer du sein a l'aide d'une quantite therapeutiquement efficace de ladite combinaison.
La presente invention concerne une combinaison pharmaceutique comprenant un inhibiteur de PI3K/mT... more La presente invention concerne une combinaison pharmaceutique comprenant un inhibiteur de PI3K/mTOR selectionne parmi des composes de formule (I) (tels que decrits dans la demande) ou des sels pharmaceutiquement acceptables ou des solvants de ceux-ci ; et au moins un agent anti-proliferatif; des compositions pharmaceutiques contenant lesdits composes de formule (I) et au moins un agent anti-proliferatif; et l'utilisation de ladite combinaison dans le traitement de maladies ou troubles anti-proliferatifs.
Glioblastoma multiforme (GBM) is a commonly diagnosed primary brain tumor with median survival of... more Glioblastoma multiforme (GBM) is a commonly diagnosed primary brain tumor with median survival of about one year. GBM is highly aggressive malignancy with limited treatment options. The standard of care for GBM is Temozolomide (TMZ), given as first-line therapy, however, 90% of recurrent gliomas acquire TMZ resistance. Treatment to GBM remains greatest challenge in the management of cancer patients worldwide due to inherent tumor heterogeneity and drug resistant nature leading to high unmet medical need. New drug development is challenging and takes an enormous amount of time, money and effort. Drug repurposing, the application of an existing therapeutic to a new disease indication holds the promise of rapid clinical impact at a lower cost than de novo drug development. This study reports the anti-cancer efficacy of Pyronaridine (PYR), a benzonaphthyridine derivative, used as an agent for the treatment of malaria. Anti-malarial action of PYR therapy is known to target haematin formation and inhibit activity of P. falciparum DNA topoisomerase II. PYR was earlier reported to show in vitro anticancer activity in combination with other drugs in breast and haematological cancer. However, in this study we report its anticancer activity in GBM with novel mechanism of action selective against GBM resistant cells. In our studies, anticancer activity of PYR was confirmed in panel of cancer cell lines comprising of kidney, pancreatic, ovarian, glioma and bladder cancer (IC50: 0.907 - 11 µM). Interestingly, PYR demonstrated dose dependant anti-proliferative and pro-apoptotic activity in both TMZ sensitive and TP53 and PTEN mutated resistant GBM cells (IC50: 1.82-4.2 µM). The 3D culture multicellular spheroid model (MCS) is important and relevant as it is predictive of in vivo antitumor efficacy, PYR inhibited MCS developed from TMZ resistant cells. Additionally, PYR also showed potent activity against glioma stem cell line and primary culture derived from high grade glioma. Cell cycle analysis indicated G1 and G2/M phase arrest and increased sub G0 population when GBM cells were treated with PYR. While deciphering the mechanism of action of PYR, we astoundingly invented a novel role for PYR as an inducer of Prostate Apoptosis Response-4 (PAR-4), a pro-apoptotic tumor suppressor protein. The expression of PAR-4 is known to be downregulated in most tumors including GBM. Here, we demonstrate that PYR modulates microenvironment by inducing PAR-4 in normal fibroblast cells in a dose dependent manner. The conditioned medium from these cells induced profound cytotoxicity against cancer cells. PYR is relatively safe to use in humans and our data clearly indicates its anti-cancer potential and hence is a promising therapeutic candidate. The detailed studies focusing on its novel mechanism of action alone and in combination in drug resistant models will be discussed. Citation Format: Jeevan D. Ghosalkar, Vinay R. Sonawane, Siddhika R. Raut, Mariamma Thekkumpurath, Radha Pujari, Padma Shastri, Geena Malhotra, Kalpana S. Joshi. Anti-malarial drug Pyronaridine modulates microenvironment leading to potent anti-cancer activity against GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 251.
Cytochrome P450 family 1 (CYP1) enzymes catalyze the metabolic activation of environmental procar... more Cytochrome P450 family 1 (CYP1) enzymes catalyze the metabolic activation of environmental procarcinogens such as benzo[a]pyrene, B[a]P, into carcinogens, which initiate the process of carcinogenesis. Thus, stopping the metabolic activation of procarcinogens can possibly prevent the onset of cancer. Several natural products have been reported to show unique ability in inhibiting CYP1 enzymes. We found that khellin, a naturally occurring furanochromone from Ammi visnaga, inhibits CYP1A1 enzyme with an IC50 value of 4.02 µM in CYP1A1-overexpressing human HEK293 suspension cells. To further explore this natural product for discovery of more potent and selective CYP1A1 inhibitors, two sets of semisynthetic derivatives were prepared. Treatment of khellin with alkali results in opening of a pyrone ring, yielding khellinone (2). Claisen-Schmidt condensation of khellinone (2) with various aldehydes in presence of potassium hydroxide, at room temperature, provide a series of furanochalcones 3a-v (khellinochalcones). Treatment of khellinone (2) with aryl aldehydes in presence of piperidine, under reflux, affords the flavanone series of compounds 4a-p (khellinoflavanones). The khellinoflavanone 4l potently inhibited CYP1A1 with IC50 value of 140 nM in live cells, with 170-fold selectivity over CYP1B1 (IC50 for CYP1B1 = 23.8 µM). Compound 4l at 3 x IC50 concentration for inhibition of CYP1A1, completely protected HEK293 cells from CYP1A1mediated B[a]P toxicity. Lung cancer cells, A549 (p53 +) and Calu-1 (p53-null), blocked in growth at the S-phase by B[a]P were restored into the cell cycle by compound 4l. The results presented herein strongly indicate the potential of these khellin derivatives for further development as cancer chemopreventive agents.
&NA; Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yea... more &NA; Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yeast cells, are extensively used for drug metabolism studies. However, they may not always portray how a developmental drug would behave in human cells with intact intracellular transport mechanisms. This study emphasizes the usefulness of human HEK293 kidney cells, grown in ‘suspension’ for expression of CYPs, in finding potent CYP1A1/CYP1B1 inhibitors, as possible anticancer agents. With live cell‐based assays, quinazolinones 9i/9b were found to be selective CYP1A1/CYP1B1 inhibitors with IC50 values of 30/21 nM, and > 150‐fold selectivity over CYP2/3 enzymes, whereas they were far less active using commercially‐available CYP1A1/CYP1B1 microsomal enzymes (IC50, >10/1.3–1.7 &mgr;M). Compound 9i prevented CYP1A1‐mediated benzo[a]pyrene‐toxicity in normal fibroblasts whereas 9b completely reversed cisplatin resistance in PC‐3/prostate, COR‐L23/lung, MIAPaCa‐2/pancreatic and LS174T/colon cancer cells, underlining the human‐cell‐assays' potential. Our results indicate that the most potent CYP1A1/CYP1B1 inhibitors would not have been identified if one had relied merely on microsomal enzymes.
, a quinazolinone-based dual inhibitor of Cdk4 and tubulin polymerization, identified via ligand-... more , a quinazolinone-based dual inhibitor of Cdk4 and tubulin polymerization, identified via ligand-based virtual screening, for efficient anticancer therapy,
Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is indu... more Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is induced (a) by polycyclic aromatic hydrocarbons (PAHs)which can be metabolized by CYP1A1 to carcinogens, and (b) in majority of breast cancers. Hence, phytochemicals or dietary flavonoids, if identified as CYP1A1 inhibitors, may help in preventing PAH-mediated carcinogenesis and breast cancer. Herein, we have investigated the cancer chemopreventive potential of a flavonoid-rich Indian medicinal plant, Pongamia pinnata (L.) Pierre. Methanolic extract of its seeds inhibits CYP1A1 in CYP1A1-overexpressing normal human HEK293 cells, with IC 50 of 0.6 µg/mL. Its secondary metabolites, the furanoflavonoids pongapin/ lanceolatin B, inhibit CYP1A1 with IC 50 of 20 nM. Although the furanochalcone pongamol inhibits CYP1A1 with IC 50 of only 4.4 µM, a semisynthetic pyrazole-derivative P5b, has ~10-fold improved potency (IC 50 , 0.49 M). Pongapin/ lanceolatin B and the methanolic extract of P. pinnata seeds protect CYP1A1-overexpressing HEK293 cells from B[a]P-mediated toxicity. Remarkably, they also block the cell cycle of CYP1A1-overexpressing MCF-7 breast cancer cells, at the G 0-G 1 phase, repress cyclin D1 levels and induce cellular-senescence. Molecular modeling studies demonstrate the interaction pattern of pongapin/lanceolatin B with CYP1A1. The results strongly indicate the potential of methanolic seed-extract and pongapin/lanceolatin B for further development as cancer chemopreventive agents.
S2 CONTENTS S1. Experimental procedures S2. CYP1A1 screening results in Sacchrosomes TM S3. HPLC ... more S2 CONTENTS S1. Experimental procedures S2. CYP1A1 screening results in Sacchrosomes TM S3. HPLC purity of selected in-house natural product library hits. S4. Karanjin is a competitive inhibitor of CYP1A1 S5. MD simulation and interactions of isopimpinellin (IN-475) with CYP1A1 S6. Interaction of isopimpinellin (IN-475) with CYP1, CYP2 and CYP3 isoforms S7. Interactions of karanjin (IN-195) with CYP2 and CYP3 isoforms S8. NMR, HPLC and HRMS data scans of IN-195 and IN-475
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