RESUMO
Our screen for tubulin-binding small molecules that do not depolymerize bulk cellular microtubules, but based upon structural features of well known microtubule-depolymerizing colchicine and podophyllotoxin, revealed tubulin binding anti-cancer property of noscapine (Ye et al. in Proc Natl Acad Sci USA 95:2280-2286, 1998). Guided by molecular modelling calculations and structure-activity relationships we conjugated at C9 of noscapine, a folate group-a ligand for cellular folate receptor alpha (FRα). FRα is over-expressed on some solid tumours such as ovarian epithelial cancers. Molecular docking experiments predicted that a folate conjugated noscapine (Targetin) accommodated well inside the binding cavity (docking score -11.295 kcal/mol) at the interface between α- and ß-tubulin. The bulky folate moiety of Targetin is extended toward lumen of microtubules. The binding free energy (ΔG (bind)) computed based on molecular mechanics energy minimization was -221.01 kcal/mol that revealed favourable interaction of Targetin with the receptor. Chemical synthesis, tubulin-binding experiments, and anti-cancer activity in vitro corroborate fully well with the molecular modelling experiments. Targetin binds tubulin with a dissociation constant (K (d) value) of 149 ± 3.0 µM and decreases the transition frequencies between growth and shortening phases of microtubule assembly dynamics at concentrations that do not alter the total polymer mass. Cancer cells in general were more sensitive to Targetin compared with the founding compound noscapine (IC(50) in the range of 15-40 µM). Quite strikingly, ovarian cancer cells (SKOV3 and A2780), known to overexpress FRα, were much more sensitive to targetin (IC(50) in the range of 0.3-1.5 µM).
Assuntos
Anticarcinógenos/química , Anticarcinógenos/farmacologia , Ácido Fólico/química , Noscapina/química , Noscapina/farmacologia , Tubulina (Proteína)/metabolismo , Anticarcinógenos/síntese química , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Receptor 1 de Folato/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Microtúbulos/química , Microtúbulos/efeitos dos fármacos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Noscapina/síntese química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/efeitos dos fármacosRESUMO
Noscapine and its derivatives are important microtubule-interfering agents shown to have potent anti-tumor activity. The binding free energies (ΔG (bind)) of noscapinoids computed using linear interaction energy (LIE) method with a surface generalized Born (SGB) continuum solvation model were in agreement with the experimental ΔG (bind) with average root mean square error of 0.082 kcal/mol. This LIE-SGB model guided us in designing a novel derivative of noscapine, amino-noscapine [(S)-3-((R)-9-amino-4-methoxy-6-methyl-5,6,7,8-tetrahydro [1, 3] dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxy isobenzo-furan-1(3H)-one] that has higher tubulin binding activity (predicted ΔG (bind) = -6.438 kcal/mol and experimental ΔG (bind) = -6.628 kcal/mol) than noscapine, but does not significantly change the total extent of the tubulin subunit/polymer ratio. The modes of interaction of amino-noscapine with the binding pocket of tubulin involved three hydrogen bonds and are distinct compared to noscapine which involved only one hydrogen bond. Also the patterns of non-bonded interactions are albeit different between both the lignads. The 'blind docking' approach (docking of ligand with different binding sites of a protein and their evaluations) as well as the reasonable accuracy of calculating ΔG (bind) using LIE-SGB model constitutes the first evidence that this class of compounds binds to tubulin at a site overlapping with colchicine-binding site or close to it. Our results revealed that amino-noscapine has better anti-tumor activity than noscapine.
Assuntos
Antineoplásicos/química , Colchicina/química , Noscapina/análogos & derivados , Noscapina/química , Tubulina (Proteína)/química , Antitussígenos , Sítios de Ligação , Desenho de Fármacos , Ligação de Hidrogênio , Ligantes , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Químicos , Estrutura Molecular , Noscapina/síntese química , Polimerização , Ligação Proteica , Termodinâmica , Tubulina (Proteína)/metabolismoRESUMO
We studied in silico docking of noscapine onto tubulin, combined with calculations of surface charge, pi-pi, van der Waals, and hydrogen bonding interactions, to rationally design a new compound, EM015. This tubulin-binding semisynthetic compound is a selective and potent anti-breast cancer agent and displays a 20-fold lower IC(50) against many tumor cells compared with our founding compound, (S)-6,7-dimethoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro[1,3]-dioxolo-[4,5-g]isoquinolin-5-yl)isobenzo-furan-1(3H)-one (noscapine). Furthermore, EM015 is also effective against a variety of drug-resistant cells. Surprisingly, the cell cycle profile of nontumorigenic normal cells is not affected. Many antimicrotubule cancer drugs in clinic today, particularly taxanes and Vincas, face challenges including frequent visits to the hospital for prolonged i.v. infusions, toxicities, and tumor recurrences due to drug resistance. EM015, on the other hand, is orally available, regresses breast tumor xenografts in nude mice models, and increases longevity. Furthermore, we have failed to observe any detectable toxicity in tissues, such as liver, kidney, spleen, lung, heart, and brain, as well as neurons, which are common targets of antimicrotubule drug therapy. Thus, EM015 has a great promise in the clinic.
Assuntos
Antineoplásicos , Neoplasias da Mama/tratamento farmacológico , Microtúbulos/efeitos dos fármacos , Noscapina/análogos & derivados , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Sistema Hematopoético/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Cinética , Camundongos , Camundongos Nus , Modelos Moleculares , Noscapina/farmacologia , Fuso Acromático/efeitos dos fármacos , Tubulina (Proteína)/metabolismoRESUMO
We have previously identified the naturally occurring non-toxic antitussive phthalideisoquinoline alkaloid, noscapine as a tubulin-binding agent that arrests mitosis and induces apoptosis. Here we present high-yield efficient synthetic methods and an evaluation of anticancer activity of halogenated noscapine analogs. Our results show that all analogs display higher tubulin-binding activity than noscapine and inhibit proliferation of human cancer cells (MCF-7, MDA-MB-231 and CEM). Surprisingly, the bromo-analog is approximately 40-fold more potent than noscapine in inhibiting cellular proliferation of MCF-7 cells. The ability of these analogs to inhibit cellular proliferation is mediated by cell cycle arrest at the G2/M phase, in that all analogs except 9-iodonoscapine, caused selective mitotic arrest with a higher efficiency than noscapine followed by apoptotic cell death as shown by immunofluorescence and quantitative FACS analyses. Furthermore, our results reveal the appearance of numerous fragmented nuclei as evidenced by DAPI staining. Thus, our data indicate a great potential of these compounds for studying microtubule-mediated processes and as chemotherapeutic agents for the management of human cancers.
Assuntos
Apoptose/efeitos dos fármacos , Halogênios/química , Mitose/efeitos dos fármacos , Noscapina/síntese química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Feminino , Fase G2/efeitos dos fármacos , Humanos , Masculino , Noscapina/análogos & derivados , Noscapina/farmacologia , Fuso Acromático/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologiaRESUMO
Several DNA- and microtubule-binding agents are used to manage hematologic malignancies in the clinic. However, drug resistance has been a challenge, perhaps due to a few surviving cancer stem cells. Toxicity is another major impediment to successful chemotherapy, leading to an impoverished quality of life. Here, we show that a semisynthetic nontoxic tubulin-binding agent, 9-bromonoscapine (EM011), effectively inhibits growth and regresses multidrug resistance-associated protein (MRP)-overexpressing teniposide-resistant T-cell lymphoma xenografts and prolongs longevity. As expected, teniposide treatment failed to regress teniposide-resistant xenografts, rather, treated mice suffered tremendous body weight loss. Mechanistically, EM011 displays significant antiproliferative activity, perturbs cell cycle progression by arresting mitosis, and induces apoptosis in teniposide-resistant lymphoblastoid T cells both in vitro and in vivo. EM011-induced apoptosis has a mitochondrially-mediated component, which was attenuated by pretreatment with cyclosporin A. We also observed alterations of apoptosis-regulatory molecules such as inactivation of Bcl2, translocation of BAX to the mitochondrial membrane, cytochrome c release, and activation of downstream apoptotic signaling. EM011 caused DNA degradation as evident by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling staining of the increased concentration of 3'-DNA ends. Furthermore, the apoptotic induction was caspase dependent as shown by cleavage of the caspase substrate, poly(ADP)ribose polymerase. In addition, EM011 treatment caused a suppression of natural survival pathways such as the phosphatidylinositol-3'-kinase/Akt signaling. These preclinical findings suggest that EM011 is an excellent candidate for clinical evaluation.
Assuntos
Apoptose , Dioxóis/farmacologia , Regulação Neoplásica da Expressão Gênica , Isoquinolinas/farmacologia , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Teniposídeo/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Linfoma/patologia , Camundongos , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação ProteicaRESUMO
We present here a novel semi-synthetic cyclic ether fluorinated noscapine analog (CEFNA) that shows potent antiproliferative and anticancer activity in both hormone-responsive (MCF-7) and hormone non-responsive (MDA-MB-231) breast cancer cells. Interestingly, it is also effective against MCF-7/Adr, an adriamycin-resistant variant of MCF-7 cells. Immunofluorescence experiments showed numerous micronuclei, indicative of apoptotic cell death triggered by this novel analog. Mechanistically, CEFNA exerts a strong antimitotic effect as revealed by cell-cycle studies that show a dose-dependent increase in G2/M population preceding a rising sub-G1 population, suggesting apoptosis.
Assuntos
Antitussígenos/síntese química , Antitussígenos/farmacologia , Noscapina/síntese química , Noscapina/farmacologia , Antibióticos Antineoplásicos/efeitos adversos , Antitussígenos/química , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Doxorrubicina/efeitos adversos , Resistencia a Medicamentos Antineoplásicos , Citometria de Fluxo , Imunofluorescência , Humanos , Noscapina/química , Fuso Acromático/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Infectious diseases have haunted human populations for millennia. Still we are struggling with this scourge because of multiple complexities. These include the emergence of new pathogenic species due to mutations owing to the naturally ticking evolutionary clock. In addition, under the selective pressure of existing powerful antibiotics, which have been helpful in effectively managing many infectious diseases for a long time, resistant species arise quite commonly. Therefore, there is always an urgent need to invent new strategies to fight new strains of these pathogens. We review here some important patents that have been licensed for drug development pertaining to protease and kinase inhibitors. Instead of being comprehensive, we have been selective in our choices. We apologize to those whose patents we could not cite. Our goal is to inform public at large of the new inventions in the pipeline and their status toward development of these technologies as drugs in the clinic.
Assuntos
Anti-Infecciosos , Inibidores Enzimáticos/farmacologia , Infecções/tratamento farmacológico , Fosfotransferases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Infecções/microbiologia , Patentes como Assunto , Inibidores de Proteases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinases raf/antagonistas & inibidoresRESUMO
We have shown previously that an antitussive plant alkaloid, noscapine, binds tubulin, displays anticancer activity, and has a safe pharmacological profile in humans. Structure-function analyses pointed to a proton at position-9 of the isoquinoline ring that can be modified without compromising tubulin binding activity. Thus, many noscapine analogs with different functional moieties at position-9 were synthesized. Those analogs that kill human cancer cells resistant to other antimicrotubule agents, vincas and taxanes, were screened. Here, we present one such analog, 9-nitro-noscapine (9-nitro-nos), which binds tubulin and induces apoptosis selectively in tumor cells (ovarian and T-cell lymphoma) resistant to paclitaxel, vinblastine, and teniposide. 9-Nitro-nos treatment at doses as high as 100 microM did not affect the cell cycle profile of normal human fibroblasts. This selectivity of 9-nitro-nos for cancer cells represents a unique edge over the other available antimitotics. 9-Nitro-nos perturbs the progression of cell cycle by mitotic arrest, followed by apoptotic cell death associated with increased caspase-3 activation and appearance of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells. Thus, we conclude that 9-nitro-nos has great potential to be a novel therapeutic agent for ovarian and T-cell lymphoma cancers, even those that have become drug-resistant to currently available chemotherapeutic drugs.
Assuntos
Antimitóticos/uso terapêutico , Apoptose , Resistencia a Medicamentos Antineoplásicos , Linfoma de Células T/tratamento farmacológico , Noscapina/análogos & derivados , Neoplasias Ovarianas/tratamento farmacológico , Antimitóticos/síntese química , Antimitóticos/farmacologia , Antineoplásicos/uso terapêutico , Caspase 3 , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Fragmentação do DNA , Ativação Enzimática , Feminino , Humanos , Noscapina/síntese química , Noscapina/química , Noscapina/farmacologia , Noscapina/uso terapêutico , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismoRESUMO
We have shown previously that EM011, a synthetic compound, binds tubulin with a higher affinity than the founding compound, noscapine, without changing total microtubule polymer mass. Now we show that EM011 is potently effective against vinblastine-resistant human lymphoblastoid line CEM/VLB100 and its parental vinblastine-sensitive line CEM. The cytotoxicity is mediated by cell cycle arrest at G2/M phase and subsequent apoptosis, as indicated by altered plasma membrane asymmetry, loss of mitochondrial transmembrane potential, activation of caspase-3, and increased DNA fragmentation. Furthermore, oral EM011 treatment of nude mice bearing human lymphoma xenografts results in pronounced tumor regression by triggering apoptosis and significantly lengthens the survival time of mice. EM011 treatment does not have obvious side effects in tissues with frequently dividing cells, such as the spleen and duodenum. In addition, EM011 does not show any toxicity in the liver, lung, heart, brain, and sciatic nerve. More importantly, EM011 does not affect hematopoiesis as determined by complete blood count profiles. These findings suggest that EM011 may be a safe and effective chemotherapeutic agent for oral treatment of drug-resistant human lymphomas.