RESUMO
Despite more than three decades of intense effort, no anti-Ras therapies have reached clinical application. Contributing to this failure has been an underestimation of Ras complexity and a dearth of structural information. In this regard, recent studies have revealed the highly dynamic character of the Ras surface and the existence of transient pockets suitable for small-molecule binding, opening up new possibilities for the development of Ras modulators. Herein, a novel Ras inhibitor (compound 12) is described that selectively impairs mutated Ras activity in a reversible manner without significantly affecting wild-type Ras, reduces the Ras-guanosine triphosphate (GTP) levels, inhibits the activation of the mitogen-activated protein kinase (MAPK) pathway, and exhibits remarkable cytotoxic activity in Ras-driven cellular models. The use of molecular dynamics simulations and NMR spectroscopy experiments has enabled the molecular bases responsible for the interactions between compound 12 and Ras protein to be explored. The new Ras inhibitor binds partially to the GTP-binding region and extends into the adjacent hydrophobic pocket delimited by switchâ II. Hence, Ras inhibitor 12 could represent a new compound for the development of more efficacious drugs to target Ras-driven cancers; a currently unmet clinical need.
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Determination of the targets of a compound remains an essential aspect in drug discovery. A complete understanding of all binding interactions is critical to recognize in advance both therapeutic effects and undesired consequences. However, the complete polypharmacology of many drugs currently in clinical development is still unknown, especially in the case of Gâ protein-coupled receptor (GPCR) ligands. In this work we have developed a chemoproteomic platform based on the use of chemical probes to explore the target profile of a compound in biological systems. As proof of concept, this methodology has been applied to selected ligands of the therapeutically relevant serotonin 5-HT1A and 5-HT6 receptors, and we have identified and validated some of their off-targets. This approach could be extended to other drugs of interest to study the targeted proteome in disease-relevant systems.
Assuntos
Receptor 5-HT1A de Serotonina/química , Receptores Acoplados a Proteínas G/química , Receptores de Serotonina/química , Desenho de Fármacos , Descoberta de Drogas , Humanos , Ligantes , Receptor 5-HT1A de Serotonina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Serotonina/metabolismoRESUMO
The human microbiota plays an important role in human health and disease, through the secretion of metabolites that regulate key biological functions. We propose that microbiota metabolites represent an unexplored chemical space of small drug-like molecules in the search of new hits for drug discovery. Here, we describe the generation of a set of complex chemotypes inspired on selected microbiota metabolites, which have been synthesized using asymmetric organocatalytic reactions. Following a primary screening in CSC models, we identified the novel compound UCM-13369 (4b) whose cytotoxicity was mediated by NPM1. This protein is one of the most frequent mutations of AML, and NPM1-mutated AML is recognized by the WHO as a distinct hematopoietic malignancy. UCM-13369 inhibits NPM1 expression, downregulates the pathway associated with mutant NPM1 C+, and specifically recognizes the C-end DNA-binding domain of NPM1 C+, avoiding the nucleus-cytoplasm translocation involved in the AML tumorological process. The new NPM1 inhibitor triggers apoptosis in AML cell lines and primary cells from AML patients and reduces tumor infiltration in a mouse model of AML with NPM1 C+ mutation. The disclosed phenotype-guided discovery of UCM-13369, a novel small molecule inspired on microbiota metabolites, confirms that CSC death induced by NPM1 inhibition represents a promising therapeutic opportunity for NPM1-mutated AML, a high-mortality disease.
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Cannabidiol (CBD), the main non-psychotomimetic component of the plant Cannabis sativa, exerts therapeutically promising effects on human mental health such as inhibition of psychosis, anxiety and depression. However, the mechanistic bases of CBD action are unclear. Here we investigate the potential involvement of hippocampal neurogenesis in the anxiolytic effect of CBD in mice subjected to 14 d chronic unpredictable stress (CUS). Repeated administration of CBD (30 mg/kg i.p., 2 h after each daily stressor) increased hippocampal progenitor proliferation and neurogenesis in wild-type mice. Ganciclovir administration to GFAP-thymidine kinase (GFAP-TK) transgenic mice, which express thymidine kinase in adult neural progenitor cells, abrogated CBD-induced hippocampal neurogenesis. CBD administration prevented the anxiogenic effect of CUS in wild type but not in GFAP-TK mice as evidenced in the novelty suppressed feeding test and the elevated plus maze. This anxiolytic effect of CBD involved the participation of the CB1 cannabinoid receptor, as CBD administration increased hippocampal anandamide levels and administration of the CB1-selective antagonist AM251 prevented CBD actions. Studies conducted with hippocampal progenitor cells in culture showed that CBD promotes progenitor proliferation and cell cycle progression and mimics the proliferative effect of CB1 and CB2 cannabinoid receptor activation. Moreover, antagonists of these two receptors or endocannabinoid depletion by fatty acid amide hydrolase overexpression prevented CBD-induced cell proliferation. These findings support that the anxiolytic effect of chronic CBD administration in stressed mice depends on its proneurogenic action in the adult hippocampus by facilitating endocannabinoid-mediated signalling.
Assuntos
Ansiolíticos/uso terapêutico , Canabidiol/uso terapêutico , Hipocampo/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Estresse Psicológico/tratamento farmacológico , Animais , Ansiolíticos/farmacologia , Bromodesoxiuridina/metabolismo , Canfanos/farmacologia , Canabidiol/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Comportamento Alimentar/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurogênese/fisiologia , Piperidinas/farmacologia , Pirazóis/farmacologia , Rimonabanto , Estresse Psicológico/patologia , Timidina Quinase/metabolismoRESUMO
The global spread of bacterial antimicrobial resistance is associated to millions of deaths from bacterial infections per year, many of which were previously treatable. This, combined with slow antibiotic deployment, has created an urgent need for developing new antibiotics. A still clinically unexploited mode of action consists in suppressing bacterial cell division. FtsZ, an assembling GTPase, is the key protein organizing division in most bacteria and an attractive target for antibiotic discovery. Nevertheless, developing effective antibacterial inhibitors targeting FtsZ has proven challenging. Here we review our decade-long multidisciplinary research on small molecule inhibitors of bacterial division, in the context of global efforts to discover FtsZ-targeting antibiotics. We focus on methods to characterize synthetic inhibitors that either replace bound GTP from the FtsZ nucleotide binding pocket conserved across diverse bacteria or selectively bind into the allosteric site at the interdomain cleft of FtsZ from Bacillus subtilis and the pathogen Staphylococcus aureus. These approaches include phenotype screening combined with fluorescence polarization screens for ligands binding into each site, followed by detailed cytological profiling, and biochemical and structural studies. The results are analyzed to design an optimized workflow to identify effective FtsZ inhibitors, and new approaches for the discovery of FtsZ-targeting antibiotics are discussed.
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Aging is considered the main risk factor for many chronic diseases that frequently appear at advanced ages. However, the inevitability of this process is being questioned by recent research that suggests that senescent cells have specific features that differentiate them from younger cells and that removal of these cells ameliorates senescent phenotype and associated diseases. This opens the door to the design of tailored therapeutic interventions aimed at reducing and delaying the impact of senescence in life, that is, extending healthspan and treating aging as another chronic disease. Although these ideas are still far from reaching the bedside, it is conceivable that they will revolutionize the way we understand aging in the next decades. In this review, we analyze the main and well-validated cellular pathways and targets related to senescence as well as their implication in aging-associated diseases. In addition, the most relevant small molecules with senotherapeutic potential, with a special emphasis on their mechanism of action, ongoing clinical trials, and potential limitations, are discussed. Finally, a brief overview of alternative strategies that go beyond the small molecule field, together with our perspectives for the future of the field, is provided.
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Spinal cord injuries (SCIs) irreversibly disrupt spinal connectivity, leading to permanent neurological disabilities. Current medical treatments for reducing the secondary damage that follows the initial injury are limited to surgical decompression and anti-inflammatory drugs, so there is a pressing need for new therapeutic strategies. Inhibition of the type 2 lysophosphatidic acid receptor (LPA2) has recently emerged as a new potential pharmacological approach to decrease SCI-associated damage. Toward validating this receptor as a target in SCI, we have developed a new series of LPA2 antagonists, among which compound 54 (UCM-14216) stands out as a potent and selective LPA2 receptor antagonist (Emax = 90%, IC50 = 1.9 µM, KD = 1.3 nM; inactive at LPA1,3-6 receptors). This compound shows efficacy in an in vivo mouse model of SCI in an LPA2-dependent manner, confirming the potential of LPA2 inhibition for providing a new alternative for treating SCI.
Assuntos
Receptores de Ácidos Lisofosfatídicos , Traumatismos da Medula Espinal , Animais , Camundongos , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Medula Espinal , Traumatismos da Medula Espinal/tratamento farmacológicoRESUMO
Tolerance development caused by dopamine replacement with l-DOPA and therapeutic drawbacks upon activation of dopaminergic receptors with orthosteric agonists reveal a significant unmet need for safe and effective treatment of Parkinson's disease. In search for selective modulators of the D1 receptor, the screening of a chemical library and subsequent medicinal chemistry program around an identified hit resulted in new synthetic compound 26 [UCM-1306, 2-(fluoromethoxy)-4'-(S-methanesulfonimidoyl)-1,1'-biphenyl] that increases the dopamine maximal effect in a dose-dependent manner in human and mouse D1 receptors, is inactive in the absence of dopamine, modulates dopamine affinity for the receptor, exhibits subtype selectivity, and displays low binding competition with orthosteric ligands. The new allosteric modulator potentiates cocaine-induced locomotion and enhances l-DOPA recovery of decreased locomotor activity in reserpinized mice after oral administration. The behavior of compound 26 supports the interest of a positive allosteric modulator of the D1 receptor as a promising therapeutic approach for Parkinson's disease.
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Cocaína , Doença de Parkinson , Animais , Compostos de Bifenilo , Dopamina/metabolismo , Dopaminérgicos , Agonistas de Dopamina/farmacologia , Humanos , Indazóis , Levodopa , Ligantes , Camundongos , Nitrofuranos , Doença de Parkinson/tratamento farmacológico , Receptores Dopaminérgicos , Receptores de Dopamina D1/agonistasRESUMO
Peptidic agonists of the glucagon-like peptide-1 receptor (GLP-1R) have gained a prominent role in the therapy of type-2 diabetes and are being considered for reducing food intake in obesity. Potential advantages of small molecules acting as positive allosteric modulators (PAMs) of GLP-1R, including oral administration and reduced unwanted effects, could improve the utility of this class of drugs. Here, we describe the discovery of compound 9 (4-{[1-({3-[4-(trifluoromethyl)phenyl]-1,2,4-oxadiazol-5-yl}methyl)piperidin-3-yl]methyl}morpholine, V-0219) that exhibits enhanced efficacy of GLP-1R stimulation, subnanomolar potency in the potentiation of insulin secretion, and no significant off-target activities. The identified GLP-1R PAM shows a remarkable in vivo activity, reducing food intake and improving glucose handling in normal and diabetic rodents. Enantioselective synthesis revealed oral efficacy for (S)-9 in animal models. Compound 9 behavior bolsters the interest of a small-molecule PAM of GLP-1R as a promising therapeutic approach for the increasingly prevalent obesity-associated diabetes.
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Diabetes Mellitus Tipo 2 , Receptor do Peptídeo Semelhante ao Glucagon 1 , Administração Oral , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Obesidade/tratamento farmacológico , Peptídeos/uso terapêuticoRESUMO
Bacterial resistance to antibiotics makes previously manageable infections again disabling and lethal, highlighting the need for new antibacterial strategies. In this regard, inhibition of the bacterial division process by targeting key protein FtsZ has been recognized as an attractive approach for discovering new antibiotics. Binding of small molecules to the cleft between the N-terminal guanosine triphosphate (GTP)-binding and the C-terminal subdomains allosterically impairs the FtsZ function, eventually inhibiting bacterial division. Nonetheless, the lack of appropriate chemical tools to develop a binding screen against this site has hampered the discovery of FtsZ antibacterial inhibitors. Herein, we describe the first competitive binding assay to identify FtsZ allosteric ligands interacting with the interdomain cleft, based on the use of specific high-affinity fluorescent probes. This novel assay, together with phenotypic profiling and X-ray crystallographic insights, enables the identification and characterization of FtsZ inhibitors of bacterial division aiming at the discovery of more effective antibacterials.
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Antibacterianos/química , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Sítio Alostérico , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , Cristalografia por Raios X , Proteínas do Citoesqueleto/antagonistas & inibidores , Polarização de Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ligantes , Testes de Sensibilidade Microbiana , Ligação Proteica , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Relação Estrutura-AtividadeRESUMO
Neuropathic pain (NP) is a complex chronic pain state with a prevalence of almost 10% in the general population. Pharmacological options for NP are limited and weakly effective, so there is a need to develop more efficacious NP attenuating drugs. Activation of the type 1 lysophosphatidic acid (LPA1) receptor is a crucial factor in the initiation of NP. Hence, it is conceivable that a functional antagonism strategy could lead to NP mitigation. Here we describe a new series of LPA1 agonists among which derivative (S)-17 (UCM-05194) stands out as the most potent and selective LPA1 receptor agonist described so far (Emax = 118%, EC50 = 0.24 µM, KD = 19.6 nM; inactive at autotaxin and LPA2-6 receptors). This compound induces characteristic LPA1-mediated cellular effects and prompts the internalization of the receptor leading to its functional inactivation in primary sensory neurons and to an efficacious attenuation of the pain perception in an in vivo model of NP.
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Analgésicos/química , Analgésicos/uso terapêutico , Neuralgia/tratamento farmacológico , Receptores de Ácidos Lisofosfatídicos/agonistas , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Descoberta de Drogas , Feminino , Humanos , Hidrocarbonetos Aromáticos/química , Hidrocarbonetos Aromáticos/uso terapêutico , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neuralgia/metabolismo , Percepção da Dor/efeitos dos fármacos , Ratos Wistar , Receptores de Ácidos Lisofosfatídicos/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismoRESUMO
Metabotropic glutamate type 7 (mGlu7) receptor is a member of the group III family of mGlu receptors. It is widely distributed in the central nervous system (CNS) and is preferentially expressed on presynaptic nerve terminals where it is thought to play a critical role in modulating normal neuronal function and synaptic transmission, making it particularly relevant in neuropharmacology. The lack of small-molecule mGlu7 ligands with adequate potency, selectivity and drug-like properties has resulted in difficulties in the preclinical validation of mGlu7 modulation in disease models. In the last decade, allosteric modulators of mGlu7 receptors have emerged as valuable tools with good potency, selectivity and physicochemical properties to study and unleash the therapeutic potential of mGlu7 receptors. This review focusses on the medicinal chemistry of mGlu7 receptor allosteric ligands discovered since 2008.
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Serotonin 5-HT6 receptor has been proposed as a promising therapeutic target for cognition enhancement though the development of new antagonists is still needed to validate these molecules as a drug class for the treatment of Alzheimer's disease and other pathologies associated with memory deficiency. As part of our efforts to target the 5-HT6 receptor, new benzimidazole-based compounds have been designed and synthesized. Site-directed mutagenesis and homology models show the importance of a halogen bond interaction between a chlorine atom of the new class of 5-HT6 receptor antagonists identified herein and a backbone carbonyl group in transmembrane domain 4. In vitro pharmacological characterization of 5-HT6 receptor antagonist 7 indicates high affinity and selectivity over a panel of receptors including 5-HT2B subtype and hERG channel, which suggests no major cardiac issues. Compound 7 exhibited in vivo procognitive activity (1 mg/kg, ip) in the novel object recognition task as a model of memory deficit.
Assuntos
Cognição/efeitos dos fármacos , Halogênios/química , Receptores de Serotonina/metabolismo , Antagonistas da Serotonina/farmacologia , Animais , Benzimidazóis/síntese química , Benzimidazóis/química , Benzimidazóis/farmacologia , Humanos , Ligantes , Microssomos Hepáticos/metabolismo , Mutagênese Sítio-Dirigida , Ratos , Receptores de Serotonina/química , Antagonistas da Serotonina/síntese química , Antagonistas da Serotonina/química , Homologia Estrutural de Proteína , Análise e Desempenho de TarefasRESUMO
FtsZ is a widely conserved tubulin-like GTPase that directs bacterial cell division and a new target for antibiotic discovery. This protein assembly machine cooperatively polymerizes forming single-stranded filaments, by means of self-switching between inactive and actively associating monomer conformations. The structural switch mechanism was proposed to involve a movement of the C-terminal and N-terminal FtsZ domains, opening a cleft between them, allosterically coupled to the formation of a tight association interface between consecutive subunits along the filament. The effective antibacterial benzamide PC190723 binds into the open interdomain cleft and stabilizes FtsZ filaments, thus impairing correct formation of the FtsZ ring for cell division. We have designed fluorescent analogs of PC190723 to probe the FtsZ structural assembly switch. Among them, nitrobenzoxadiazole probes specifically bind to assembled FtsZ rather than to monomers. Probes with several spacer lengths between the fluorophore and benzamide moieties suggest a binding site extension along the interdomain cleft. These probes label FtsZ rings of live Bacillus subtilis and Staphylococcus aureus, without apparently modifying normal cell morphology and growth, but at high concentrations they induce impaired bacterial division phenotypes typical of benzamide antibacterials. During the FtsZ assembly-disassembly process, the fluorescence anisotropy of the probes changes upon binding and dissociating from FtsZ, thus reporting open and closed FtsZ interdomain clefts. Our results demonstrate the structural mechanism of the FtsZ assembly switch, and suggest that the probes bind into the open clefts in cellular FtsZ polymers preferably to unassembled FtsZ in the bacterial cytosol.
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The 5-HT2CR agonist lorcaserin, clinically approved for the treatment of obesity, causes important side effects mainly related to subtype selectivity. In the search for 5-HT2CR allosteric modulators as safer antiobesity drugs, a chemical library from Vivia Biotech was screened using ExviTech platform. Structural modifications of identified hit VA240 in synthesized analogues 6-41 afforded compound 11 (N-[(1-benzyl-1H-indol-3-yl)methyl]pyridin-3-amine, VA012), which exhibited dose-dependent enhancement of serotonin efficacy, no significant off-target activities, and low binding competition with serotonin or other orthosteric ligands. PAM 11 was very active in feeding inhibition in rodents, an effect that was not related to the activation of 5-HT2AR. A combination of 11 with the SSRI sertraline increased the anorectic effect. Subchronic administration of 11 reduced food intake and body weight gain without causing CNS-related malaise. The behavior of compound 11 identified in this work supports the interest of a serotonin 5-HT2CR PAM as a promising therapeutic approach for obesity.
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Fármacos Antiobesidade/química , Fármacos Antiobesidade/farmacologia , Regulação do Apetite/efeitos dos fármacos , Obesidade/tratamento farmacológico , Receptor 5-HT2C de Serotonina/metabolismo , Agonistas do Receptor 5-HT2 de Serotonina/química , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Humanos , Masculino , Camundongos , Piridinas/química , Piridinas/farmacologia , Ratos Wistar , Serotonina/metabolismo , Percepção Gustatória/efeitos dos fármacosRESUMO
A basal tone of the endocannabinoid 2-arachidonoylglycerol (2-AG) enhances late oligodendrocyte progenitor cell (OPC) differentiation. Here, we investigated whether endogenous 2-AG may also promote OPC proliferation in earlier stages. We found that the blockade of 2-AG synthesizing enzymes, sn-1-diacylglycerol lipases α and ß (DAGLs), with RHC-80267 or the antagonism of either CB1 or CB2 cannabinoid receptors with AM281 and AM630, respectively, impaired early OPC proliferation stimulated by platelet-derived growth factor (PDGF-AA) and basic fibroblast growth factor (bFGF). On the contrary, increasing the levels of endogenous 2-AG by blocking the degradative enzyme monoacylglycerol lipase (MAGL) with JZL-184, significantly increased OPC proliferation as did agonists of cannabinoid receptor CB1 (ACEA), CB2 (JWH133) or both (HU-210). To elucidate signaling pathways underlying OPC proliferation, we studied the involvement of phosphatidylinositol 3-kinase (PI3K)/Akt and its downstream target mammalian target of rapamycin (mTOR). We show that phosphorylation of Akt and mTOR is required for OPC proliferation stimulated by growth factors (PDGF-AA and bFGF) or by CB1/CB2 agonists (ACEA/JWH133), since it was strongly decreased after LY294002 or rapamycin treatment. In line with this, blockade of CB1 (AM281), CB2 (AM630) or DAGLs (RHC-80267), decreased phosphorylation of Akt, mTOR and 4E-BP1, diminished cyclin E-cdk2 complex association and increased p27(kip1) levels. Our data suggest that proliferation of early OPCs stimulated by PDGF-AA and bFGF depends on the tonic activation of cannabinoid receptors by endogenous 2-AG and provide further evidence on the role of endocannabinoids in oligodendrocyte development, being important for the maintenance and self-renewal of the OPCs. The results highlight the therapeutic potential of the endocannabinoid signaling in the emerging field of brain repair.
Assuntos
Ácidos Araquidônicos/farmacologia , Endocanabinoides/farmacologia , Glicerídeos/farmacologia , Oligodendroglia/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células-Tronco/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Agonistas de Receptores de Canabinoides/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Humanos , Camundongos , Oligodendroglia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células-Tronco/efeitos dos fármacosRESUMO
Angiogenesis is a requirement for the sustained growth and proliferation of solid tumors, and the development of new compounds that induce a sustained inhibition of the proangiogenic signaling generated by tumor hypoxia still remains as an important unmet need. In this work, we describe a new antiangiogenic compound (22) that inhibits proangiogenic signaling under hypoxic conditions in breast cancer cells. Compound 22 blocks the MAPK pathway, impairs cellular migration under hypoxic conditions, and regulates a set of genes related to angiogenesis. These responses are mediated by HIF-1α, since the effects of compound 22 mostly disappear when its expression is knocked-down. Furthermore, administration of compound 22 in a xenograft model of breast cancer produced tumor growth reductions ranging from 46 to 55% in 38% of the treated animals without causing any toxic side effects. Importantly, in the responding tumors, a significant reduction in the number of blood vessels was observed, further supporting the mechanism of action of the compound. These findings provide a rationale for the development of new antiangiogenic compounds that could eventually lead to new drugs suitable for the treatment of some types of tumors either alone or in combination with other agents.
Assuntos
Inibidores da Angiogênese/química , Benzamidas/química , Carbamatos/química , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacologia , Animais , Benzamidas/síntese química , Benzamidas/farmacologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carbamatos/síntese química , Carbamatos/farmacologia , Hipóxia Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transplante de Neoplasias , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Essential cell division protein FtsZ is considered an attractive target in the search for antibacterials with novel mechanisms of action to overcome the resistance problem. FtsZ undergoes GTP-dependent assembly at midcell to form the Z-ring, a dynamic structure that evolves until final constriction of the cell. Therefore, molecules able to inhibit its activity will eventually disrupt bacterial viability. In this work, we report a new series of small molecules able to replace GTP and to specifically inhibit FtsZ, blocking the bacterial division process. These new synthesized inhibitors interact with the GTP-binding site of FtsZ (Kd = 0.4-0.8 µM), display antibacterial activity against Gram-positive pathogenic bacteria, and show selectivity against tubulin. Biphenyl derivative 28 stands out as a potent FtsZ inhibitor (Kd = 0.5 µM) with high antibacterial activity [MIC (MRSA) = 7 µM]. In-depth analysis of the mechanism of action of compounds 22, 28, 33, and 36 has revealed that they act as effective inhibitors of correct FtsZ assembly, blocking bacterial division and thus leading to filamentous undivided cells. These findings provide a compelling rationale for the development of compounds targeting the GTP-binding site as antibacterial agents and open the door to antibiotics with novel mechanisms of action.
Assuntos
Antibacterianos/síntese química , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Compostos de Bifenilo/síntese química , Proteínas do Citoesqueleto/antagonistas & inibidores , Guanosina Trifosfato/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Naftalenos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Bacillus subtilis/química , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/química , Sítios de Ligação , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Proteínas do Citoesqueleto/química , Cinética , Staphylococcus aureus Resistente à Meticilina/química , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Modelos Moleculares , Naftalenos/química , Naftalenos/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Pharmacological activation of cannabinoid receptors elicits antitumoral responses in different cancer models. However, the biological role of these receptors in tumor physio-pathology is still unknown. METHODS: We analyzed CB2 cannabinoid receptor protein expression in two series of 166 and 483 breast tumor samples operated in the University Hospitals of Kiel, Tübingen, and Freiburg between 1997 and 2010 and CB2 mRNA expression in previously published DNA microarray datasets. The role of CB2 in oncogenesis was studied by generating a mouse line that expresses the human V-Erb-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog 2 (HER2) rat ortholog (neu) and lacks CB2 and by a variety of biochemical and cell biology approaches in human breast cancer cells in culture and in vivo, upon modulation of CB2 expression by si/shRNAs and overexpression plasmids. CB2-HER2 molecular interaction was studied by colocalization, coimmunoprecipitation, and proximity ligation assays. Statistical tests were two-sided. RESULTS: We show an association between elevated CB2 expression in HER2+ breast tumors and poor patient prognosis (decreased overall survival, hazard ratio [HR] = 0.29, 95% confidence interval [CI] = 0.09 to 0.71, P = .009) and higher probability to suffer local recurrence (HR = 0.09, 95% CI = 0.049 to 0.54, P = .003) and to develop distant metastases (HR = 0.33, 95% CI = 0.13 to 0.75, P = .009). We also demonstrate that genetic inactivation of CB2 impairs tumor generation and progression in MMTV-neu mice. Moreover, we show that HER2 upregulates CB2 expression by activating the transcription factor ELK1 via the ERK cascade and that an increased CB2 expression activates the HER2 pro-oncogenic signaling at the level of the tyrosine kinase c-SRC. Finally, we show HER2 and CB2 form heteromers in cancer cells. CONCLUSIONS: Our findings reveal an unprecedented role of CB2 as a pivotal regulator of HER2 pro-oncogenic signaling in breast cancer, and they suggest that CB2 may be a biomarker with prognostic value in these tumors.