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1.
PLoS One ; 12(12): e0190251, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29287118

RESUMO

OBJECTIVE: Cartilage homeostasis relies on a balance of catabolism and anabolism of cartilage matrix. Our goal was to evaluate the burden of radiographic osteoarthritis and serum levels of type IIA procollagen amino terminal propeptide (sPIIANP), a biomarker representing type II collagen synthesis, in osteoarthritis. METHODS: OA burden was quantified on the basis of radiographic features as total joint faces with an osteophyte, joint space narrowing, or in the spine, disc space narrowing. sPIIANP was measured in 1,235 participants from the Genetics of Generalized Osteoarthritis study using a competitive enzyme-linked immunosorbent assay. Separate multivariable linear regression models, adjusted for age, sex, and body mass index and additionally for ipsilateral osteophytes or joint/disc space narrowing, were used to assess the independent association of sPIIANP with osteophytes and with joint/disc space narrowing burden in knees, hips, hands and spine, individually and together. RESULTS: After full adjustment, sPIIANP was significantly associated with a lesser burden of hip joint space narrowing and knee osteophytes. sPIIANP was associated with a lesser burden of hand joint space narrowing but a greater burden of hand osteophytes; these results were only evident upon adjustment for osteoarthritic features in all other joints. There were no associations of sPIIANP and features of spine osteoarthritis. CONCLUSIONS: Higher cartilage collagen synthesis, as reflected in systemic PIIANP concentrations, was associated with lesser burden of osteoarthritic features in lower extremity joints (knees and hips), even accounting for osteoarthritis burden in hands and spine, age, sex and body mass index. These results suggest that pro-anabolic agents may be appropriate for early treatment to prevent severe lower extremity large joint osteoarthritis.


Assuntos
Proteínas de Ligação ao Cálcio/sangue , Colágeno Tipo II/sangue , Osteoartrite/diagnóstico por imagem , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Osteoartrite/sangue , Osteoartrite/patologia
2.
J Org Chem ; 81(21): 10302-10320, 2016 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-27447195

RESUMO

We report a second-generation synthesis of the exceedingly potent antimitotic agent N14-desacetoxytubulysin H (1) as well as the preparation of nine analogues of this lead structure. Highlights of our synthetic efforts include an efficient late-stage functionalization that allows for the preparation of new side-chain- and backbone-modified analogues. We also discovered C-terminal modifications that preserve the exquisite biological activity of acid 1 and offer the opportunity for effective conjugation to cell type-targeting moieties. All analogues had antiproliferative activities in the high picomolar to low nanomolar range and caused apoptosis and mitotic arrest as measured in a high content nuclear morphology assay. The ten synthetic agents described herein spanned a range of almost 4 orders of magnitude in biological activity and illustrate the continued potential to discover extraordinarily potent antiproliferative compounds based on natural product leads.


Assuntos
Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Apoptose/efeitos dos fármacos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Mitose/efeitos dos fármacos , Oligopeptídeos/química , Espectroscopia de Prótons por Ressonância Magnética
3.
Eur J Med Chem ; 96: 504-18, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25951294

RESUMO

Tubulin-interacting agents, like vinca alkaloid and taxanes, play a fundamental role in cancer chemotherapy, making cellular microtubules (MT), one of the few validated anticancer targets. Cancer resistance to classical MT inhibitors has motivated the development of novel molecules with increased efficacy and lower toxicity. Aiming at designing structurally-simple inhibitors of MT assembly, we synthesized a series of thirty-one 3,4,5-trimethoxy-hydrazones and twenty-five derivatives or analogs. Docking simulations suggested that a representative N-acylhydrazone could adopt an appropriate stereochemistry inside the colchicine-binding domain of tubulin. Several of these compounds showed anti-leukemia effects in the nanomolar concentration range. Interference with MT polymerization was validated by the compounds' ability to inhibit MT assembly at the biochemical and cellular level. Selective toxicity investigations done with the most potent compound, a 3,4,5-trimethoxy-hydrazone with a 1-naphthyl group, showed remarkably selective toxicity against leukemia cells in comparison with stimulated normal lymphocytes, and no acute toxicity in vivo. Finally, this molecule was as active as vincristine in a murine model of human acute lymphoblastic leukemia at a weekly dose of 1 mg/kg.


Assuntos
Anisóis/farmacologia , Antineoplásicos/farmacologia , Hidrazonas/farmacologia , Microtúbulos/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Animais , Anisóis/síntese química , Anisóis/química , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microtúbulos/metabolismo , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas
4.
Arthritis Rheumatol ; 67(4): 956-65, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25544994

RESUMO

OBJECTIVE: To evaluate the ability of the macrophage markers CD163 and CD14 to predict different osteoarthritis (OA) phenotypes defined by severity of joint inflammation, radiographic features and progression, and joint pain. METHODS: We evaluated 2 different cohorts totaling 184 patients with radiographic knee OA. These included 25 patients from a cross-sectional imaging study for whom there were data on activated macrophages in the knee joint, and 159 patients (134 with 3-year longitudinal data) from the longitudinal Prediction of Osteoarthritis Progression study. Multivariable linear regression models with generalized estimating equations were used to assess the association of CD163 and CD14 in synovial fluid (SF) and blood with OA phenotypic outcomes. Models were adjusted for age, sex, and body mass index. P values less than or equal to 0.05 were considered significant. RESULTS: SF CD14, SF CD163, and serum CD163 were associated with the abundance of activated macrophages in the knee joint capsule and synovium. SF CD14 was positively associated with severity of joint space narrowing and osteophytes in both cohorts. SF and plasma CD14 were positively associated with self-reported knee pain severity in the imaging study. Both SF CD14 and SF CD163 were positively associated with osteophyte progression. CONCLUSION: Soluble macrophage biomarkers reflected the abundance of activated macrophages and appeared to mediate structural progression (CD163 and CD14) and pain (CD14) in OA knees. These data support the central role of inflammation as a determinant of OA severity, progression risk, and clinical symptoms, and they suggest a means of readily identifying a subset of patients with an active inflammatory state and worse prognosis.


Assuntos
Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Inflamação/sangue , Receptores de Lipopolissacarídeos/análise , Macrófagos/metabolismo , Osteoartrite do Joelho/sangue , Receptores de Superfície Celular/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Progressão da Doença , Feminino , Humanos , Inflamação/diagnóstico por imagem , Articulação do Joelho/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/diagnóstico por imagem , Fenótipo , Radiografia , Índice de Gravidade de Doença , Líquido Sinovial/química
5.
Eur J Med Chem ; 63: 501-10, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23524161

RESUMO

Based on classical colchicine site ligands and a computational model of the colchicine binding site on beta tubulin, two classes of chalcone derivatives were designed, synthesized and evaluated for inhibition of tubulin assembly and toxicity in human cancer cell lines. Docking studies suggested that the chalcone scaffold could fit the colchicine site on tubulin in an orientation similar to that of the natural product. In particular, a 3,4,5-trimethoxyphenyl ring adjacent to the carbonyl group appeared to benefit the ligand-tubulin interaction, occupying the same subcavity as the corresponding moiety in colchicine. Consistent with modeling predictions, several 3,4,5-trimethoxychalcones showed improved cytotoxicity to murine acute lymphoblastic leukemia cells compared with a previously described parent compound, and inhibited tubulin assembly in vitro as potently as colchicine. The most potent chalcones inhibited the growth of human leukemia cell lines at nanomolar concentrations, caused microtubule destabilization and mitotic arrest in human cervical cancer cells, and inhibited human breast cancer cell migration in scratch wound and Boyden chamber assays.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Chalconas/síntese química , Chalconas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Chalconas/química , Relação Dose-Resposta a Droga , Humanos , Células Jurkat , Camundongos , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Células NIH 3T3 , Polimerização/efeitos dos fármacos , Tubulina (Proteína)/metabolismo
6.
Biometals ; 26(1): 151-65, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23344786

RESUMO

Complexes [Ga(2Ac4pFPh)(2)]NO(3) (1), [Ga(2Ac4pClPh)(2)]NO(3) (2), [Ga(2Ac4pIPh)(2)]NO(3) (3), [Ga(2Ac4pNO(2)Ph)(2)]NO(3)·3H(2)O (4) and [Ga(2Ac4pT)(2)]NO(3) (5) were obtained with 2-acetylpyridine N(4)-para-fluorophenyl-(H2Ac4pFPh), 2-acetylpyridine N(4)-para-chlorophenyl-(H2Ac4pClPh), 2-acetylpyridine N(4)-para-iodophenyl-(H2Ac4pIPh), 2-acetylpyridine N(4)-para-nitrophenyl-(H2Ac4pNO(2)Ph) and 2-acetylpyridine N(4)-para-tolyl-(H2Ac4pT) thiosemicarbazone. 1-5 presented antimicrobial and cytotoxic properties. Coordination to gallium(III) proved to be an effective strategy for activity improvement against Pseudomonas aeruginosa and Candida albicans. The complexes were highly cytotoxic against malignant glioblastoma and breast cancer cells at nanomolar concentrations. The compounds induced morphological changes characteristic of apoptotic death in tumor cells and showed no toxicity against erythrocytes. 2 partially inhibited tubulin assembly at high concentrations and induced cellular microtubule disorganization, but this does not appear to be the main mechanism of cytotoxic activity.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Gálio/química , Tiossemicarbazonas/química , Tubulina (Proteína)/química , Antibacterianos/química , Antifúngicos/química , Antineoplásicos/química , Candida albicans/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Complexos de Coordenação/química , Cristalografia por Raios X , Eritrócitos/efeitos dos fármacos , Células HeLa , Humanos , Concentração Inibidora 50 , Cinética , Células MCF-7 , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Multimerização Proteica/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Piridinas/química , Staphylococcus aureus/efeitos dos fármacos , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia
7.
Mol Cell ; 48(4): 655-61, 2012 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-23063524

RESUMO

Despite the crucial impact of leptin signaling on metabolism and body weight, little is known about the structure of the liganded leptin receptor (LEP-R) complex. Here, we applied single-particle electron microscopy (EM) to characterize the architecture of the extracellular region of LEP-R alone and in complex with leptin. We show that unliganded LEP-R displays significant flexibility in a hinge region within the cytokine homology region 2 (CHR2) that is connected to rigid membrane-proximal FnIII domains. Leptin binds to CHR2 in order to restrict the flexible hinge and the disposition of the FnIII "legs." Through a separate interaction, leptin engages the Ig-like domain of a second liganded LEP-R, resulting in the formation of a quaternary signaling complex. We propose that the membrane proximal domain rigidification in the context of a liganded cytokine receptor dimer is a key mechanism for the transactivation of Janus kinases (Jaks) bound at the intracellular receptor region.


Assuntos
Leptina/farmacologia , Receptores para Leptina/química , Receptores para Leptina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Humanos , Leptina/química , Leptina/metabolismo , Ligantes , Microscopia Eletrônica , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Receptores para Leptina/isolamento & purificação , Receptores para Leptina/ultraestrutura
8.
Assay Drug Dev Technol ; 10(1): 46-60, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21919741

RESUMO

Agonist-induced glucocorticoid receptor [GR] transport from the cytoplasm to the nucleus was used as a model to identify dynein-mediated cargo transport inhibitors. Cell-based screening of the library of pharmacologically active compound (LOPAC)-1280 collection identified several small molecules that stalled the agonist-induced transport of GR-green fluorescent protein (GFP) in a concentration-dependent manner. Fluorescent images of microtubule organization, nuclear DNA staining, expression of GR-GFP, and its subcellular distribution were inspected and quantified by image analysis to evaluate the impact of compounds on cell morphology, toxicity, and GR transport. Given the complexity of the multi-protein complex involved in dynein-mediated cargo transport and the variety of potential mechanisms for interruption of that process, we therefore developed and validated a panel of biochemical assays to investigate some of the more likely intracellular target(s) of the GR transport inhibitors. Although the apomorphine enantiomers exhibited the most potency toward the ATPase activities of cytoplasmic dynein, myosin, and the heat-shock proteins (HSPs), their apparent lack of specificity made them unattractive for further study in our quest. Other molecules appeared to be nonspecific inhibitors that targeted reactive cysteines of proteins. Ideally, specific retrograde transport inhibitors would either target dynein itself or one of the other important proteins associated with the transport process. Although the hits from the cell-based screen of the LOPAC-1280 collection did not exhibit this desired profile, this screening platform provided a promising phenotypic system for the discovery of dynein/HSP modulators.


Assuntos
Núcleo Celular/metabolismo , Dineínas do Citoplasma/fisiologia , Avaliação Pré-Clínica de Medicamentos/métodos , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/metabolismo , Animais , Apomorfina/metabolismo , Apomorfina/farmacologia , Bovinos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Transporte Proteico/fisiologia
9.
Mol Cancer Ther ; 10(6): 994-1006, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21490306

RESUMO

The natural product (--)-dictyostatin is a microtubule-stabilizing agent that potently inhibits the growth of human cancer cells, including paclitaxel-resistant clones. Extensive structure-activity relationship studies have revealed several regions of the molecule that can be altered without loss of activity. The most potent synthetic dictyostatin analogue described to date, 6-epi-dictyostatin, has superior in vivo antitumor activity against human breast cancer xenografts compared with paclitaxel. In spite of their encouraging activities in preclinical studies, the complex chemical structure of the dictyostatins presents a major obstacle for their development into novel antineoplastic therapies. We recently reported a streamlined synthesis of 16-desmethyl-25,26-dihydrodictyostatins and found several agents that, when compared with 6-epi-dictyostatin, retained nanomolar activity in cellular microtubule-bundling assays but had lost activity against paclitaxel-resistant cells with mutations in ß-tubulin. Extending these studies, we applied the new, highly convergent synthesis to generate 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin. Both compounds were potent microtubule-perturbing agents that induced mitotic arrest and microtubule assembly in vitro and in intact cells. In vitro radioligand binding studies showed that 25,26-dihydrodictyostatin and its C6-epimer were capable of displacing [3H]paclitaxel and [14C]epothilone B from microtubules with potencies comparable to (--)-dictyostatin and discodermolide. Both compounds inhibited the growth of paclitaxel- and epothilone B-resistant cell lines at low nanomolar concentrations, synergized with paclitaxel in MDA-MB-231 human breast cancer cells, and had antiangiogenic activity in transgenic zebrafish larvae. These data identify 25,26-dihydrodictyostatin and 6-epi-25,26-dihydrodictyostatin as candidates for scale-up synthesis and further preclinical development.


Assuntos
Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Macrolídeos/síntese química , Macrolídeos/farmacologia , Inibidores da Angiogênese/química , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Epotilonas/farmacologia , Células HeLa , Humanos , Macrolídeos/química , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Paclitaxel/farmacologia , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Peixe-Zebra
10.
Sensors (Basel) ; 10(11): 9630-46, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-22163431

RESUMO

Biosensors have been used extensively in the scientific community for several purposes, most notably to determine association and dissociation kinetics, protein-ligand, protein-protein, or nucleic acid hybridization interactions. A number of different types of biosensors are available in the field, each with real or perceived benefits over the others. This review discusses the basic theory and operational arrangements of four commercially available types of optical biosensors: surface plasmon resonance, resonant mirror, resonance waveguide grating, and dual polarization interferometry. The different applications these techniques offer are discussed from experiments and results reported in recently published literature. Additionally, recent advancements or modifications to the current techniques are also discussed.


Assuntos
Técnicas Biossensoriais/instrumentação , Interferometria/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Técnicas Biossensoriais/métodos , Interferometria/métodos , Dispositivos Ópticos , Ressonância de Plasmônio de Superfície/métodos
11.
Biosens Bioelectron ; 25(1): 136-41, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19595587

RESUMO

Dual polarization interferometry (DPI) and resonant mirror (RM) methods were used to characterize the growth of microtubules (MTs) on biosensor surfaces. The structure and dynamics of MTs play an important role in cell division and are a target for many anti-cancer drugs. Evidence from DPI demonstrated the growth of MTs on streptavidin-biotinylated-tubulin surfaces from the increase in mass and thickness, with a simultaneous decrease in density. The initial increase in thickness of 0.236 nm/min suggested the elongation of protofilaments before they join laterally to form the MT, where the rate of growth increased to 0.436 nm/min. Continuous mass increases were also observed when tubulin was added to a similar underlying RM surface. Tubulin binding to these surfaces was also temperature dependent, increasing the absolute response with MT stabilizers, while inhibiting binding with destabilizers when temperature was changed from 15 to 37 degrees C. Finally, the initial rates of tubulin assembly (mean+/-SD, n=3) with MT-stabilizer agents were significantly higher at 1.50+/-0.27 and 1.04+/-0.13 arcseconds/s, respectively, compared to 0.37+/-0.11 arcseconds/s for tubulin containing GTP only. In the presence of the MT destabilizers, colchicine and dolastatin 10, the slopes of initial rates were lower than in their absence at 0.05+/-0.01 and 0.27+/-0.08 arcseconds/s, respectively. This provides evidence for the ability of surface-based optical sensors to distinguish between MT stabilizers and destabilizers, while also paving the path to develop other methods to screen for MT-perturbing agents using the same underlying surface engineering.


Assuntos
Técnicas Biossensoriais/métodos , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Bovinos , Interferometria , Luz , Ligação Proteica , Propriedades de Superfície , Temperatura
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