RESUMO
BACKGROUND: Optimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease. METHODS: In this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett's multiple comparison test. RESULTS: We demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p < 0.05) and invasion (n ≥ 3, p < 0.001) and this was translated in vivo where AD-01 inhibited breast cancer metastasis in MDA-MB-231-lucD3H1 in vivo model (p < 0.05). In ER+ MCF-7 cells and primary breast tumour samples, we demonstrated that ALM201 inhibits endocrine therapy resistant mammospheres, representative of CSC content (n ≥ 3, p < 0.05). Whilst an in vivo limiting dilution assay, using SCID mice, demonstrated that ALM201 alone or in combination with tamoxifen was very effective at delaying tumour recurrence by 12 (p < 0.05) or 21 days (p < 0.001), respectively, by reducing the number of CSCs. The potential mechanism of action, in addition to CD44, involves downregulation of DLL4 and Notch4. CONCLUSION: This study demonstrates, for the first time, the pre-clinical activity of novel systemic anti-cancer therapeutic peptides, ALM201 and AD-01, in the metastatic setting, and highlights their impact on endocrine therapy resistant CSCs; both areas of unmet clinical need.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Imunofilinas/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Mama/patologia , Neoplasias da Mama/patologia , Proteínas de Ligação ao Cálcio , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunofilinas/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos SCID , Recidiva Local de Neoplasia/prevenção & controle , Células-Tronco Neoplásicas/patologia , Peptídeos/uso terapêutico , Receptor Notch4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Ligação a Tacrolimo , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This Letter describes the further development and SAR exploration of a novel series of Legumain inhibitors. Based upon a previously identified Legumain inhibitor from our group, we explored the SAR of the carbamate phenyl ring system to probe the P3 pocket of the enzyme. This led to the identification of a sub-nanomolar inhibitor of Legumain.
Assuntos
Compostos de Bifenilo/farmacologia , Carbamatos/farmacologia , Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/química , Carbamatos/síntese química , Carbamatos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The serine/threonine protein kinase AKT plays a pivotal role within the PI3K pathway in regulating cellular proliferation and apoptotic cellular functions, and AKT hyper-activation via gene amplification and/or mutation has been implicated in multiple human malignancies. There are 3 AKT isoenzymes (AKT1-3) which mediate critical, non-redundant functions. We present the discovery and development of ALM301, a novel, allosteric, sub-type selective inhibitor of AKT1/2. ALM301 binds in an allosteric pocket created by the combined movement of the PH domain and the catalytic domain, resulting in a DFG out conformation. ALM301 was shown to be highly selective against a panel of over 450 kinases and potently inhibited cellular proliferation. These effects were particularly pronounced in MCF-7 cells containing a PI3KCA mutation. Subsequent cellular downstream pathway analysis in this sensitive cell line revealed potent inhibition of pAKT signalling up to 48 h post dosing. ALM301 treatment was well tolerated in an MCF-7 xenograft model and led to a dose-dependent reduction in tumour growth. Enhanced efficacy was observed in combination with tamoxifen. In summary, ALM301 is a highly specific AKT 1/2 inhibitor with an excellent pharmacological profile suitable for further clinical development.
Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Inibidores da Angiogênese , Humanos , Isoenzimas , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina , Tamoxifeno , TreoninaRESUMO
Breast cancer remains a leading cause of cancer death in women, representing a significant unmet medical need. Here, we disclose our discovery efforts culminating in a clinical candidate, 35 (GDC-9545 or giredestrant). 35 is an efficient and potent selective estrogen receptor degrader (SERD) and a full antagonist, which translates into better antiproliferation activity than known SERDs (1, 6, 7, and 9) across multiple cell lines. Fine-tuning the physiochemical properties enabled once daily oral dosing of 35 in preclinical species and humans. 35 exhibits low drug-drug interaction liability and demonstrates excellent in vitro and in vivo safety profiles. At low doses, 35 induces tumor regressions either as a single agent or in combination with a CDK4/6 inhibitor in an ESR1Y537S mutant PDX or a wild-type ERα tumor model. Currently, 35 is being evaluated in Phase III clinical trials.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Carbolinas/uso terapêutico , Antagonistas do Receptor de Estrogênio/uso terapêutico , Receptor alfa de Estrogênio/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Carbolinas/química , Carbolinas/farmacocinética , Cães , Antagonistas do Receptor de Estrogênio/química , Antagonistas do Receptor de Estrogênio/farmacocinética , Feminino , Humanos , Células MCF-7 , Macaca fascicularis , Camundongos , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: A number of cytotoxic chemotherapy agents tested at low concentrations show antiangiogenic properties with limited cytotoxicity, e.g., cyclophosphamide, tirapazamine, and mitoxantrone. AQ4N is a bioreductive alkylaminoanthraquinone that is cytotoxic when reduced to AQ4; hence, it can be used to target hypoxic tumor cells. AQ4N is structurally similar to mitoxantrone and was evaluated for antiangiogenic properties without the need for bioreduction. EXPERIMENTAL DESIGN: The effect of AQ4N and fumagillin on human microvascular endothelial cells (HMEC-1) was measured using a variety of in vitro assays, i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound scrape, tubule formation, rat aortic ring, and invasion assays. Low-dose AQ4N (20 mg/kg) was also given in vivo to mice bearing a tumor in a dorsal skin flap. RESULTS: AQ4N (10(-11) to 10(-5) mol/L) had no effect on HMEC-1 viability. AQ4N (10(-9) to 10(-5)mol/L) caused a sigmoidal dose-dependent inhibition of endothelial cell migration in the wound scrape model. Fumagillin showed a similar response over a lower dose range (10(-13) to 10(-9) mol/L); however, the maximal inhibition was less (25% versus 43% for AQ4N). AQ4N inhibited HMEC-1 cell contacts on Matrigel (10(-8) to 10(-5) mol/L), HMEC-1 cell invasion, and sprouting in rat aorta explants. Immunofluorescence staining with tubulin, vimentim, dynein, and phalloidin revealed that AQ4N caused disruption to the cell cytoskeleton. When AQ4N (20 mg/kg) was given in vivo for 5 days, microvessels disappeared in LNCaP tumors grown in a dorsal skin flap. CONCLUSIONS: This combination of assays has shown that AQ4N possesses antiangiogenic effects in normoxic conditions, which could potentially contribute to antitumor activity.
Assuntos
Inibidores da Angiogênese/farmacologia , Antraquinonas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Pele/irrigação sanguínea , Animais , Antineoplásicos/farmacologia , Aorta/efeitos dos fármacos , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Neoplasias do Colo/tratamento farmacológico , Cicloexanos/farmacologia , Citoesqueleto/metabolismo , Combinação de Medicamentos , Ácidos Graxos Insaturados/farmacologia , Humanos , Laminina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microcirculação , Neovascularização Fisiológica , Neoplasias da Próstata/tratamento farmacológico , Proteoglicanas/metabolismo , Ratos , Ratos Wistar , Sesquiterpenos/farmacologia , Retalhos Cirúrgicos , Cicatrização/efeitos dos fármacosRESUMO
FKBP-like (FKBPL) protein is a novel immunophilin-like protein that plays a role in the cellular stress response. Its three tetratricopeptide repeat motifs are homologous to the heat shock protein 90 interaction sites of other immunophilins that have roles in steroid hormone receptor signaling. In this study, using biomolecular complementation and coimmunoprecipitation techniques, we show that FKBPL also colocalizes and interacts with the components of the heat shock protein 90-glucocorticoid receptor (GR) complex and demonstrate that the PPIase domain of FKBPL is important for the interaction between this complex and the dynein motor protein, dynamitin. Treatment of DU145 cells with the GR ligand, dexamethasone, induced a rapid and coordinated translocation of both GR and FKBPL to the nucleus; this response was perturbed when FKBPL was knocked down with a targeted small interfering RNA. Furthermore, overexpression of FKBPL increased GR protein levels and transactivation of a luciferase reporter gene in response to dexamethasone in DU145 cells. However, these responses were cell line dependent. In summary, these data suggest that FKBPL can be classed as a new member of the FKBP protein family with a role in steroid receptor complexes and signaling.
Assuntos
Imunofilinas/metabolismo , Imunofilinas/fisiologia , Receptores de Glucocorticoides/metabolismo , Receptores de Esteroides/fisiologia , Núcleo Celular/metabolismo , Humanos , Imunofilinas/antagonistas & inibidores , Imunofilinas/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/farmacologia , Receptores de Esteroides/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Ligação a Tacrolimo , Ativação Transcricional/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
Synthetic bradykinin antagonist peptides/peptoids have been powerful tools for delineating the roles of kinins in both normal physiology and in pathological states. Here, we report the identification of a novel, naturally occurring bradykinin B2 receptor antagonist peptide, helokinestatin, isolated and structurally characterized from the venoms of helodermatid lizards-the Gila monster (Heloderma suspectum) and the Mexican beaded lizard (Heloderma horridum). The primary structure of the peptide was established by a combination of microsequencing and mass spectroscopy as Gly-Pro-Pro-Tyr-Gln-Pro-Leu-Val-Pro-Arg (Mr 1122.62). A synthetic replicate of helokinestatin was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle, mediated by the B2 receptor sub-type, in a dose-dependent manner. Natural selection, that generates functional optimization of predatory reptile venom peptides, can potentially provide new insights for drug lead design or for normal physiological or pathophysiological processes.
Assuntos
Antagonistas de Receptor B2 da Bradicinina , Músculo Liso Vascular/efeitos dos fármacos , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Peçonhas/química , Animais , Artérias/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão/métodos , Lagartos , Masculino , Oligopeptídeos/isolamento & purificação , Ratos , Ratos Wistar , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fatores de Tempo , Peçonhas/isolamento & purificação , Peçonhas/farmacologiaRESUMO
Previous peptidomic analyses of the defensive skin secretion from the North American pickerel frog, Rana palustris, have established the presence of canonical bradykinin and multiple bradykinin-related peptides (BRPs). As a consequence of the multiplicity of peptides identified and their diverse primary structures, it was speculated that they must represent the products of expression of multiple genes. Here, we present unequivocal evidence that the majority of BRPs (11/13) identified in skin secretion by the peptidomic approach can be generated by differential site-specific protease cleavage from a single common precursor of 321 amino acid residues, named skin kininogen 1, whose primary structure was deduced from cloned skin secretion-derived cDNA. The organization of skin kininogen 1 consists of a hydrophobic signal peptide followed by eight non-identical domains each encoding a single copy of either canonical bradykinin or a BRP. Two additional splice variants, encoding precursors of 233 (skin kininogen 2) or 189 amino acid residues (skin kininogen 3), were also cloned and were found to lack BRP-encoding domains 5 and 6 or 4, 5 and 6, respectively. Thus, generation of peptidome diversity in amphibian defensive skin secretions can be achieved in part by differential protease cleavage of relatively large and multiple-encoding domain precursors reflecting a high degree of transcriptional economy.
Assuntos
Secreções Corporais/química , Peptídeos/química , Ranidae/classificação , Ranidae/metabolismo , Pele/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bradicinina/química , Bradicinina/genética , Clonagem Molecular , DNA Complementar/genética , Cininogênios/química , Cininogênios/genética , Dados de Sequência Molecular , Peptídeos/genética , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Proteoma/genética , Ranidae/genética , Transcrição GênicaRESUMO
Multiple bradykinin-related peptides including a novel bradykinin structural variant, (Val(1))-bradykinin, have been identified from the defensive skin secretion of Guenther's frog, Hylarana guentheri by a tandem mass spectrometry method. Subsequently, four different preprobradykinin cDNAs, which encoded multiple bradykinin copies and its structural variants, were consistently cloned from a skin derived cDNA library. These preprobradykinin cDNAs showed little structural similarity with mammalian kininogens and the kininogens from the skin of toads, but have regions that are highly conserved in the kininogens from another ranid frog, Odorrana schmackeri. Alignment of these preprobradykinins revealed that preprobradykinin 1, 2 and 3 may derive from a single gene by alternative exon splicing.
Assuntos
Anuros/metabolismo , Bradicinina/isolamento & purificação , Peptídeos/isolamento & purificação , Pele/metabolismo , Sequência de Aminoácidos , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/isolamento & purificação , Proteínas de Anfíbios/farmacologia , Animais , Anuros/genética , Sequência de Bases , Bradicinina/genética , Bradicinina/farmacologia , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Relação Dose-Resposta a Droga , Técnicas In Vitro , Cininogênios/química , Masculino , Dados de Sequência Molecular , Músculo Liso/efeitos dos fármacos , Peptídeos/genética , Peptídeos/farmacologia , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Ratos , Ratos Wistar , Análise de Sequência de DNA , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: Pericytes, located in close proximity to the underlying endothelium, form an integral component of the microvasculature. These cells are intimately involved in angiogenesis, which is of fundamental importance in many physiological and pathological processes. We evaluated the influence of pericyte-conditioned medium (PCM) on endothelial cell growth characteristics and modulation of endothelial gene expression. METHODS: Migration and tubule formation assays were performed in vitro to determine the effect of PCM on endothelial growth characteristics. cDNA microarray analysis was used to identify alterations in gene expression following exposure of human microvascular endothelial cells (HMEC-1) to PCM. Overexpression of PAI-1 using recombinant protein or transient transfection, and inhibition using an inhibitory antibody against PAI-1, were used to determine whether up- or down-regulation of this gene was responsible for the changes in endothelial cell characteristics observed in response to PCM exposure. RESULTS: We have shown that PCM exerts a dramatic inhibitory influence on endothelial cell migration in vitro. In addition, endothelial cells cultured on Matrigel and exposed to PCM were found to generate significantly fewer angiogenic branches. Microarray analysis of endothelial cells exposed to PCM identified PAI-1 as the gene showing the greatest level of differential expression (3.4-fold induction). Studies using an inhibitory antibody to PAI-1 suggest that induction of this protein by PCM is pivotal to the observed inhibitory influence on the migratory and angiogenic potential of HMEC-1. We further investigated this by overexpressing PAI-1, which was shown to have a potent inhibitory influence on EC migration and angiogenic branching, although the concentration of PAI-1 was clearly important. CONCLUSION: Collectively, these findings suggest that PCM contains a bioactive element(s) that controls both endothelial cell migration and tubule formation in vitro and that these responses may be partially controlled by increased endothelial cell expression of PAI-1.
Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Neovascularização Fisiológica , Pericitos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Northern Blotting/métodos , Western Blotting/métodos , Movimento Celular , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/citologia , Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Inibidor 1 de Ativador de Plasminogênio/metabolismoRESUMO
Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.
Assuntos
Aorta/efeitos dos fármacos , Aorta/fisiologia , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Quinase de Cadeia Leve de Miosina/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Aorta/metabolismo , Carcinoma de Células Renais/irrigação sanguínea , Humanos , Neoplasias Renais/irrigação sanguínea , Masculino , Camundongos , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/deficiência , Ratos , Ratos Wistar , Vasodilatação/efeitos dos fármacosRESUMO
Bradykinin and (Thr(6))-bradykinin have been identified in the defensive skin secretion of the fire-bellied toad, Bombina orientalis. The homologous cDNAs for both peptides were cloned from a skin library using a 3'- and 5'-RACE strategy. Kininogen-1 (BOK-1) contained an open-reading frame of 167 amino acid residues encoding four repeats of bradykinin, and kininogen-2 (BOK-2) contained an open-reading frame of 161 amino acid residues encoding two repeats of (Thr(6))-bradykinin. Alignment of both precursor nucleotide and amino acid sequences revealed a high degree of structural similarity. These amphibian skin kininogens/preprobradykinins are not biologically analogous to mammalian kininogens.
Assuntos
Anuros/metabolismo , Bradicinina/química , Pele/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Bradicinina/genética , Cromatografia Líquida , Clonagem Molecular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Cininogênios/química , Cininogênios/metabolismo , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Músculo Liso/metabolismo , Fases de Leitura Aberta , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Raios UltravioletaRESUMO
Structural homologues of vertebrate regulatory peptides found in defensive skin secretions of anuran amphibians often display enhanced bioactivity and receptor binding when compared with endogenous mammalian peptide ligands. Maximakinin, a novel N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the skin venom of a Chinese toad (Bombina maxima), displays such activity enhancement when compared with bradykinin but is additionally highly selective for mammalian arterial smooth muscle bradykinin receptors displaying a 50-fold increase in molar potency in this smooth muscle type. In contrast, a 100-fold decrease in molar potency was observed at bradykinin receptors in intestinal and uterine smooth muscle preparations. Maximakinin has thus evolved as a "smart" defensive weapon in the toad with receptor/tissue selective targeting. Natural selection of amphibian skin venom peptides for antipredator defence, through inter-species delivery by an exogenous secretory mode, produces subtle structural stabilisation modifications that can potentially provide new insights for the design of selectively targeted peptide therapeutics.
Assuntos
Venenos de Anfíbios/farmacologia , Anuros , Artérias/efeitos dos fármacos , Bradicinina/química , Músculo Liso Vascular/efeitos dos fármacos , Peçonhas/química , Sequência de Aminoácidos , Venenos de Anfíbios/síntese química , Venenos de Anfíbios/química , Animais , Bioensaio , Antagonistas dos Receptores da Bradicinina , Dados de Sequência Molecular , Músculo Liso Vascular/fisiologia , Ratos , Receptores da Bradicinina/metabolismo , Especificidade por Substrato , Fatores de TempoRESUMO
We have isolated a novel bradykinin B(2)-receptor antagonist peptide, kinestatin, from toad (Bombina maxima) defensive skin secretion. Mass spectroscopy established a molecular mass of 931.56 Da and a provisional structure: pGlu-Leu/Ile-Pro-Gly-Leu/Ile-Gly-Pro-Leu/Ile-Arg.amide. The unmodified sequence, -QIPGLGPLRG-, was located at the C-terminus of a 116-amino-acid residue open-reading frame following interrogation of a sequenced B. maxima skin cDNA library database. This confirmed the presence of appropriate primary structural attributes for the observed post-translational modifications present on the mature peptide and established residue 2 as Ile and residues 5/8 as Leu. Kinestatin represents a prototype novel peptide from amphibian skin.
Assuntos
Anuros , Antagonistas de Receptor B2 da Bradicinina , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Pele/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Bradicinina/farmacologia , DNA Complementar/análise , DNA Complementar/genética , Masculino , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Fenilefrina/farmacologia , Ratos , Ratos Wistar , Receptor B2 da Bradicinina/metabolismo , Alinhamento de Sequência , Pele/metabolismo , Vasodilatação/efeitos dos fármacosRESUMO
Tryptophyllins are a heterogenous group of amphibian skin peptides originally identified in skin extracts of Neotropical leaf frogs, Phyllomedusa sp., by chemical means. Until now, biosynthetic precursor structure and biological activity remain unreported. Here we describe the isolation of a novel, post-translationally modified tryptophyllin, Lys-Pro-Hyp-Ala-Trp-Val-Pro.amide (PdT-1), from the skin secretion of the Mexican leaf frog, Pachymedusa dacnicolor. Using a 3'- and 5'-RACE strategy and an in vitro skin cDNA library, the PdT-1-encoding precursor was cloned and found to consist of an open-reading frame of 62 amino acids with a single copy of PdT-1 located towards the C-terminus. A synthetic replicate of PdT-1 was found to be a potent myoactive agent, relaxing mammalian arterial smooth muscle and contracting small intestinal smooth muscle at nanomolar concentrations. PdT-1 is thus the first amphibian skin tryptophyllin to be pharmacologically characterized and the first whose precursor cDNA has been cloned.
Assuntos
Músculo Liso/efeitos dos fármacos , Oligopeptídeos/genética , Pele/química , Sequência de Aminoácidos , Animais , Anuros , Sequência de Bases , Bradicinina/genética , Bradicinina/metabolismo , Clonagem Molecular , DNA Complementar/análise , Relação Dose-Resposta a Droga , Dados de Sequência Molecular , Músculo Liso/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , RatosRESUMO
PURPOSE: Antiangiogenic therapies can be an important adjunct to the management of many malignancies. Here we investigated a novel protein, FKBPL, and peptide derivative for their antiangiogenic activity and mechanism of action. EXPERIMENTAL DESIGN: Recombinant FKBPL (rFKBPL) and its peptide derivative were assessed in a range of human microvascular endothelial cell (HMEC-1) assays in vitro. Their ability to inhibit proliferation, migration, and Matrigel-dependent tubule formation was determined. They were further evaluated in an ex vivo rat model of neovascularization and in two in vivo mouse models of angiogenesis, that is, the sponge implantation and the intravital microscopy models. Antitumor efficacy was determined in two human tumor xenograft models grown in severe compromised immunodeficient (SCID) mice. Finally, the dependence of peptide on CD44 was determined using a CD44-targeted siRNA approach or in cell lines of differing CD44 status. RESULTS: rFKBPL inhibited endothelial cell migration, tubule formation, and microvessel formation in vitro and in vivo. The region responsible for FKBPL's antiangiogenic activity was identified, and a 24-amino acid peptide (AD-01) spanning this sequence was synthesized. It was potently antiangiogenic and inhibited growth in two human tumor xenograft models (DU145 and MDA-231) when administered systemically, either on its own or in combination with docetaxel. The antiangiogenic activity of FKBPL and AD-01 was dependent on the cell-surface receptor CD44, and signaling downstream of this receptor promoted an antimigratory phenotype. CONCLUSION: FKBPL and its peptide derivative AD-01 have potent antiangiogenic activity. Thus, these agents offer the potential of an attractive new approach to antiangiogenic therapy.
Assuntos
Inibidores da Angiogênese/farmacologia , Imunofilinas/uso terapêutico , Neoplasias/irrigação sanguínea , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Inibidores da Angiogênese/química , Inibidores da Angiogênese/uso terapêutico , Animais , Western Blotting , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Docetaxel , Células Endoteliais/efeitos dos fármacos , Receptores de Hialuronatos/genética , Imunofilinas/química , Imunofilinas/farmacologia , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias/tratamento farmacológico , Fragmentos de Peptídeos/uso terapêutico , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas de Ligação a Tacrolimo , Taxoides/farmacologia , Taxoides/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The HSP90 chaperone and immunophilin FKBPL is an estrogen-responsive gene that interacts with estogen receptor alpha (ERalpha) and regulates its levels. In this study, we explored the effects of FKBPL on breast cancer proliferation. Breast cancer cells stably overexpressing FKBPL became dependent on estrogen for their growth and were dramatically more sensitive to the antiestrogens tamoxifen and fulvestrant, whereas FKBPL knockdown reverses this phenotype. FKBPL knockdown also decreased the levels of the cell cycle inhibitor p21WAF1 and increased ERalpha phosphorylation on Ser(118) in response to 17beta-estradiol and tamoxifen. In support of the likelihood that these effects explained FKBPL-mediated cell growth inhibition and sensitivity to endocrine therapies, FKBPL expression was correlated with increased overall survival and distant metastasis-free survival in breast cancer patients. Our findings suggest that FKBPL may have prognostic value based on its impact on tumor proliferative capacity and sensitivity to endocrine therapies, which improve outcome.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Imunofilinas/fisiologia , Transdução de Sinais/fisiologia , Tamoxifeno/farmacologia , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Catepsina D/genética , Catepsina D/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacologia , Estrogênios/farmacologia , Fulvestranto , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Imunofilinas/genética , Imunofilinas/metabolismo , Imunoprecipitação , Estimativa de Kaplan-Meier , Metanálise como Assunto , Fosforilação/efeitos dos fármacos , Ligação Proteica , Interferência de RNA , Serina/metabolismo , Transdução de Sinais/genética , Proteínas de Ligação a Tacrolimo , TransfecçãoRESUMO
PURPOSE: Poly(ADP-ribose) polymerase (PARP) plays an important role in DNA repair, and PARP inhibitors can enhance the activity of DNA-damaging agents in vitro and in vivo. AG014699 is a potent PARP inhibitor in phase II clinical development. However, the range of therapeutics with which AG014699 could interact via a DNA-repair based mechanism is limited. We aimed to investigate a novel, vascular-based activity of AG014699, underlying in vivo chemosensitization, which could widen its clinical application. EXPERIMENTAL DESIGN: Temozolomide response was analyzed in vitro and in vivo. Vessel dynamics were monitored using "mismatch" following the administration of perfusion markers and real-time analysis of fluorescently labeled albumin uptake in to tumors established in dorsal window chambers. Further mechanistic investigations used ex vivo assays of vascular smooth muscle relaxation, gut motility, and myosin light chain kinase (MLCK) inhibition. RESULTS: AG014699 failed to sensitize SW620 cells to temozolomide in vitro but induced pronounced enhancement in vivo. AG014699 (1 mg/kg) improved tumor perfusion comparably with the control agents nicotinamide (1 g/kg) and AG14361 (forerunner to AG014699; 10 mg/kg). AG014699 and AG14361 relaxed preconstricted vascular smooth muscle more potently than the standard agent, hydralazine, with no impact on gut motility. AG014699 inhibited MLCK at concentrations that relaxed isolated arteries, whereas AG14361 had no effect. CONCLUSION: Increased vessel perfusion elicited by AG014699 could increase tumor drug accumulation and therapeutic response. Vasoactive concentrations of AG014699 do not cause detrimental side effects to gut motility and may increase the range of therapeutics with which AG014699 could be combined with for clinical benefit.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Vasos Sanguíneos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacologia , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Azulenos/administração & dosagem , Azulenos/farmacologia , Benzodiazepinas/administração & dosagem , Benzodiazepinas/farmacologia , Vasos Sanguíneos/fisiopatologia , Permeabilidade Capilar/efeitos dos fármacos , Dacarbazina/administração & dosagem , Dacarbazina/análogos & derivados , Feminino , Células HT29 , Humanos , Indóis/administração & dosagem , Camundongos , Camundongos Nus , Neoplasias/patologia , Temozolomida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.
Assuntos
Bradicinina/antagonistas & inibidores , Venenos de Crotalídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Viperidae , Sequência de Aminoácidos , Animais , Bradicinina/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Masculino , Dados de Sequência Molecular , Peptídeos/química , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Cauda/efeitos dos fármacosRESUMO
Two novel bradykinin-related peptides (Ala3,Thr6)-bradykinin and (Val1,Thr3,Thr6)-bradykinin, were identified by a systematic sequencing study of peptides in the defensive skin secretion of the yellow-bellied toad, Bombina variegata. These peptides are the first amphibian skin bradykinins to exhibit amino acid substitutions at the Pro3 position of the bradykinin nonapeptide. Previously reported bradykinins from other Bombina species were not detected. Respective precursor cDNAs, designated BVK-1 and BVK-2, respectively, were cloned from a skin library by 3'- and 5'-RACE reactions. BVK-1 contained an open-reading frame of 97 amino acids encoding a single copy of (Ala3,Thr6)-bradykinin and similarly, the open-reading frame of BVK-2 consisted of 96 amino acids encoding a single copy of (Val1,Thr3,Thr6)-bradykinin. Synthetic replicates of each novel bradykinin were found to be active on mammalian arterial and small intestinal smooth muscle preparations. The structural diversity of bradykinins in amphibian defensive skin secretions may be related to defence against specific predators.