RESUMO
Exatecan and deruxtecan are antineoplastic camptothecin derivatives in development as tumor-targeted-delivery warheads in various formulations including peptides, liposomes, polyethylene glycol nanoparticles, and antibody-drug conjugates. Here, we report the molecular pharmacology of exatecan compared with the clinically approved topoisomerase I (TOP1) inhibitors and preclinical models for validating biomarkers and the combination of exatecan with ataxia telangiectasia and Rad3-related kinase (ATR) inhibitors. Modeling exatecan binding at the interface of a TOP1 cleavage complex suggests two novel molecular interactions with the flanking DNA base and the TOP1 residue N352, in addition to the three known interactions of camptothecins with the TOP1 residues R364, D533, and N722. Accordingly, exatecan showed much stronger TOP1 trapping, higher DNA damage, and apoptotic cell death than the classical TOP1 inhibitors used clinically. We demonstrate the value of SLFN11 expression and homologous recombination (HR) deficiency (HRD) as predictive biomarkers of response to exatecan. We also show that exatecan kills cancer cells synergistically with the clinical ATR inhibitor ceralasertib (AZD6738). To establish the translational potential of this combination, we tested CBX-12, a clinically developed pH-sensitive peptide-exatecan conjugate that selectively targets cancer cells and is currently in clinical trials. The combination of CBX-12 with ceralasertib significantly suppressed tumor growth in mouse xenografts. Collectively, our results demonstrate the potency of exatecan as a TOP1 inhibitor and its clinical potential in combination with ATR inhibitors, using SLFN11 and HRD as predictive biomarkers.
Assuntos
DNA Topoisomerases Tipo I , Neoplasias , Inibidores da Topoisomerase I , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Camptotecina/análogos & derivados , DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores da Topoisomerase I/farmacologiaRESUMO
Aims: To determine whether antigen-independent targeting of the TOP1 inhibitor exatecan to tumor with a pH-sensitive peptide (CBX-12) produces superior synergy with immunotherapy compared with unconjugated exatecan. Materials & methods: In vitro and ex vivo functional assays were performed via FACS and ELISA assays. In vivo efficacy was evaluated in the syngeneic CT26 model. Results: CBX-12 combined with anti-PD-1 or anti-CTLA4 results in delayed tumor growth and complete response, with cured animals displaying long-term antitumor immunity. CBX-12 stimulates expression of MHC 1 and PD-L1 and is an inducer of immunogenic cell death, producing long-term immune recognition of tumor cells and resultant antitumor immunity. Conclusion: The authors' data provide the rationale for exploring immunotherapy combinations with CBX-12 in clinical trials.
Although combinations of chemotherapy and immunotherapy have shown great promise for cancer treatment, they have also demonstrated significant safety concerns that require dose reductions. Targeting chemotherapy to the tumor can avoid these safety issues, thereby enhancing efficacy of combination therapies. CBX-12 is a novel peptide-drug agent targeting the TOP1-inhibiting drug exatecan to tumor via pH-sensitive peptide. Unlike tumor targeting via antibody, CBX-12 universally targets all solid tumors. CBX-12 avoids the immune cell toxicity of nontumor-targeted exatecan and safely synergizes with immunotherapies. CBX-12 treatment causes tumor cells to express and secrete molecules that result in activation of immune components to recognize and eliminate tumor cells. These data support the upcoming clinical trials of CBX-12 in combination with immunotherapy.
Assuntos
Neoplasias , Animais , Neoplasias/tratamento farmacológico , Camptotecina/uso terapêutico , Imunoterapia/métodos , Imunidade , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
[This corrects the article DOI: 10.1093/narcan/zcab021.].
RESUMO
Topoisomerase inhibitors are potent DNA damaging agents which are widely used in oncology, and they demonstrate robust synergistic tumor cell killing in combination with DNA repair inhibitors, including poly(ADP)-ribose polymerase (PARP) inhibitors. However, their use has been severely limited by the inability to achieve a favorable therapeutic index due to severe systemic toxicities. Antibody-drug conjugates address this issue via antigen-dependent targeting and delivery of their payloads, but this approach requires specific antigens and yet still suffers from off-target toxicities. There is a high unmet need for a more universal tumor targeting technology to broaden the application of cytotoxic payloads. Acidification of the extracellular milieu arises from metabolic adaptions associated with the Warburg effect in cancer. Here we report the development of a pH-sensitive peptide-drug conjugate to deliver the topoisomerase inhibitor, exatecan, selectively to tumors in an antigen-independent manner. Using this approach, we demonstrate potent in vivo cytotoxicity, complete suppression of tumor growth across multiple human tumor models, and synergistic interactions with a PARP inhibitor. These data highlight the identification of a peptide-topoisomerase inhibitor conjugate for cancer therapy that provides a high therapeutic index, and is applicable to all types of human solid tumors in an antigen-independent manner.
RESUMO
AIMS: Bone morphogenetic proteins (BMPs) are involved in the development and homeostasis of multiple organs and tissues. There has been a significant focus on understanding the role of BMPs in pancreatic ß-cell dysfunction associated with type 2 diabetes (T2D). Our objective was to investigate the relationship between BMP6 and glucose homeostasis. METHODS: Ob/ob mice were treated with BMP6 for 6 days and analyzed for insulin release, body weight, lipid parameters and glucose tolerance. Quantitative real-time PCR, chromatin immunoprecipitation and glucose output assays were used to assess BMP6 effect on gluconeogenesis in rat hepatoma H4IIE cells. Specificity of BMP6 receptors was characterized by the utilization of various receptor Fc fusion proteins in luciferase reporter gene and glucose output assays in INS1 and H4IIE cells. RESULTS: Treatment of ob/ob mice with BMP6 for 6 days resulted in a reduction of circulating glucose and lipid levels, followed by a significantly elevated plasma insulin level in a dose-dependent manner. In addition, BMP6 improved the glucose excursion during an oral glucose tolerance test, lowering the total glycemic response by 21%. In rat H4IIE hepatoma cells, BMP6 inhibited gluconeogenesis and glucose output via downregulation the PepCK expression. Moreover, BMP6 inhibited glucose production regardless of the presence of cAMP, antagonizing its glycogenolytic effect. BMP6 acted on pancreatic and liver cells utilizing Alk3, Alk6 and ActRIIA serine/threonine kinase receptors. CONCLUSIONS: Collectively, we demonstrate that BMP6 improves glycaemia in T2D mice and regulates glucose metabolism in hepatocytes representing an exciting prospect for future treatments of diabetes.
Assuntos
Proteína Morfogenética Óssea 6/farmacologia , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Animais , Glicemia/genética , Glicemia/metabolismo , Proteína Morfogenética Óssea 6/fisiologia , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Homeostase/genética , Insulina/metabolismo , Masculino , Camundongos , Camundongos Obesos , Ratos , Proteínas Recombinantes/farmacologiaRESUMO
As a follow-up to the discovery of our spirocyclic proline-based TPH1 inhibitor lead, we describe the optimization of this scaffold. Through a combination of X-ray co-crystal structure guided design and an in vivo screen, new substitutions in the lipophilic region of the inhibitors were identified. This effort led to new TPH1 inhibitors with in vivo efficacy when dosed as their corresponding ethyl ester prodrugs. In particular, 15b (KAR5585), the prodrug of the potent TPH1 inhibitor 15a (KAR5417), showed robust reduction of intestinal serotonin (5-HT) levels in mice. Furthermore, oral administration of 15b generated high and sustained systemic exposure of the active parent 15a in rats and dogs. KAR5585 was selected for further pharmacological evaluation in disease models associated with a dysfunctional peripheral 5-HT system.
Assuntos
Pró-Fármacos/química , Prolina/análogos & derivados , Pirimidinas/química , Compostos de Espiro/química , Triptofano Hidroxilase/antagonistas & inibidores , Triptofano Hidroxilase/metabolismo , Animais , Sítios de Ligação , Cães , Meia-Vida , Humanos , Concentração Inibidora 50 , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Prolina/metabolismo , Prolina/farmacologia , Estrutura Terciária de Proteína , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Ratos , Serotonina/metabolismo , Compostos de Espiro/metabolismo , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
Pulmonary arterial hypertension (PAH) is a progressive disease defined by a chronic elevation in pulmonary arterial pressure with extensive pulmonary vascular remodeling and perivascular inflammation characterized by an accumulation of macrophages, lymphocytes, dendritic cells, and mast cells. Although the exact etiology of the disease is unknown, clinical as well as preclinical data strongly implicate a role for serotonin (5-HT) in the process. Here, we investigated the chronic effects of pharmacological inhibition of tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in peripheral 5-HT biosynthesis, in two preclinical models of pulmonary hypertension (PH), the monocrotaline (MCT) rat and the semaxanib (SUGEN, Medinoah, Suzhou, China)-hypoxia rat. In both PH models, ethyl (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1'-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate and ethyl (S)-8-(2-amino-6-((R)-1-(3',4'-dimethyl-3-(3-methyl-1 H-pyrazol-1-yl)-[1,1'-biphenyl]-4-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate, novel orally active TPH1 inhibitors with nanomolar in vitro potency, decreased serum, gut, and lung 5-HT levels in a dose-dependent manner and significantly reduced pulmonary arterial pressure, and pulmonary vessel wall thickness and occlusion in male rats. In the MCT rat model, decreases in lung 5-HT significantly correlated with reductions in histamine levels and mast cell number (P < 0.001, r2 = 0.88). In contrast, neither ambrisentan nor tadalafil, which are vasodilators approved for the treatment of PAH, reduced mast cell number or 5-HT levels, nor were they as effective in treating the vascular remodeling as were the TPH1 inhibitors. When administered in combination with ambrisentan, the TPH1 inhibitors showed an additive effect on pulmonary vascular remodeling and pressures. These data demonstrate that in addition to reducing vascular remodeling, TPH1 inhibition has the added benefit of reducing the perivascular mast cell accumulation associated with PH.
Assuntos
Inibidores Enzimáticos/farmacologia , Hipertensão Pulmonar/fisiopatologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiopatologia , Triptofano Hidroxilase/antagonistas & inibidores , Remodelação Vascular/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Serotonina/sangue , Serotonina/metabolismo , Triptofano Hidroxilase/metabolismoRESUMO
An increasing number of diseases have been linked to a dysfunctional peripheral serotonin system. Given that tryptophan hydroxylase 1 (TPH1) is the rate limiting enzyme in the biosynthesis off serotonin, it represents an attractive target to regulate peripheral serotonin. Following up to our first disclosure, we report a new chemotype of TPH1 inhibitors where-by the more common central planar heterocycle has been replaced with an open-chain, acyl guanidine surrogate. Through our work, we found that compounds of this nature provide highly potent TPH1 inhibitors with favorable physicochemical properties that were effective in reducing murine intestinal 5-HT in vivo. Furthermore, we obtained a high resolution (1.90Å) X-ray structure crystal structure of one of these inhibitors (compound 51) that elucidated the active conformation along with revealing a dimeric form of TPH1 for the first time.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Guanidina/farmacologia , Triptofano Hidroxilase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Guanidina/síntese química , Guanidina/química , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Triptofano Hidroxilase/metabolismoRESUMO
Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, ß-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)2D3 decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin.
Assuntos
Doenças Ósseas/sangue , Diabetes Mellitus Tipo 2/complicações , Serotonina/sangue , Animais , Doenças Ósseas/metabolismo , Doenças Ósseas/patologia , Doenças Ósseas/fisiopatologia , Remodelação Óssea , Feminino , Masculino , Camundongos , Tamanho do Órgão , Osteoblastos/patologia , Osteoclastos/patologia , Fenótipo , RatosRESUMO
The central role of the biogenic monoamine serotonin (5-hydroxytryptamine, 5-HT) as a neurotransmitter with important cognitive and behavioral functions is well known. However, 5-HT produced in the brain only accounts for approximately 5% of the total amount of 5-HT generated in the body. At the onset of our work, it appeared that substituted phenylalanine derivatives or related aryl amino acids were required to produce potent inhibitors of TPH1, as significant losses of inhibitory activity were noted in the absence of this structural element. We disclose herein the discovery of a new class of TPH1 inhibitors that significantly lower peripherally 5-HT.
Assuntos
Prolina/análogos & derivados , Triptofano Hidroxilase/antagonistas & inibidores , Animais , Sítios de Ligação , Encéfalo/metabolismo , Desenho de Fármacos , Meia-Vida , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Prolina/síntese química , Prolina/farmacocinética , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Serotonina/metabolismo , Compostos de Espiro/química , Relação Estrutura-Atividade , Triptofano Hidroxilase/metabolismoRESUMO
Bone morphogenetic proteins (BMPs) participate in organ regeneration through autocrine and paracrine actions, but the existence and effects of these proteins in the systemic circulation is unknown. Using liquid chromatography-mass spectrometry, we identified BMP6, GDF15, and the BMP1-3 isoform of the Bmp1 gene in plasma samples from healthy volunteers and patients with CKD. We isolated the endogenous BMP1-3 protein and demonstrated that it circulates as an active enzyme, evidenced by its ability to cleave dentin matrix protein-1 in vitro. In rats with CKD, administration of recombinant BMP1-3 increased renal fibrosis and reduced survival. In contrast, administration of a BMP1-3-neutralizing antibody reduced renal fibrosis, preserved renal function, and increased survival. In addition, treating with the neutralizing antibody was associated with low plasma levels of TGFß1 and connective tissue growth factor. In HEK293 cells and remnant kidneys, BMP1-3 increased the transcription of collagen type I, TGFß1, ß-catenin, and BMP7 via a BMP- and Wnt-independent mechanism that involved signaling through an integrin ß1 subunit. The profibrotic effect of BMP1-3 may, in part, be a result of the accompanied decrease in decorin (DCN) expression. Taken together, inhibition of circulating BMP1-3 reduces renal fibrosis, suggesting that this pathway may be a therapeutic target for CKD.
Assuntos
Proteína Morfogenética Óssea 1/sangue , Proteína Morfogenética Óssea 2/sangue , Proteína Morfogenética Óssea 3/sangue , Nefropatias/patologia , Rim/patologia , Adulto , Idoso , Animais , Proteína Morfogenética Óssea 7/metabolismo , Células Cultivadas , Doença Crônica , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Células HEK293 , Humanos , Rim/metabolismo , Nefropatias/metabolismo , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/metabolismo , beta Catenina/metabolismoRESUMO
PF-04840082 is a humanized prototype anti-Dickkopf-1 (Dkk-1) immunoglobulin isotype G(2) (IgG(2)) antibody for the treatment of osteoporosis. In vitro, PF-04840082 binds to human, monkey, rat, and mouse Dkk-1 with high affinity. After administration of PF-04840082 to rat and monkey, free Dkk-1 concentrations decreased rapidly and returned to baseline in a dose-dependent manner. In rat and monkey, PF-04840082 exhibited nonlinear pharmacokinetics (PK) and a target-mediated drug disposition (TMDD) model was used to characterize PF-04840082 versus Dkk-1 concentration response relationship. PK/pharmacodynamic (PK/PD) modeling enabled estimation of antibody non-target-mediated elimination, Dkk-1 turnover, complex formation, and complex elimination. The TMDD model was translated to human to predict efficacious dose and minimum anticipated biological effect level (MABEL) by incorporating information on typical IgG(2) human PK, antibody-target association/dissociation rates, Dkk-1 expression, and turnover rates. The PK/PD approach to MABEL was compared with the standard "no adverse effect level" (NOAEL) approach to calculating clinical starting doses and a pharmacological equilibrium method. The NOAEL method gave estimates of dose that were too high to ensure safety of clinical trials. The pharmacological equilibrium approach calculated receptor occupancy (RO) based on equilibrium dissociation constant alone and did not take into account rate of turnover of the target or antibody-target complex kinetics and, as a result, it likely produced a substantial overprediction of RO at a given dose. It was concluded that the calculation of MABEL according to the TMDD model was the most appropriate means for ensuring safety and efficacy in clinical studies.
Assuntos
Anticorpos Monoclonais/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Modelos Biológicos , Osteoporose/sangue , Adulto , Idoso , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Doenças Ósseas Metabólicas/sangue , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Macaca fascicularis , Masculino , Camundongos , Pessoa de Meia-Idade , Osteoporose/tratamento farmacológico , Osteoporose Pós-Menopausa/sangue , Ratos , Ratos Sprague-DawleyRESUMO
As part of a strategy to deliver short-acting calcium-sensing receptor (CaSR) antagonists, the metabolically labile thiomethyl functionality was incorporated into the zwitterionic amino alcohol derivative 3 with the hope of increasing human clearance through oxidative metabolism, while delivering a pharmacologically inactive sulfoxide metabolite. The effort led to the identification of thioanisoles 22 and 23 as potent and orally active CaSR antagonists with a rapid onset of action and short pharmacokinetic half-lives, which led to a rapid and transient stimulation of parathyroid hormone in a dose-dependent fashion following oral administration to rats. On the basis of the balance between target pharmacology, safety, and human disposition profiles, 22 and 23 were advanced as clinical candidates for the treatment of osteoporosis.
RESUMO
Synthesis and structure-activity relationship (SAR) studies on 5-trifluoromethylpyrido[4,3-d]pyrimidin-4(3H)-ones, a novel class of calcium receptor antagonists is described with particular emphasis on optimization of the pharmacokinetic/pharmacodynamic parameters required for a short duration of action compound. Orally-active compounds were identified which displayed the desired animal pharmacology (rapid and transient stimulation of parathyroid hormone) essential for bone anabolic effects.
Assuntos
Anabolizantes/química , Pirimidinonas/química , Receptores de Detecção de Cálcio/antagonistas & inibidores , Administração Oral , Anabolizantes/administração & dosagem , Anabolizantes/farmacocinética , Animais , Masculino , Hormônio Paratireóideo/metabolismo , Pirimidinonas/administração & dosagem , Pirimidinonas/farmacocinética , Ratos , Ratos Sprague-Dawley , Receptores de Detecção de Cálcio/metabolismo , Relação Estrutura-AtividadeRESUMO
The design and profile of a series of zwitterionic calcium sensing receptor negative allosteric modulators is described. Evaluation of key analogues using a rat model demonstrate a robust response, significantly improved potency over ronacaleret and have the potential as an oral, anabolic treatment for osteoporosis.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Ciclopropanos/química , Indanos/química , Fenilpropionatos/química , Receptores de Detecção de Cálcio/antagonistas & inibidores , Administração Oral , Anabolizantes , Animais , Linhagem Celular , Ciclopropanos/farmacocinética , Ciclopropanos/farmacologia , Descoberta de Drogas , Humanos , Indanos/farmacocinética , Indanos/farmacologia , Microssomos Hepáticos , Osteoporose/tratamento farmacológico , Fenilpropionatos/farmacocinética , Fenilpropionatos/farmacologia , RatosRESUMO
Sulfonamides, exemplified by 3a, were identified as highly selective EP(2) agonists. Lead optimization led to the identification of CP-533536, 7f, a potent and selective EP(2) agonist. CP-533536 demonstrated the ability to heal fractures when administered locally as a single dose in rat models of fracture healing.
Assuntos
Osteogênese/efeitos dos fármacos , Piridinas/química , Receptores de Prostaglandina E/agonistas , Animais , Masculino , Piridinas/administração & dosagem , Piridinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP2 , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2-/- mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2-/- mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.
Assuntos
Quinase 2 de Adesão Focal/fisiologia , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteogênese/fisiologia , Osteoporose/terapia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Diferenciação Celular , Células Cultivadas , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Knockout , Ovariectomia , Ratos , Ratos Sprague-DawleyRESUMO
Growth differentiation factor (GDF15) is a distant member of the transforming growth factor-beta superfamily, a diverse group of structurally related proteins that exert multiple effects on cell fate such as on cell growth and differentiation but little is known about GDF15 in these processes. Previously we observed the mature GDF15 to be associated with human prostate carcinogenesis hence prompting us to study GDF15 further. Here we report gdf15 expression both at the RNA and protein levels, in normal prostatic tissues of wild type (wt) and prostatic intraepithelial neoplasia (PIN) of transgenic (Tg) 12T-7s model mice during embryonic, postnatal, and adult prostate formation up to 15 weeks after birth. Dynamic changes in expression, at both the mRNA and protein level, correlated with cell proliferation and differentiation during distinct phases of normal mouse prostate development and alterations in the dynamics of gdf15 expression correlated with the changes in development resulting in PIN formation. Most notably mature gdf15 protein was significantly elevated during hyperplasia and PIN development. Changes in the protein levels did not always correlate well with the mRNA levels. This was more prominent during PIN than during normal prostate development suggesting that this may also be an indicator of disturbed regulation of gdf15 in PIN. We propose that gdf15 is a growth factor with dual function either promoting proliferation or growth arrest and differentiation due most likely to differences in cellular differentiation. Because of the differentiation defect in PIN its epithelium no longer responds to gdf15 by cellular growth arrest as does the normal epithelium and gdf may even stimulate proliferation. The data supports our hypothesis that GDF15 plays a role in the early stages of human prostate cancer.
Assuntos
Citocinas/metabolismo , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Citocinas/genética , Modelos Animais de Doenças , Feminino , Fator 15 de Diferenciação de Crescimento , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neoplasia Prostática Intraepitelial/genética , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
UNLABELLED: CP432 is a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 agonist. CP432 stimulates trabecular and cortical bone formation and restores bone mass and bone strength in aged ovariectomized rats with established osteopenia. INTRODUCTION: The purpose of this study was to determine whether a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 (PGE2) agonist, CP432, could produce bone anabolic effects in aged, ovariectomized (OVX) rats with established osteopenia. MATERIALS AND METHODS: CP432 at 0.3, 1, or 3 mg/kg/day was given for 6 weeks by subcutaneous injection to 12-month-old rats that had been OVX for 8.5 months. The effects on bone mass, bone formation, bone resorption, and bone strength were determined. RESULTS: Total femoral BMD increased significantly in OVX rats treated with CP432 at all doses. CP432 completely restored trabecular bone volume of the third lumbar vertebral body accompanied with a dose-dependent decrease in osteoclast number and osteoclast surface and a dose-dependent increase in mineralizing surface, mineral apposition rate, and bone formation rate-tissue reference in OVX rats. CP432 at 1 and 3 mg/kg/day significantly increased total tissue area, cortical bone area, and periosteal and endocortical bone formation in the tibial shafts compared with both sham and OVX controls. CP432 at all doses significantly and dose-dependently increased ultimate strength in the fifth lumber vertebral body compared with both sham and OVX controls. At 1 and 3 mg/kg/day, CP432 significantly increased maximal load in a three-point bending test of femoral shaft compared with both sham and OVX controls. CONCLUSIONS: CP432 completely restored trabecular and cortical bone mass and strength in established osteopenic, aged OVX rats by stimulating bone formation and inhibiting bone resorption on trabecular and cortical surfaces.
Assuntos
Envelhecimento/fisiologia , Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/tratamento farmacológico , Dinoprostona/agonistas , Osteogênese/efeitos dos fármacos , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E/metabolismo , Animais , Peso Corporal , Densidade Óssea/fisiologia , Modelos Animais de Doenças , Feminino , Fêmur/anatomia & histologia , Vértebras Lombares/anatomia & histologia , Estrutura Molecular , Tamanho do Órgão/efeitos dos fármacos , Ovariectomia , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E Subtipo EP4 , Especificidade por Substrato , Tíbia/anatomia & histologiaRESUMO
Heptanoic acid lactams, exemplified by 2, were identified as highly selective EP4 agonists via high throughput screening. Lead optimization led to the identification of lactams with a 30-fold increase in EP4 potency in vitro. Compounds demonstrated robust bone anabolic effects when administered in vivo in rat models of osteoporosis.