RESUMO
Small molecules and antibodies each have advantages and limitations as therapeutics. Here, we present for the first time to our knowledge, the structure-guided design of "chemibodies" as small molecule-antibody hybrids that offer dual recognition of a single target by both a small molecule and an antibody, using DPP-IV enzyme as a proof of concept study. Biochemical characterization demonstrates that the chemibodies present superior DPP-IV inhibition compared to either small molecule or antibody component alone. We validated our design by successfully solving a co-crystal structure of a chemibody in complex with DPP-IV, confirming specific binding of the small molecule portion at the interior catalytic site and the Fab portion at the protein surface. The discovery of chemibodies presents considerable potential for novel therapeutics that harness the power of both small molecule and antibody modalities to achieve superior specificity, potency, and pharmacokinetic properties.
Assuntos
Dipeptidil Peptidase 4/química , Inibidores Enzimáticos/síntese química , Imunoconjugados/farmacologia , Animais , Anticorpos Monoclonais/química , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Imunoconjugados/química , Modelos Moleculares , Ratos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
PURPOSE: Although angle surgeries show good success in primary congenital glaucoma, reported success in glaucoma following cataract surgery (GFCS) and juvenile open-angle glaucoma (JOAG) is variable and with relatively short follow-up. We evaluated longer-term outcomes of 360-degree trabeculotomy for medically refractory GFCS and JOAG. DESIGN: Retrospective case series. METHODS: First operated eyes of consecutive patients with medically refractory GFCS and JOAG in a single-surgeon pediatric glaucoma practice who underwent illuminated microcatheter-assisted 360-degree trabeculotomy from February 2008 to June 2015 were reviewed. Baseline characteristics, time to failure or last visit, surgical details, final intraocular pressure (IOP), and complications were recorded. Success required IOP ≤22 mm Hg and 20% reduction without additional glaucoma surgery or devastating complication. RESULTS: Thirty-five eyes (35 patients) were included: 25 GFCS and 10 JOAG (mean age at surgery 5.6 vs 16.7 years, respectively, P < .001). Success for GFCS and JOAG was 18 of 25 (72%) vs 6 of 10 (60%) eyes at mean follow-up of 31.9 ± 26.1 vs 24.5 ± 19.7 months, respectively. IOP was significantly reduced from baseline for both GFCS and JOAG (31.5 ± 7.5 mm Hg vs 19.2 ± 7.7 mm Hg, P < .001; and 29.5 ± 10.3 mm Hg vs 15.8 ± 6.6 mm Hg, P < .001, respectively). Fewer glaucoma medications were needed after surgery (P = .01) for GFCS but not JOAG. Complications (all but 2 spontaneously resolving) included choroidal effusion (1), vitreous hemorrhage (3), Descemet detachment (1), and persistent hyphema (2). Three-year Kaplan-Meier success for GFCS vs JOAG was 75.3% vs 53.3%, respectively. CONCLUSIONS: Illuminated microcatheter-assisted 360-degree trabeculotomy is a useful, low-risk, modestly successful initial surgical treatment for both medically refractory GFCS and JOAG.
Assuntos
Extração de Catarata/efeitos adversos , Catarata/complicações , Glaucoma de Ângulo Aberto/cirurgia , Pressão Intraocular/fisiologia , Trabeculectomia/métodos , Acuidade Visual , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Seguimentos , Glaucoma de Ângulo Aberto/etiologia , Glaucoma de Ângulo Aberto/fisiopatologia , Gonioscopia , Humanos , Lactente , Masculino , Complicações Pós-Operatórias , Estudos Retrospectivos , Fatores de Tempo , Tonometria Ocular , Resultado do Tratamento , Adulto JovemRESUMO
The structure-based design and optimization of a novel series of selective PERK inhibitors are described resulting in the identification of 44 as a potent, highly selective, and orally active tool compound suitable for PERK pathway biology exploration both in vitro and in vivo.
Assuntos
Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , eIF-2 Quinase/antagonistas & inibidores , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-Atividade , eIF-2 Quinase/metabolismoRESUMO
The Cell division cycle 7 (Cdc7) protein kinase is essential for DNA replication and maintenance of genome stability. We systematically explored thiazole-based compounds as inhibitors of Cdc7 kinase activity in cancer cells. Our studies resulted in the identification of a potent, selective Cdc7 inhibitor that decreased phosphorylation of the direct substrate MCM2 in vitro and in vivo, and inhibited DNA synthesis and cell viability in vitro.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tiazóis/síntese química , Tiazóis/farmacologia , Animais , Domínio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Técnicas de Química Sintética , Feminino , Células HCT116 , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Simulação de Acoplamento Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Aphakic and juvenile open-angle glaucoma (JOAG) cases often prove to be challenging to manage, frequently requiring surgical intervention. Angle surgery has some reported success in these cases. PURPOSE: The purpose of this study was to evaluate 360-degree trabeculotomy, facilitated by iTrack, for refractory aphakic glaucoma and JOAG. PATIENTS AND METHODS: This study was conducted to evaluate the success and complication rates of illuminated microcatheter-assisted 360-degree trabeculotomy for aphakic glaucoma and JOAG (2 surgeons/2 sites, 2008 to 2011). The success of this surgery was defined as intraocular pressure ≤22 mm Hg with >30% reduction, without disease progression, oral glaucoma medications, or additional glaucoma surgery. One eye per subject was analyzed. All had gonioscopically open angles preoperatively. RESULTS: A total of 23 eyes status post iTrack-facilitated 360-degree trabeculotomy, 13 aphakic glaucoma cases (mean age 3.1 y at surgery), and 10 JOAG cases (mean age, 18.6 y) were included in the study. Complete cannulation/opening of the Schlemm canal occurred intraoperatively in 8 aphakic and in all JOAG cases. Success rates achieved at last follow-up were as follows: 8/13 (62%) aphakic glaucoma cases and 9/10 (90%) JOAG cases. Preoperative versus final intraocular pressure decreased for all surgically successful eyes (35.5±3.9 vs. 17.3±4.6 mm Hg for aphakic glaucoma, P<0.0001, after mean 30 mo and 30.7±7.4 vs. 13.4±2.8 mm Hg for JOAG, P=0.0001, after mean 10 mo). All trabeculotomy failures (n=5) occurred within 5 months. Complications included vitreous hemorrhage (2 aphakic eyes) and transient choroidal effusion (1 aphakic eye). CONCLUSIONS: iTrack-facilitated 360-degree trabeculotomy shows early promise for initial surgical treatment of medically refractory aphakic glaucoma and JOAG, with few complications and without affecting future surgical options.
Assuntos
Afacia Pós-Catarata/cirurgia , Catéteres , Glaucoma de Ângulo Aberto/cirurgia , Trabeculectomia/instrumentação , Adolescente , Adulto , Afacia Pós-Catarata/diagnóstico , Afacia Pós-Catarata/fisiopatologia , Criança , Pré-Escolar , Feminino , Seguimentos , Glaucoma de Ângulo Aberto/diagnóstico , Glaucoma de Ângulo Aberto/fisiopatologia , Gonioscopia , Humanos , Lactente , Pressão Intraocular/fisiologia , Iluminação , Masculino , Estudos Retrospectivos , Tonometria Ocular , Trabeculectomia/métodos , Resultado do Tratamento , Adulto JovemRESUMO
The human hypoxia-inducible factor prolyl hydroxylases 1, 2, and 3 (HIF-PHD1, -2, and -3) are thought to act as proximal sensors of cellular hypoxia by virtue of their mechanism-based dependence on molecular oxygen. These 2-oxoglutarate (2-OG) and non-heme iron-dependent oxygenases constitutively hydroxylate HIF, resulting in high-affinity binding to Von Hippel-Lindau protein (pVHL). Some reported affinities for the HIF-PHDs for 2-OG and iron approach the estimated physiological concentrations for these cofactors, suggesting that the system as described is not catalytically optimal. Here we report the enzymatic characterization of full-length recombinant human HIF-PHD2 using a novel and sensitive catalytic assay. We demonstrated submicromolar affinities for 2-OG and ferrous iron and HIF-PHD2 Km values for oxygen that are greater than atmospheric oxygen levels, suggesting that molecular oxygen is indeed the key regulator of this pathway. In addition, we observed enhancement of HIF-PHD2 catalytic activity in the presence of ascorbic acid with only minor modifications of HIF-PHD2 requirements for 2-OG, and a detailed pH study demonstrated optimal HIF-PHD2 catalytic activity at pH 6.0. Lastly, we used this sensitive and facile assay to rapidly perform a large high-throughput screen of a chemical library to successfully identify and characterize novel 2-OG competitive inhibitors of HIF-PHD2.
Assuntos
Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência/métodos , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/química , Ácido Ascórbico/metabolismo , Inibidores Enzimáticos/análise , Humanos , Concentração de Íons de Hidrogênio , Prolina Dioxigenases do Fator Induzível por Hipóxia , Ácidos Cetoglutáricos/metabolismo , Cinética , Modelos Biológicos , Oxigênio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
We report the structure-based design and synthesis of a novel series of aza-benzimidazoles as PHD2 inhibitors. These efforts resulted in compound 22, which displayed highly potent inhibition of PHD2 function in vitro.
Assuntos
Compostos Aza/síntese química , Compostos Aza/farmacologia , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Compostos Aza/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Benzimidazóis/química , Técnicas de Química Combinatória , Cristalografia por Raios X , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Inhibition of the PHD2 enzyme has been associated with increased red blood cell levels. From a screening hit, a series of novel hydroxyl-thiazoles were developed as potent PHD2 inhibitors.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/síntese química , Tiazóis/química , Química Farmacêutica/métodos , Diabetes Mellitus/tratamento farmacológico , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Eritrócitos/enzimologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Concentração Inibidora 50 , Modelos Químicos , Modelos Moleculares , Infarto do Miocárdio/tratamento farmacológico , Doenças Vasculares Periféricas/terapia , Acidente Vascular Cerebral/tratamento farmacológico , Relação Estrutura-Atividade , Tiazóis/farmacologiaRESUMO
A series of (4-piperidinylphenyl)aminoethyl amides based on dipeptide anilines were synthesized and tested against cathepsin K, cathepsin L and cathepsin B. These new non-covalent inhibitors exhibited single-digit nM inhibition of the cysteine proteases. Compounds 3 and 7 demonstrated potency in both mouse and human osteoclast resorption assays.