RESUMO
The G2019S variant of LRRK2, which causes an increase in kinase activity, is associated with the occurrence of Parkinson's disease (PD). Potent, mutation-selective, and brain penetrant inhibitors of LRRK2 can suppress the biological effects specific to G2019S-LRRK2 that cause pathogenicity. We report the discovery of a series of cyanoindane and cyanotetralin kinase inhibitors culminating in compound 34 that demonstrated selective inhibition of phosphorylation of LRRK2 in the mouse brain. These novel inhibitors may further enable the precision medicine path for future PD therapeutics.
RESUMO
It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody-drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addition to the DAR and site of conjugation, linker composition also plays an important role in the properties of an ADC. We have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chemistry results in a stable C-C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled us to undertake a systematic evaluation of the impact of conjugation site and linker composition on ADC properties. Here, we describe the production and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-iso-Pictet-Spengler (HIPS) chemistry. We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.
Assuntos
Aldeídos/química , Anticorpos Monoclonais/química , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Receptor ErbB-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Neoplasias da Mama/patologia , Feminino , Humanos , Imunoconjugados/farmacologia , Camundongos , Camundongos SCID , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of [1,2,4]triazolo[4,3-b]pyridazines that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays and show an unprecedented selectivity towards the G2019S mutant. A structural rational for the observed selectivity is proposed.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridazinas/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Piridazinas/síntese química , Piridazinas/química , Relação Estrutura-AtividadeRESUMO
Leucine rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of cinnoline-3-carboxamides that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays. These compounds are also shown to be potent inhibitors in a cellular assay and to have good to excellent CNS penetration.
Assuntos
Compostos Heterocíclicos com 2 Anéis/química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Amidas/metabolismo , Animais , Sítios de Ligação , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , TransfecçãoRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familial Parkinson's disease (PD). The kinase activity of this complex protein is increased by pathogenic mutations. Inhibition of LRRK2 kinase activity has therefore emerged as a promising approach for the treatment of PD. Herein we report our findings on a series of 4-alkylamino-7-aryl-3-cyanoquinolines that exhibit kinase inhibitory activity against both wild type and G2019S mutant LRRK2. Activity was determined in both biochemical and cellular assays. Compound 14 was further evaluated in an in vivo pharmacodynamic study and found to significantly inhibit Ser935 phosphorylation after oral dosing.
Assuntos
Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinolinas/farmacologia , Animais , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Moleculares , Estrutura Molecular , Mutação , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
Structure-activity relationship (SAR) of a novel, potent and metabolically stable series of benzo [3.2.1] bicyclic sulfonamide-pyrazoles as γ-secretase inhibitors are described. Compounds that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via oral dose, as well as those with high selectivity over Notch, are highlighted.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Camundongos , Pirazóis/química , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
The structure-activity relationship (SAR) of a novel, potent and metabolically stable series of sulfonamide-pyrazoles that attenuate ß-amyloid peptide synthesis via γ-secretase inhibition is detailed herein. Sulfonamide-pyrazoles that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via a single PO dose, as well as sulfonamide-pyrazoles that exhibit selectivity for inhibition of APP versus Notch processing by γ-secretase, are highlighted.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/síntese química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Sulfonamidas/síntese química , Sulfonamidas/farmacologia , Peptídeos beta-Amiloides/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Compostos Heterocíclicos com 3 Anéis/química , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
Utilizing a pharmacophore hypothesis, previously described gamma-secretase inhibiting HTS hits were evolved into novel tricyclic sulfonamide-pyrazoles, with high in vitro potency, good brain penetration, low metabolic stability, and high clearance.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Animais , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Pirazóis/química , Pirazóis/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMO
Discovery of a series of pyrazolopiperidine sulfonamide based gamma-secretase inhibitors and its SAR evolution is described. Significant increases in APP potency on the pyrazolopiperidine scaffold over the original N-bicyclic sulfonamide scaffold were achieved and this potency increase translated in an improved in vivo efficacy.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Piperidinas/química , Piperidinas/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Doença de Alzheimer/tratamento farmacológico , Animais , Humanos , Camundongos , Modelos Moleculares , Piperidinas/uso terapêutico , Relação Estrutura-Atividade , Sulfonamidas/uso terapêuticoRESUMO
Significant improvement in metabolic stability on the pyrazolopiperidine scaffold over the original series were achieved and this stability improvement translated in an improved in vivo efficacy.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Piperidinas/química , Sulfonamidas/química , Animais , Descoberta de Drogas , Estabilidade de Medicamentos , Microssomos Hepáticos/metabolismo , Piperidinas/síntese química , Piperidinas/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Sulfonamidas/síntese química , Sulfonamidas/farmacologiaRESUMO
Pathogenic variants in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified that increase the risk for developing Parkinson's disease in a dominantly inherited fashion. These pathogenic variants, of which G2019S is the most common, cause abnormally high kinase activity, and compounds that inhibit this activity are being pursued as potentially disease-modifying therapeutics. Because LRRK2 regulates important cellular processes, developing inhibitors that can selectively target the pathogenic variant while sparing normal LRRK2 activity could offer potential advantages in heterozygous carriers. We conducted a high-throughput screen and identified a single selective compound that preferentially inhibited G2019S-LRRK2. Optimization of this scaffold led to a series of novel, potent, and highly selective G2019S-LRRK2 inhibitors.
Assuntos
Indazóis/farmacologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tetrazóis/farmacologia , Animais , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Indazóis/síntese química , Indazóis/farmacocinética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos , Estrutura Molecular , Mutação , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Tetrazóis/síntese química , Tetrazóis/farmacocinéticaRESUMO
The structural modification of a series of [3.3.1] bicyclic sulfonamide based gamma-secretase inhibitors is described. Appropriate substitution on the bicyclic scaffold provides a significant increase in the metabolic stability of the compounds resulting in an improved in vivo metabolic profile.
Assuntos
Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Compostos Bicíclicos com Pontes/química , Inibidores de Proteases/química , Sulfonamidas/química , Doença de Alzheimer/tratamento farmacológico , Compostos Bicíclicos com Pontes/farmacologia , Compostos Bicíclicos com Pontes/uso terapêutico , Humanos , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêuticoRESUMO
In this Letter, we report our strategy to design potent and metabolically stable gamma-secretase inhibitors that are efficacious in reducing the cortical Abetax-40 levels in FVB mice via a single PO dose.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Inibidores de Proteases/síntese química , Pirazóis/química , Quinolinas/química , Sulfonamidas/química , Administração Oral , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Desenho de Fármacos , Camundongos , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Quinolinas/síntese química , Quinolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
The B1 receptor is an attractive target for the treatment of pain and inflammation. A series of 3-carboxamido-5-phenacylamino pyrazole B1 receptor antagonists are described that exhibit good potency against B1 and high selectivity over B2. Initially, N-unsubstituted pyrazoles were studied, but these compounds suffered from extensive glucuronidation in primates. This difficulty could be surmounted by the use of N-substituted pyrazoles. Optimization efforts culminated in compound 41, which has high receptor potency and metabolic stability.
Assuntos
Benzamidas/síntese química , Antagonistas de Receptor B1 da Bradicinina , Pirazóis/síntese química , Benzamidas/química , Benzamidas/farmacologia , Cristalografia por Raios X , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Pulmão/citologia , Estrutura Molecular , Pirazóis/química , Pirazóis/farmacologia , Ensaio Radioligante , Relação Estrutura-AtividadeRESUMO
Expanded ligation techniques are sorely needed to generate unique linkages for the growing field of functionally enhanced proteins. To address this need, we present a unique chemical ligation that involves the double addition of a pyrazolone moiety with an aldehyde-labeled protein. This ligation occurs via a tandem Knoevenagel condensation-Michael addition. A pyrazolone reacts with an aldehyde to generate an enone, which undergoes subsequent attack by a second pyrazolone to generate a bis-pyrazolone species. This rapid and facile ligation technique is performed under mild conditions in the absence of catalyst to generate new architectures that were previously inaccessible via conventional ligation reactions. Using this unique ligation, we generated three site-specifically labeled antibody-drug conjugates (ADCs) with an average of four drugs to one antibody. The in vitro and in vivo efficacies along with pharmacokinetic data of the site-specific ADCs are reported.
RESUMO
There is a need for facile chemistries that allow for chemo- and regioselectivity in bioconjugation reactions. To address this need, we are pioneering site-specific bioconjugation methods that use formylglycine as a bioorthogonal handle on a protein surface. Here we introduce aldehyde-specific bioconjugation chemistry, the trapped-Knoevenagel ligation. The speed and stability of the trapped-Knoevenagel ligation further advances the repertoire of aldehyde-based bioconjugations and expands the toolbox for site-specific protein modifications. The trapped-Knoevenagel ligation reaction can be run at near neutral pH in the absence of catalysts to produce conjugates that are stable under physiological conditions. Using this new ligation, we generated an antibody-drug conjugate that demonstrates excellent efficacy in vitro and in vivo.
Assuntos
Carbono/química , Proteínas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/toxicidade , Brentuximab Vedotin , Catálise , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Imunoconjugados/química , Pirazóis/química , Trastuzumab/químicaRESUMO
The delta-oxygenated allylic stannanes 4.4 and 4.5, prepared through addition of Bu(3)SnLi to gamma-OTBS crotonaldehyde 4.3c followed by etherification of the adduct with TBS-Cl or MOM-Cl, undergo transmetalation with InCl(3) and in situ addition to aldehydes leading to mainly anti adducts 5.1 or 5.2, accompanied by varying amounts of syn diastereomers. Selectivities of >95:5 can be realized with the MOM reagent 4.5 and ynals 4.3d and 4.3e or cyclohexanecarboxaldehyde 4.3a. With enals 4.3b and 4.3c, 80:20 mixtures of anti and syn adducts are formed. The S enantiomer 10.1 of stannane 4.5 has also been prepared as a reagent for carbohydrate synthesis. Accordingly, addition to alpha-ODPS acetaldehyde 10.2 in the presence of InCl(3) leads to the adduct 10.3 as an inseparable 90:10 mixture of anti and syn diastereomers. Dihydroxylation of the OTBS derivative 10.4 affords the potential altrose precursor 10.5 in 81% yield.
RESUMO
In the context of antibody-drug conjugates (ADCs), noncleavable linkers provide a means to deliver cytotoxic small molecules to cell targets while reducing systemic toxicity caused by nontargeted release of the free drug. Additionally, noncleavable linkers afford an opportunity to change the chemical properties of the small molecule to improve potency or diminish affinity for multidrug transporters, thereby improving efficacy. We employed the aldehyde tag coupled with the hydrazino-iso-Pictet-Spengler (HIPS) ligation to generate a panel of site-specifically conjugated ADCs that varied only in the noncleavable linker portion. The ADC panel comprised antibodies carrying a maytansine payload ligated through one of five different linkers. Both the linker-maytansine constructs alone and the resulting ADC panel were characterized in a variety of in vitro and in vivo assays measuring biophysical and functional properties. We observed that slight differences in linker design affected these parameters in disparate ways, and noted that efficacy could be improved by selecting for particular attributes. These studies serve as a starting point for the exploration of more potent noncleavable linker systems.
Assuntos
Anticorpos/química , Antineoplásicos/química , Imunoconjugados/química , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoconjugados/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos SCID , Conformação MolecularRESUMO
Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aß generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds 30 (ELND006) and 34 (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aß generation in vivo between 30, 34, Semagacestat 41, Begacestat 42, and Avagacestat 43 in mice is made. 30 lowered Aß in the CSF of healthy human volunteers.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Pirazóis/farmacologia , Quinolinas/farmacologia , Receptores Notch/antagonistas & inibidores , Sulfonamidas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Peptídeos beta-Amiloides/metabolismo , Animais , Área Sob a Curva , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cães , Relação Dose-Resposta a Droga , Desenho de Fármacos , Estabilidade de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/química , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Químicos , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/farmacocinética , Quinolinas/síntese química , Quinolinas/farmacocinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Notch/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Fatores de Tempo , Fatores de Transcrição HES-1RESUMO
INTRODUCTION: Inhibition of gamma-secretase presents a direct target for lowering Aß production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy. METHODS: In vitro assays monitoring inhibitor potencies at APP γ-site cleavage (equivalent to Aß40), and Notch ε-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of Aß production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain Aß was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain Aß reduction vs. Notch signaling endpoints in periphery. RESULTS: The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting Aß production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain Aß in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain Aß was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested. CONCLUSIONS: The discordant in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.