RESUMO
Aß, the product of APP (amyloid precursor protein), has been implicated in the pathophysiology of Alzheimer's disease (AD). ß-Site APP cleaving enzyme1 (BACE1) is the enzyme initiating the processing of the APP to Aß peptides. Small molecule BACE1 inhibitors are expected to decrease Aß-peptide generation and thereby reduce amyloid plaque formation in the brain, a neuropathological hallmark of AD. BACE1 inhibition thus addresses a key mechanism in AD and its potential as a therapeutic target is currently being addressed in clinical studies. Here, we report the discovery and the pharmacokinetic and pharmacodynamic properties of BACE1 inhibitor AZ-4217, a high potency compound (IC50 160 pM in human SH-SY5Y cells) with an excellent in vivo efficacy. Central efficacy of BACE1 inhibition was observed after a single dose in C57BL/6 mice, guinea pigs, and in an APP transgenic mouse model of cerebral amyloidosis (Tg2576). Furthermore, we demonstrate that in a 1 month treatment paradigm BACE1 inhibition of Aß production does lower amyloid deposition in 12-month-old Tg2576 mice. These results strongly support BACE1 inhibition as concretely impacting amyloid deposition and therefore potentially an important approach for therapeutic intervention in AD.
Assuntos
Doença de Alzheimer/patologia , Amiloide/metabolismo , Inibidores Enzimáticos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Células Cultivadas , Córtex Cerebral/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Feminino , Cobaias , Humanos , Isoindóis/farmacologia , Isoindóis/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Fragmentos de Peptídeos/metabolismo , Piridonas/farmacologia , Piridonas/uso terapêutico , Fatores de TempoRESUMO
ß-Site amyloid precursor protein cleaving enzyme1 (BACE1) is one of the key enzymes involved in the processing of the amyloid precursor protein (APP) and formation of amyloid ß peptide (Aß) species. Because cerebral deposition of Aß species might be critical for the pathogenesis of Alzheimer disease, BACE1 has emerged as a key target for the treatment of this disease. Here, we report the discovery and comprehensive preclinical characterization of AZD3839, a potent and selective inhibitor of human BACE1. AZD3839 was identified using fragment-based screening and structure-based design. In a concentration-dependent manner, AZD3839 inhibited BACE1 activity in a biochemical fluorescence resonance energy transfer (FRET) assay, Aß and sAPPß release from modified and wild-type human SH-SY5Y cells and mouse N2A cells as well as from mouse and guinea pig primary cortical neurons. Selectivity against BACE2 and cathepsin D was 14 and >1000-fold, respectively. AZD3839 exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aß levels in mouse, guinea pig, and non-human primate. Pharmacokinetic/pharmacodynamic analyses of mouse and guinea pig data showed a good correlation between the potency of AZD3839 in primary cortical neurons and in vivo brain effects. These results suggest that AZD3839 effectively reduces the levels of Aß in brain, CSF, and plasma in several preclinical species. It might, therefore, have disease-modifying potential in the treatment of Alzheimer disease and related dementias. Based on the overall pharmacological profile and its drug like properties, AZD3839 has been progressed into Phase 1 clinical trials in man.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Indóis/farmacologia , Pirimidinas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Biomarcadores/metabolismo , Encéfalo/metabolismo , Catepsina D/metabolismo , Linhagem Celular , Progressão da Doença , Relação Dose-Resposta a Droga , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Transferência Ressonante de Energia de Fluorescência/métodos , Cobaias , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Resultado do TratamentoRESUMO
The evaluation of a series of bicyclic aminoimidazoles as potent BACE-1 inhibitors is described. The crystal structures of compounds 14 and 23 in complex with BACE-1 reveal hydrogen bond interactions with the protein important for achieving potent inhibition. The optimization of permeability and efflux properties of the compounds is discussed as well as the importance of these properties for attaining in vivo brain efficacy. Compound (R)-25 was selected for evaluation in vivo in wild type mice and 1.5h after oral co-administration of 300µmol/kg (R)-25 and efflux inhibitor GF120918 the brain Aß40 level was reduced by 17% and the plasma Aß40 level by 76%.
Assuntos
Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Encéfalo/efeitos dos fármacos , Imidazóis/química , Imidazóis/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Aminas/química , Aminas/farmacocinética , Aminas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/enzimologia , Encéfalo/metabolismo , Linhagem Celular , Cristalografia por Raios X , Imidazóis/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Fragmentos de Peptídeos/metabolismoRESUMO
Benzothiazole amides were identified as TRPV1 antagonists from high throughput screening using recombinant human TRPV1 receptor and structure-activity relationships were explored to pinpoint key pharmacophore interactions. By increasing aqueous solubility, through the attachment of polar groups to the benzothiazole core, and enhancing metabolic stability, by blocking metabolic sites, the drug-like properties and pharmokinetic profiles of benzothiazole compounds were sufficiently optimized such that their therapeutic potential could be verified in rat pharmacological models of pain.
Assuntos
Amidas/farmacologia , Benzotiazóis/farmacologia , Dor/tratamento farmacológico , Canais de Cátion TRPV/antagonistas & inibidores , Amidas/administração & dosagem , Amidas/química , Animais , Benzotiazóis/administração & dosagem , Benzotiazóis/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Inflamação/tratamento farmacológico , Estrutura Molecular , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Solubilidade , Relação Estrutura-AtividadeRESUMO
beta-Secretase (BACE) is an aspartyl protease, which proteolytically processes amyloid precursor protein, making BACE an interesting pharmacological target in Alzheimer's disease. To study the enzymatic function of BACE, we mutated either of the two aspartic acid residues in the active site of BACE. This rendered BACE functionally inactive without affecting the degree of glycosylation or endosomal localization. In contrast, substituting both active site aspartic acid residues produced a functionally inactive, endoplasmic reticulum-retained and partially glycosylated BACE. Interestingly, co-expression of the two single active site mutants partially restored beta-site cleavage of amyloid precursor protein, and the restored activity was inhibited with similar dose-dependency and potency as wildtype BACE by a small molecule inhibitor raised against BACE. In sum, our data suggest that two different active site mutants can complement each other in a partially functional BACE dimer and mediate APP processing.
Assuntos
Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/genética , Domínio Catalítico/genética , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Knockout , Mutação , Multimerização ProteicaRESUMO
Approaches to the design of libraries for fragment screening are illustrated with reference to a 20 k generic fragment screening library and a 1.2 k generic NMR screening library. Tools and methods for library design that have been developed within AstraZeneca are described, including Foyfi fingerprints and the Flush program for neighborhood characterization. It will be shown how Flush and the BigPicker, which selects maximally diverse sets of compounds, are used to apply the Core and Layer method for library design. Approaches to partitioning libraries into cocktails are also described.
Assuntos
Descoberta de Drogas , Ligantes , Terapia de Alvo Molecular , Bibliotecas de Moléculas Pequenas/química , Sítios de Ligação , Técnicas de Química Combinatória , Desenho Assistido por Computador , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Conformação Proteica , Bibliotecas de Moléculas Pequenas/uso terapêutico , Software , Relação Estrutura-AtividadeRESUMO
BACE1 is responsible for the first step in APP proteolysis, leading to toxic Aß production, and has been indicated to play a key role in the pathogenesis of Alzheimer's disease. The related isoform BACE2 is thought to be involved in processing of the pigment cell-specific melanocyte protein. To avoid potential effects on pigmentation, we investigated the feasibility for developing isoform-selective BACE1 inhibitors. Cocrystal structures of 47 compounds were analyzed and clustered according to their selectivity profiles. Selective BACE1 inhibitors were found to exhibit two distinct conformational features proximal to the flap and the S3 subpocket. Several new molecules were designed and tested to make use of this observation. The combination of a pyrimidinyl C-ring and a methylcyclohexyl element resulted in lead molecule 28, which exhibited â¼50-fold selectivity. Compared to a nonselective BACE1/2 inhibitor, 28 showed significantly less inhibition of PMEL processing in human melanocytes, indicating good functional selectivity of this inhibitor class.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Secretases da Proteína Precursora do Amiloide/química , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/química , Encéfalo/metabolismo , Domínio Catalítico , Cães , Feminino , Humanos , Células Madin Darby de Rim Canino , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Oxazóis/síntese química , Oxazóis/química , Oxazóis/farmacocinética , Oxazóis/farmacologia , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Inibidores de Proteases/farmacocinética , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Ratos , Compostos de Espiro/síntese química , Compostos de Espiro/química , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade , Antígeno gp100 de Melanoma/metabolismoRESUMO
Fragment-based lead generation has led to the discovery of a novel series of cyclic amidine-based inhibitors of beta-secretase (BACE-1). Initial fragment hits with an isocytosine core having millimolar potency were identified via NMR affinity screening. Structure-guided evolution of these fragments using X-ray crystallography together with potency determination using surface plasmon resonance and functional enzyme inhibition assays afforded micromolar inhibitors. Similarity searching around the isocytosine core led to the identification of a related series of inhibitors, the dihydroisocytosines. By leveraging the knowledge of the ligand-BACE-1 recognition features generated from the isocytosines, the dihydroisocytosines were efficiently optimized to submicromolar potency. Compound 29, with an IC50 of 80 nM, a ligand efficiency of 0.37, and cellular activity of 470 nM, emerged as the lead structure for future optimization.
Assuntos
Amidinas/síntese química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Citosina/análogos & derivados , Modelos Moleculares , Pirimidinas/síntese química , Amidinas/química , Amidinas/farmacologia , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Linhagem Celular , Cristalografia por Raios X , Citosina/síntese química , Citosina/química , Citosina/farmacologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Pirimidinas/química , Pirimidinas/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A series of dihydrofolate reductase (DHFR) inhibitors, where the methylenamino-bridge of non-classical inhibitors was replaced with an ester function, have been prepared as potential soft drugs intended for inhalation against Pneumocystis carinii pneumonia (PCP). Several of the new ester-based inhibitors that should serve as good substrates for the ubiquitous esterases and possibly constitute safer alternatives to metabolically stable DHFR inhibitors administered orally, were found to be potent inhibitors of P. carinii DHFR (pcDHFR). Although the objectives of the present program is to achieve a favorable toxicity profile by applying the soft drug concept, a high preference for inhibition of the fungal DHFR versus the mammalian DHFR is still desirable to suppress host toxicity at the site of administration. Compounds with a slight preference for the fungal enzyme were identified. The selection of the target compounds for synthesis was partly guided by an automated docking and scoring procedure as well as molecular dynamics simulations. The modest selectivity of the synthesized inhibitors was reasonably well predicted, although a correct ranking of the relative affinities was not successful in all cases.
Assuntos
Ésteres/química , Ésteres/síntese química , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/síntese química , Pneumocystis carinii/enzimologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Tetra-Hidrofolato Desidrogenase/químicaRESUMO
In order to find optimal core structures as starting points for lead optimization, a multiparameter lead generation workflow was designed with the goal of finding BACE-1 inhibitors as a treatment for Alzheimer's disease. De novo design of core fragments was connected with three predictive in silico models addressing target affinity, permeability, and hERG activity, in order to guide synthesis. Taking advantage of an additive SAR, the prioritized cores were decorated with a few, well-characterized substituents from known BACE-1 inhibitors in order to allow for core-to-core comparisons. Prediction methods and analyses of how physicochemical properties of the core structures correlate to in vitro data are described. The syntheses and in vitro data of the test compounds are reported in a separate paper by Ginman et al. [J. Med. Chem. 2013, 56, 4181-4205]. The affinity predictions are described in detail by Roos et al. [J. Chem. Inf. 2014, DOI: 10.1021/ci400374z].
RESUMO
The evaluation of a series of aminoisoindoles as ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors and the discovery of a clinical candidate drug for Alzheimer's disease, (S)-32 (AZD3839), are described. The improvement in permeability properties by the introduction of fluorine adjacent to the amidine moiety, resulting in in vivo brain reduction of Aß40, is discussed. Due to the basic nature of these compounds, they displayed affinity for the human ether-a-go-go related gene (hERG) ion channel. Different ways to reduce hERG inhibition and increase hERG margins for this series are described, culminating in (S)-16 and (R)-41 showing large in vitro margins with BACE1 cell IC(50) values of 8.6 and 0.16 nM, respectively, and hERG IC(50) values of 16 and 2.8 µM, respectively. Several compounds were advanced into pharmacodynamic studies and demonstrated significant reduction of ß-amyloid peptides in mouse brain following oral dosing.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Indóis/síntese química , Pirimidinas/síntese química , Administração Oral , Alcinos/síntese química , Alcinos/farmacocinética , Alcinos/farmacologia , Amidas/síntese química , Amidas/farmacocinética , Amidas/farmacologia , Secretases da Proteína Precursora do Amiloide/química , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/química , Disponibilidade Biológica , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Cristalografia por Raios X , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligação de Hidrogênio , Indóis/farmacocinética , Indóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Fragmentos de Peptídeos/metabolismo , Permeabilidade , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Amino-2H-imidazoles are described as a new class of BACE-1 inhibitors for the treatment of Alzheimer's disease. Synthetic methods, crystal structures, and structure-activity relationships for target activity, permeability, and hERG activity are reported and discussed. Compound (S)-1m was one of the most promising compounds in this report, with high potency in the cellular assay and a good overall profile. When guinea pigs were treated with compound (S)-1m, a concentration and time dependent decrease in Aß40 and Aß42 levels in plasma, brain, and CSF was observed. The maximum reduction of brain Aß was 40-50%, 1.5 h after oral dosing (100 µmol/kg). The results presented highlight the potential of this new class of BACE-1 inhibitors with good target potency and with low effect on hERG, in combination with a fair CNS exposure in vivo.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Encéfalo/efeitos dos fármacos , Imidazóis/síntese química , Fragmentos de Peptídeos/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Animais , Ácido Aspártico Endopeptidases/química , Encéfalo/metabolismo , Linhagem Celular , Cristalografia por Raios X , Cães , Feminino , Cobaias , Humanos , Imidazóis/química , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/líquido cefalorraquidiano , Permeabilidade , Estereoisomerismo , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
In drug design, it is crucial to have reliable information on how a chemical entity behaves in the presence of metabolizing enzymes. This requires substantial experimental efforts. Consequently, being able to predict the likely site/s of metabolism in any compound, synthesized or virtual, would be highly beneficial and time efficient. In this work, six different methodologies for predictions of the site of metabolism (SOM) have been compared and validated using structurally diverse data sets of drug-like molecules with well-established metabolic pattern in CYP3A4, CYP2C9, or both. Three of the methods predict the SOM based on the ligand's chemical structure, two additional methods use structural information of the enzymes, and the sixth method combines structure and ligand similarity and reactivity. The SOM is correctly predicted in 50 to 90% of the cases, depending on method and enzyme, which is an encouraging rate. We also discuss the underlying mechanisms of cytochrome P450 metabolism in the light of the results from this comparison.
Assuntos
Biologia Computacional/métodos , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Algoritmos , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sítios de Ligação , Biologia Computacional/tendências , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Análise de Componente PrincipalRESUMO
Results from theoretical calculations of (16)O/(18)O equilibrium isotope effects (EIEs) on deprotonation of phosphate and methyl phosphate monoanions as well as their deuterated counterparts are reported. The EIEs are calculated from the Bigeleisen equation using harmonic vibrational frequencies from several quantum mechanical methods (HF, DFT, MP2, and AM1). All methods correctly predict the qualitative trends in the EIEs related to the different isotope substitutions. However, the calculated gas-phase values are found to be systematically higher than those experimentally observed in aqueous solution. On the other hand, the addition of explicit solvent molecules (up to 24 waters) in the first solvation shells of the phosphate ion substantially improves the calculated EIE, which approaches the experimental value with increasing size of the water cluster. The large effects of surrounding water molecules on the phosphate deprotonation EIE can be explained by the strong solute-solvent interactions, which result in solvent coupled vibrational modes of the phosphate ions.