RESUMO
Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.
Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Pró-Proteína Convertase 9/metabolismo , Proteólise/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Inibidores de Serina Proteinase/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Reported herein are a series of reverse indoles that represent novel non-steroidal mineralocorticoid receptor (MR) antagonists. The key structure-activity relationships (SAR) are presented below. This reverse indole series is exemplified by a compound that demonstrated efficacy in an acute natriuresis rodent model comparable to marketed MR antagonists, spironolactone and eplerenone.
Assuntos
Descoberta de Drogas , Indóis/farmacologia , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Receptores de Mineralocorticoides/metabolismo , Relação Dose-Resposta a Droga , Humanos , Indóis/síntese química , Indóis/química , Antagonistas de Receptores de Mineralocorticoides/síntese química , Antagonistas de Receptores de Mineralocorticoides/química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Elaboration of the oxazolidinedione series led to replacement of the exocyclic amides with substituted benzimidazoles. The structure-activity relationship (SAR) exploration resulted in the discovery of potent and selective nonsteroidal mineralocorticoid receptor (MR) antagonists with significantly improved microsomal stability and pharmacokinetic (PK) profile relative to the HTS hit 1a. One compound 2p possessed comparable efficacy as spironolactone (SPL) at 100 mg/kg (p.o.) in the rat natriuresis model. As such, this series was validated as a lead series for further optimization.
RESUMO
We report the discovery of a novel series of DGAT1 inhibitors in the benzimidazole class with a piperdinyl-oxy-cyclohexanecarboxylic acid moiety. This novel series possesses significantly improved selectivity against the A2A receptor, no ACAT1 off-target activity at 10 µM, and higher aqueous solubility and free fraction in plasma as compared to the previously reported pyridyl-oxy-cyclohexanecarboxylic acid series. In particular, 5B was shown to possess an excellent selectivity profile by screening it against a panel of more than 100 biological targets. Compound 5B significantly reduces lipid excursion in LTT in mouse and rat, demonstrates DGAT1 mediated reduction of food intake and body weight in mice, is negative in a 3-strain Ames test, and appears to distribute preferentially in the liver and the intestine in mice. We believe this lead series possesses significant potential to identify optimized compounds for clinical development.
RESUMO
Bioisosteres are integral components of modern pharmaceutical research that allow structural optimization to maximize in vivo efficacy and minimize adverse effects by selectively modifying pharmacodynamic, pharmacokinetic and physicochemical properties. A recent medicinal chemistry campaign focused on identifying small molecule inhibitors of prolylcarboxypeptidase (PrCP) initiated an investigation into the use of pyrazoles as bioisosteres for amides. The results indicate that pyrazoles are suitable bioisosteric replacements of amide functional groups. The study is an example of managing bioisosteric replacement by incorporating subsequent structural modifications to maintain potency against the selected target. A heuristic model for an embedded pharmacophore is also described.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pirazóis/farmacologia , Animais , Carboxipeptidases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Humanos , Camundongos , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Relação Estrutura-AtividadeRESUMO
Novel potent and selective mineralocorticoid receptor antagonists were identified, utilizing heterocyclic amide replacements in the oxazolidinedione series. Structure-activity relationship (SAR) efforts focused on improving lipophilic ligand efficiency (LLE) while maintaining nuclear hormone receptor selectivity and reasonable pharmacokinetic profiles.
Assuntos
Amidas/química , Compostos Heterocíclicos/química , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Oxazóis/farmacologia , Receptores de Mineralocorticoides/metabolismo , Animais , Relação Dose-Resposta a Droga , Humanos , Microssomos Hepáticos , Antagonistas de Receptores de Mineralocorticoides/síntese química , Antagonistas de Receptores de Mineralocorticoides/química , Estrutura Molecular , Oxazóis/síntese química , Oxazóis/química , Ratos , Relação Estrutura-AtividadeRESUMO
The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Cicloexanos/química , Cicloexanos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Animais , Cicloexanos/farmacocinética , Descoberta de Drogas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/tratamento farmacológico , Relação Estrutura-AtividadeRESUMO
Novel oxazolidinedione analogs were discovered as potent and selective mineralocorticoid receptor (MR) antagonists. Structure-activity relationship (SAR) studies were focused on improving the potency and microsomal stability. Selected compounds demonstrated excellent MR activity, reasonable nuclear hormone receptor selectivity, and acceptable rat pharmacokinetics.
Assuntos
Antagonistas de Receptores de Mineralocorticoides/química , Oxazóis/química , Receptores de Mineralocorticoides/metabolismo , Animais , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Microssomos/metabolismo , Antagonistas de Receptores de Mineralocorticoides/síntese química , Antagonistas de Receptores de Mineralocorticoides/farmacocinética , Simulação de Acoplamento Molecular , Oxazóis/síntese química , Oxazóis/farmacocinética , Estrutura Terciária de Proteína , Ratos , Receptores de Mineralocorticoides/química , Relação Estrutura-AtividadeRESUMO
We report the design and synthesis of a series of novel DGAT1 inhibitors in the benzimidazole class with a pyridyl-oxy-cyclohexanecarboxylic acid moiety. In particular, compound 11A is a potent DGAT1 inhibitor with excellent selectivity against ACAT1. Compound 11A significantly reduces triglyceride excursion in lipid tolerance tests (LTT) in both mice and dogs at low plasma exposure. An in vivo study in mice with des-fluoro analogue 10A indicates that this series of compounds appears to distribute in intestine preferentially over plasma. The propensity to target intestine over plasma could be advantageous in reducing potential side effects since lower circulating levels of drug are required for efficacy. However, in the preclinical species, compound 11A undergoes cis/trans epimerization in vivo, which could complicate further development due to the presence of an active metabolite.
RESUMO
A new structural class of potent prolylcarboxypeptidase (PrCP) inhibitors was discovered by high-throughput screening. The series possesses a tractable SAR profile with sub-nanomolar in vitro IC(50) values. Compared to prior inhibitors, the new series demonstrated minimal activity shifts in pure plasma and complete ex vivo plasma target engagement in mouse plasma at the 20 h post-dose time point (po). In addition, the in vivo level of CNS and non-CNS drug exposure was measured.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos , Animais , Butanóis/síntese química , Butanóis/química , Butanóis/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/tratamento farmacológico , Pirrolidinas/síntese química , Pirrolidinas/química , Pirrolidinas/farmacologiaRESUMO
A series of potent inhibitors of prolylcarboxypeptidase (PrCP) was developed by modifying a lead structure that was discovered by high-throughput screening. The tert-butyl pyrrolidine was replaced by an aminocyclopentane to reduce the metabolic liabilities of the original lead. The compounds demonstrated sub-nanomolar in vitro IC(50) values, minimal activity shifts in pure plasma and improved pharmacokinetics. Complete ex vivo plasma target engagement was achieved with low brain exposure at the 20 h time point following p.o. dosing in a mouse. The results indicate that the aminocyclopentanes are useful tools for studying the therapeutic potential of peripheral (non-CNS) PrCP inhibition.
Assuntos
Aminas/farmacologia , Carboxipeptidases/antagonistas & inibidores , Ciclopentanos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos , Aminas/síntese química , Aminas/química , Animais , Ciclização , Ciclopentanos/síntese química , Ciclopentanos/química , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/tratamento farmacológicoRESUMO
Metabolic syndrome is a combination of medical disorders that increases the risk of developing cardiovascular disease and diabetes. Constitutive overexpression of 11ß-HSD1 in adipose tissue in mice leads to metabolic syndrome. In the process of generating transgenic mice overexpressing 11ß-HSD1 in an inducible manner, we found a metabolic syndrome phenotype in control, transgenic mice, expressing the reverse tetracycline-transactivator (rtTA) in adipose tissue. The control mice exhibited all four sequelae of metabolic syndrome (visceral obesity, insulin resistance, dyslipidemia, and hypertension), a pro-inflammatory state and marked hepatic steatosis. Gene expression profiling of the adipose tissue, muscle and liver of these mice revealed changes in expression of genes involved in lipid metabolism, insulin resistance, and inflammation. Transient transfection of rtTA, but not tTS, into 3T3-L1 cells resulted in lipid accumulation. We conclude that expression of rtTA in adipose tissue causes metabolic syndrome in mice.
Assuntos
Tecido Adiposo/metabolismo , Síndrome Metabólica/genética , Transativadores/metabolismo , Ativação Transcricional , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Células 3T3-L1 , Tecido Adiposo/patologia , Animais , Pressão Sanguínea , Cromossomos de Mamíferos/genética , Cromossomos de Mamíferos/metabolismo , Fragmentação do DNA , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Perfilação da Expressão Gênica , Resistência à Insulina , Metabolismo dos Lipídeos , Masculino , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Camundongos , Camundongos Transgênicos , Músculos/metabolismo , Músculos/patologia , Fenótipo , Tetraciclina/metabolismo , Transativadores/genética , Transfecção , TransgenesRESUMO
Ezetimibe (Zetia®), a cholesterol-absorption inhibitor (CAI) approved by the FDA for the treatment of hypercholesterolemia, is believed to target the intestine protein Niemann-Pick C1-Like 1 (NPC1L1) or its pathway. A spiroimidazolidinone NPC1L1 inhibitor identified by virtual screening showed moderate binding activity but was not efficacious in an in vivo rodent model of cholesterol absorption. Synthesis of analogs established the structure-activity relationships for binding activity, and resulted in compounds with in vivo efficacy, including 24, which inhibited plasma cholesterol absorption by 67% in the mouse, thereby providing proof-of-concept that non-ß-lactams can be effective CAIs.
Assuntos
Anticolesterolemiantes/síntese química , Imidazóis/síntese química , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Animais , Anticolesterolemiantes/farmacologia , Azetidinas , Ezetimiba , Imidazóis/química , Imidazóis/farmacologia , Absorção Intestinal/efeitos dos fármacos , Camundongos , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
During our effort to design a receptor binding assay to aid in the elucidation of the molecular mechanism of ezetimibe, we prepared a sulfur-35 containing radioligand which exhibits improved potency over the glucuronide conjugate of ezetimibe in both native enterocyte brush border membranes and membranes from cells expressing recombinant NPC1L1. Herein, we describe the different synthetic strategies which were used to obtain this compound as well as its effectiveness in the aforementioned assay.
Assuntos
Anticolesterolemiantes/química , Azetidinas/química , Proteínas de Membrana/antagonistas & inibidores , Animais , Anticolesterolemiantes/síntese química , Anticolesterolemiantes/farmacologia , Azetidinas/síntese química , Azetidinas/farmacologia , Linhagem Celular , Ezetimiba , Glucuronídeos/química , Humanos , Ligantes , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Camundongos , Ligação Proteica , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Radioisótopos de Enxofre/químicaRESUMO
A series of spiroimidazolidinone NPC1L1 inhibitors was discovered by virtual screening of the Merck corporate sample repository using 3D-similarity-based screening. Selection of 330 compounds for testing in an in vitro NPC1L1 binding assay yielded six hits in six distinct chemical series. Follow-up 2D similarity searching yielded several sub- to low-micromolar leads; among these was spiroimidazolidinone 10, with an IC(50) of 2.5 microM. Compound 10 provided a useful scaffold to initiate a medicinal chemistry campaign.
Assuntos
Anticolesterolemiantes/química , Imidazolidinas/química , Proteínas de Membrana/antagonistas & inibidores , Compostos de Espiro/química , Animais , Anticolesterolemiantes/farmacologia , Cricetinae , Cães , Desenho de Fármacos , Cobaias , Humanos , Imidazolidinas/farmacologia , Macaca mulatta , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Modelos Químicos , Conformação Molecular , Ratos , Software , Compostos de Espiro/farmacologia , SuínosRESUMO
Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.
Assuntos
Azetidinas/farmacologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas/metabolismo , Animais , Azetidinas/química , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Enterócitos/citologia , Enterócitos/metabolismo , Ezetimiba , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Macaca mulatta , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Microvilosidades/metabolismo , Doenças de Niemann-Pick , Ligação Proteica , Proteínas/genética , Ratos , Ratos Sprague-Dawley , Especificidade da EspécieRESUMO
We have developed assays for the binding of nucleotide and protein substrates to p38alpha protein kinase based on time-resolved Forster resonance energy transfer. p38alpha was biotinylated by addition of a sequence that targets biotin to a single lysine when coexpressed with biotin ligase in Escherichia coli, allowing formation of a complex between a streptavidin "LANCE" europium chelate conjugate and p38alpha. When this reagent was combined with M39AF, a p38 inhibitor containing a fluorescent moiety whose excitation wavelengths match the emission wavelengths of the europium chelate, a change in ratio of light emitted at 665 nm/615 nm is detected. Less than 100pM complex was detected with a signal/background ratio of >30-fold. The complex exhibits slow, tight binding kinetics where the apparent K(d) decreases with a relaxation time of 21 min at 125 pM biotin-p38alpha. Preincubating inhibitors or ATP with biotin-p38alpha and adding M39AF as a competitor yielded IC(50)s consistent with those measured by enzyme assay for the activated form of biotin-p38alpha. The same technique was also used to measure affinity of inhibitors for the unphosphorylated and catalytically inactive form of biotin-p38alpha. To measure affinity of p38alpha for its protein substrate MK2, we incubated biotin-p38alpha with a glutathione S-transferase MK2 fusion protein. Detection of the complex after incubation with streptavidin-allophycocyanin and a LANCE-conjugated anti-GST allowed measurement of affinity of MK2 for biotin-p38alpha and detection of 0.5 nM p38alpha.MK2 complex with signal/background ratio >5-fold. Competition with unbiotinylated p38alpha yielded an IC(50) value of 5 nM. Activation of either p38alpha or MK2 had no effect on the measured K(d). M39AF was found to bind in a ternary complex with p38alpha.MK2 with lower affinity than that observed in the binary complex with p38alpha alone.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Nucleotídeos/química , Proteínas Quinases p38 Ativadas por Mitógeno/química , Animais , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/metabolismo , Humanos , Nucleotídeos/metabolismo , Ligação Proteica , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
The c-Jun terminal kinases (JNKs) are members of the mitogen-activated protein (MAP) kinase family and regulate signal transduction in response to environmental stress. Activation of JNK3, a neuronal-specific isoform, has been associated with neurological damage, and as such, JNK3 may represent an attractive target for the treatment of neurological disorders. The MAP kinases share between 50% and 80% sequence identity. In order to obtain efficacious and safe compounds, it is necessary to address the issues of potency and selectivity. We report here four crystal structures of JNK3 in complex with three different classes of inhibitors. These structures provide a clear picture of the interactions that each class of compound made with the kinase. Knowledge of the atomic interactions involved in these diverse binding modes provides a platform for structure-guided modification of these compounds, or the de novo design of novel inhibitors that could satisfy the need for potency and selectivity.