RESUMO
The ability to target specific proteins for degradation may open a new door toward developing therapeutics. Although effort in chemistry is essential for advancing this modality, i.e., one needs to generate proteolysis targeting chimeras (bifunctional molecules, also referred to as PROTACS) or "molecular glues" to accelerate protein degradation, we suspect that investigations could also benefit by directing attention toward physiological regulation surrounding protein homeostasis, including the methods that can be used to examine changes in protein kinetics. This perspective will first consider some metabolic scenarios that might be of importance when one aims to change protein abundance by increasing protein degradation. Specifically, could protein turnover impact the apparent outcome? We will then outline how to study protein dynamics by coupling stable isotope tracer methods with mass spectrometry-based detection; since the experimental conditions could have a dramatic effect on protein turnover, special attention is directed toward the application of methods for quantifying protein kinetics using in vitro and in vivo models. Our goal is to present key concepts that should enable mechanistically informed studies which test targeted protein degradation strategies.
Assuntos
Biossíntese de Proteínas/fisiologia , Proteínas/análise , Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Proteostase/fisiologia , Animais , Humanos , Marcação por Isótopo , Cinética , Espectrometria de Massas , Proteínas/químicaRESUMO
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
Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters. In contrast, experiments in primary human, rhesus, and cynomolgus hepatocytes demonstrated that selective inhibition of DGAT2 has only a modest effect. Acute and chronic inhibition of DGAT2 in rhesus primates recapitulated the in vitro data yielding no significant effects on production of plasma TG or VLDL apolipoprotein B. These results call into question whether selective inhibition of DGAT2 is sufficient for remediation of dyslipidemia.
Assuntos
Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Dislipidemias/metabolismo , Hepatócitos/metabolismo , Obesidade/metabolismo , Triglicerídeos/metabolismo , Animais , Apolipoproteínas B/metabolismo , Células Cultivadas , Diacilglicerol O-Aciltransferase/genética , Modelos Animais de Doenças , Inativação Gênica , Humanos , Lipoproteínas VLDL/metabolismo , Macaca fascicularis , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Triglyceride (TG) storage in adipose tissue provides the major reservoir for metabolic energy in mammals. During lipolysis, fatty acids (FAs) are hydrolyzed from adipocyte TG stores and transported to other tissues for fuel. For unclear reasons, a large portion of hydrolyzed FAs in adipocytes is re-esterified to TGs in a "futile," ATP-consuming, energy dissipating cycle. Here we show that FA re-esterification during adipocyte lipolysis is mediated by DGAT1, an ER-localized DGAT enzyme. Surprisingly, this re-esterification cycle does not preserve TG mass but instead functions to protect the ER from lipotoxic stress and related consequences, such as adipose tissue inflammation. Our data reveal an important role for DGAT activity and TG synthesis generally in averting ER stress and lipotoxicity, with specifically DGAT1 performing this function during stimulated lipolysis in adipocytes.
Assuntos
Adipócitos/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Estresse do Retículo Endoplasmático , Lipólise , Triglicerídeos/biossíntese , Células 3T3-L1 , Animais , Retículo Endoplasmático/enzimologia , Humanos , CamundongosRESUMO
High-throughput screening (HTS) has enabled millions of compounds to be assessed for biological activity, but challenges remain in the prioritization of hit series. While biological, absorption, distribution, metabolism, excretion, and toxicity (ADMET), purity, and structural data are routinely used to select chemical matter for further follow-up, the scarcity of historical ADMET data for screening hits limits our understanding of early hit compounds. Herein, we describe a process that utilizes a battery of in-house quantitative structure-activity relationship (QSAR) models to generate in silico ADMET profiles for hit series to enable more complete characterizations of HTS chemical matter. These profiles allow teams to quickly assess hit series for desirable ADMET properties or suspected liabilities that may require significant optimization. Accordingly, these in silico data can direct ADMET experimentation and profoundly impact the progression of hit series. Several prospective examples are presented to substantiate the value of this approach.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Preparações Farmacêuticas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Simulação por Computador , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Preparações Farmacêuticas/metabolismo , Farmacocinética , Farmacologia , Relação Quantitativa Estrutura-AtividadeRESUMO
Mass spectrometry offers significant advantages over other detection technologies in the areas of hit finding, hit validation, and medicinal chemistry compound optimization. The foremost obvious advantage is the ability to directly measure enzymatic product formation. In addition, the inherent sensitivity of the liquid chromatography/mass spectrometry (LC/MS) approach allows the execution of enzymatic assays at substrate concentrations typically at or below substrate Km. Another advantage of the LC/MS approach is the ability to assay impure enzyme systems that would otherwise be difficult to prosecute with traditional labeled methods. This approach was used to identify inhibitors of diacylglycerol O-acyltransferase-2 (DGAT2), a transmembrane enzyme involved in the triglyceride (TG) production pathway. The LC/MS approach was employed because of its increased assay window (compared with control membranes) of more than sevenfold compared with less than twofold with a conventional fluorogenic assay. The ability to generate thousands of dose-dependent IC50 data was made possible by the use of a staggered parallel LC/MS system with fast elution gradients. From the hit-deconvolution efforts, several structural scaffold series were identified that inhibit DGAT2 activity. Additional profiling of one chemotype in particular identified two promising reversible and selective compounds (compound 15 and compound 16) that effectively inhibit TG production in mouse primary hepatocytes.
Assuntos
Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/química , Animais , Linhagem Celular , Cromatografia Líquida/métodos , Diacilglicerol O-Aciltransferase/química , Ensaios Enzimáticos/métodos , Humanos , Espectrometria de Massas/métodos , Células Sf9 , Triglicerídeos/químicaRESUMO
DGAT2 plays a critical role in hepatic triglyceride production, and data suggests that inhibition of DGAT2 could prove to be beneficial in treating a number of disease states. This article documents the discovery and optimization of a selective small molecule inhibitor of DGAT2 as well as pharmacological proof of biology in a mouse model of triglyceride production.
Assuntos
Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Isoquinolinas/química , Isoquinolinas/farmacologia , Triglicerídeos/metabolismo , Animais , Diacilglicerol O-Aciltransferase/metabolismo , Descoberta de Drogas , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Humanos , Isoquinolinas/administração & dosagem , Isoquinolinas/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Triglicerídeos/sangueRESUMO
Aryl and heteroaryl sulfoxides undergo ortho allylation upon treatment with Tf(2)O and allylsilanes. The method complements the use of sulfoxides to direct ortho-metalation and reaction with electrophiles as it allows allylic carbon nucleophiles to be added ortho to the directing group in a metal-free process. The versatile sulfide adducts can be selectively manipulated using various methods including Kumada-Corriu cross-coupling of the organosulfanyl group.
Assuntos
Sulfóxidos/química , Catálise , Estrutura MolecularRESUMO
Amino-anthranilic acid derivatives have been identified as a new class of low serum shifted, high affinity full agonists of the human orphan G-protein-coupled receptor GPR109a with improved ADME properties.
Assuntos
Descoberta de Drogas , Receptores Acoplados a Proteínas G/agonistas , Animais , Flúor/química , Humanos , Concentração Inibidora 50 , Camundongos , Estrutura Molecular , Niacina/síntese química , Niacina/química , Niacina/farmacologia , Piridinas/síntese química , Piridinas/química , Ratos , Ratos Sprague-Dawley , Receptores NicotínicosRESUMO
5-Alkyl and aryl-pyrazole-acids have been identified as a new class of selective, small-molecule, agonists of the human orphan G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.
Assuntos
Pirazóis/química , Receptores Acoplados a Proteínas G/agonistas , Animais , Ácidos Graxos/metabolismo , Humanos , Camundongos , Niacina/química , Pirazóis/síntese química , Pirazóis/farmacocinética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Nicotínicos/metabolismoRESUMO
5-Alkyl and aryl-pyrazole-tetrazoles have been identified as a new class of selective, small-molecule, agonists of the human G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.
Assuntos
Pirazóis/química , Receptores Acoplados a Proteínas G/agonistas , Tetrazóis/química , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacologia , Pirazóis/farmacologia , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/fisiologia , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/fisiologia , Tetrazóis/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologiaRESUMO
Thiazolylalanine, in appropriately functionalized form, has been found to function as an enantioselective catalyst for an intramolecular Stetter reaction. Incorporation of the residue in a number of environments has resulted in a family of catalysts that promote the cyclization of a test substrate with up to 81% enantiomeric excess.
Assuntos
Peptídeos/química , Alanina/análogos & derivados , Benzoína/química , Catálise , Ciclização , Estrutura MolecularRESUMO
[reaction: see text] Nucleophile-loaded peptides and proline have been found to function synergistically as cocatalysts for the asymmetric ketone-based Baylis-Hillman reaction. Although neither compound is effective independently in terms of rate or enantioselectivity, their combination leads to catalysis where enantioselectivities up to 81% have been observed.