RESUMO
(+)-Plakevulin A (1), an oxylipin isolated from an Okinawan sponge Plakortis sp. inhibits enzymatic inhibition of DNA polymerases (pols) α and δ and exhibits cytotoxicity against murine leukemia (L1210) and human cervix carcinoma (KB) cell lines. However, the half-maximal inhibitory concentration (IC50) value for cytotoxicity significantly differed from those observed for the enzymatic inhibition of pols α and ß, indicating the presence of target protein(s) other than pols. This study demonstrated cytotoxicity against human promyelocytic leukemia (HL60), human cervix epithelioid carcinoma (HeLa), mouse calvaria-derived pre-osteoblast (MC3T3-E1), and human normal lung fibroblast (MRC-5) cell lines. This compound had selectivity to cancer cells over normal ones. Among these cell lines, HL60 exhibited the highest sensitivity to (+)-plakevulin A. (+)-Plakevulin A induced DNA fragmentation and caspase-3 activation in HL60 cells, indicating its role in apoptosis induction. Additionally, hydroxysteroid 17-ß dehydrogenase 4 (HSD17B4) was isolated from the HL60 lysate as one of its binding proteins through pull-down experiments using its biotinylated derivative and neutravidin-coated beads. Moreover, (+)-plakevulin A suppressed the activation of interleukin 6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3). Because the knockdown or inhibition of STAT3 induces apoptosis and HSD17B4 regulates STAT3 activation, (+)-plakevulin A may induce apoptosis in HL60 cell lines by suppressing STAT3 activation, potentially by binding to HSD17B4. The present findings provide valuable information for the mechanism of its action.
Assuntos
Apoptose , Interleucina-6 , Fator de Transcrição STAT3 , Humanos , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Células HL-60 , Interleucina-6/metabolismo , Animais , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Relação Dose-Resposta a Droga , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Targeted protein degradation (TPD), employing proteolysis-targeting chimeras (PROTACs) composed of ligands for both a target protein and ubiquitin ligase (E3) to redirect the ubiquitin-proteasome system (UPS) to the target protein, has emerged as a promising strategy in drug discovery. However, despite the vast number of E3 ligases, the repertoire of E3 ligands utilized in PROTACs remains limited. Here, we report the discovery of a small-molecule degron with a phenylpropionic acid skeleton, derived from a known ligand of S-phase kinase-interacting protein 2 (Skp2), an E3 ligase. We used this degron to design PROTACs inducing proteasomal degradation of HaloTag-fused proteins, and identified key structural relationships. Surprisingly, our mechanistic studies excluded the involvement of Skp2, suggesting that this degron recruits other protein(s) within the UPS.
Assuntos
Proteínas Quinases Associadas a Fase S , Bibliotecas de Moléculas Pequenas , Humanos , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Proteólise/efeitos dos fármacos , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Relação Estrutura-Atividade , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Molecular , Ligantes , Células HEK293 , DegronsRESUMO
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and competitive inhibitors targeting the catalytic domain of this enzyme, so-called statins, are widely used for the treatment of hyperlipidemia. The membrane domain mediates the sterol-accelerated degradation, a post-translational negative feedback mechanism, and small molecules triggering such degradation have been studied as an alternative therapeutic option. Such strategies are expected to provide benefits over catalytic site inhibitors, as the inhibition leads to transcriptional and post-translational upregulation of the enzyme, necessitating a higher dose of the inhibitors and concomitantly increasing the risk of serious adverse effects, including myopathies. Through our previous study on SR12813, a synthetic small molecule that induces degradation of HMG-CoA reductase, we identified a nitrogen-containing bisphosphonate ester SRP3042 as a highly potent HMG-CoA reductase degrader. Here, we performed a systematic structure-activity relationship study to optimize its activity and physicochemical properties, specifically focusing on the reduction of lipophilicity. Mono-fluorination of tert-butyl groups on the molecules was found to increase the HMG-CoA reductase degradation activity while reducing lipophilicity, suggesting the mono-fluorination of saturated alkyl groups as a useful strategy to balance potency and lipophilicity of the lead compounds.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Oxirredutases , Animais , Cricetinae , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Colesterol/metabolismo , Células CHORESUMO
Cholesterol biosynthesis is a high-cost process and, therefore, tightly regulated by both transcriptional and posttranslational negative feedback mechanisms in response to the level of cellular cholesterol. Squalene monooxygenase (SM, also known as squalene epoxidase or SQLE) is a rate-limiting enzyme in the cholesterol biosynthetic pathway and catalyzes epoxidation of squalene. The stability of SM is negatively regulated by cholesterol via its N-terminal regulatory domain (SM-N100). In this study, using a SM-luciferase fusion reporter cell line, we performed a chemical genetics screen that identified inhibitors of SM itself as up-regulators of SM. This effect was mediated through the SM-N100 region, competed with cholesterol-accelerated degradation, and required the E3 ubiquitin ligase MARCH6. However, up-regulation was not observed with statins, well-established cholesterol biosynthesis inhibitors, and this pointed to the presence of another mechanism other than reduced cholesterol synthesis. Further analyses revealed that squalene accumulation upon treatment with the SM inhibitor was responsible for the up-regulatory effect. Using photoaffinity labeling, we demonstrated that squalene directly bound to the N100 region, thereby reducing interaction with and ubiquitination by MARCH6. Our findings suggest that SM senses squalene via its N100 domain to increase its metabolic capacity, highlighting squalene as a feedforward factor for the cholesterol biosynthetic pathway.
Assuntos
Esqualeno Mono-Oxigenase/metabolismo , Esqualeno/metabolismo , Regulação Alostérica , Benzilaminas , Colesterol/biossíntese , Retículo Endoplasmático/enzimologia , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Proteostase , Esqualeno Mono-Oxigenase/antagonistas & inibidores , Tiofenos , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Juglorubin is a natural dye isolated from the culture of Streptomyces sp. 3094, 815, and GW4184. It has been previously synthesized via the biomimetic dimerization of juglomycin C, a plausible genetic precursor. In this study, the derivatives of juglorubin, 1-O-acetyljuglorubin dimethyl ester and juglorubin dimethyl ester, were found to exhibit antiviral activity against hepatitis C virus (HCV) without exerting any remarkable cytotoxicity against host Huh-7 cells. They also inhibited liver X receptor α activation and lipid droplet accumulation in Huh-7 cells. These findings suggest that 1-O-acetyljuglorubin dimethyl ester and juglorubin dimethyl ester targeted the host factors required for HCV production.
Assuntos
Hepacivirus , Hepatite C , Humanos , Hepacivirus/genética , Linhagem Celular , Ésteres , Replicação Viral , Antivirais/farmacologiaRESUMO
We have previously reported that neoechinulin B (1a), a prenylated indole diketopiperazine alkaloid, shows antiviral activities against hepatitis C virus (HCV) via the inactivation of the liver X receptors (LXRs) and the resultant disruption of double-membrane vesicles. In this study, a two-step synthesis of the diketopiperazine scaffold of 1a was achieved by the base-induced coupling of 1,4-diacetyl-3-{[(tert-butyldimethylsilyl)oxy]methyl}piperazine-2,5-dione with aldehydes, followed by the treatment of the resultant coupling products with tetra-n-butylammonium fluoride. Compound 1a and its 16 derivatives 1b-q were prepared using this method. Furthermore, variecolorin H, a related alkaloid, was obtained by the acid treatment of 1a in MeOH. The antiviral evaluation of 1a and its derivatives revealed that 1a, 1c, 1d, 1h, 1j, 1l, and 1o exhibited both anti-HCV and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activities. The results of this study indicate that the exomethylene moiety on the diketopiperazine ring is important for the antiviral activities. The antiviral compounds can inhibit the production of HCV and SARS-CoV-2 by inactivating LXRs.
Assuntos
Alcaloides/farmacologia , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Piperazinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , Alcaloides/síntese química , Alcaloides/química , Antivirais/síntese química , Antivirais/química , Linhagem Celular Tumoral , Dicetopiperazinas/química , Dicetopiperazinas/farmacologia , Humanos , Receptores X do Fígado/antagonistas & inibidores , Estrutura Molecular , Piperazinas/síntese química , Piperazinas/química , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacosRESUMO
Secondary metabolites in plants influence the health of herbivores such as Japanese rock ptarmigans that feed on the leaves and fruits of alpine plants. Thus, it is important to understand the secondary metabolites of alpine plants and their biological activities for conserving Japanese rock ptarmigans. We isolated C-methylflavone from the leaves of Kalmia procumbens, on which Japanese rock ptarmigans feed. Although its structure was deduced to be 8-demethyleucalyptin by comparing its nuclear magnetic resonance (NMR) data with the reported ones, the possibility that the isolated compound is 6-demethyleucalyptin cannot be ruled out. Thus, both isomers were synthesized. The isolated compound was unambiguously determined to be 8-demethyleucalyptin by comparing its NMR data with those of the synthetic ones. Cytotoxic evaluation of 8- and 6-demethyleucalyptins revealed that only the former showed cytotoxicity against HCT116 and MRC-5 cells. The present study provides not only easy access to 8- and 6-demethyleucalyptins, but also their biological information.
Assuntos
Antineoplásicos , Ericaceae , Folhas de Planta/químicaRESUMO
Alkynes were employed as tags to observe small molecules in cells by Raman microscopy. Herein, simple deuteration was found to shift the vibrational frequency of the alkyne by 135 cm-1. Two-color Raman imaging of D-alkynes and H-alkynes made it possible to distinguish between and observe similar small molecules in live cells.
Assuntos
AlcinosRESUMO
Epo-C12 is a synthetic derivative of epolactaene, isolated from Penicillium sp. BM 1689-P. Epo-C12 induces apoptosis in human acute lymphoblastoid leukemia BALL-1 cells. In our previous studies, seven proteins that bind to Epo-C12 were identified by a combination of pull-down experiments using biotinylated Epo-C12 (Bio-Epo-C12) and mass spectrometry. In the present study, the effect of Epo-C12 on peroxiredoxin 1 (Prx 1), one of the proteins that binds to Epo-C12, was investigated. Epo-C12 inhibited Prx 1 peroxidase activity. However, it did not suppress its chaperone activity. Binding experiments between Bio-Epo-C12 and point-mutated Prx 1s suggest that Epo-C12 binds to Cys52 and Cys83 in Prx 1. The present study revealed that Prx 1 is one of the target proteins through which Epo-C12 exerts an apoptotic effect in BALL-1 cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Peroxirredoxinas/antagonistas & inibidores , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Inibidores Enzimáticos , Compostos de Epóxi/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Estrutura Molecular , Mutação , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Polienos/químicaRESUMO
HMG-CoA reductase (HMGCR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and is the target of cholesterol-lowering drugs, statins. Previous studies have demonstrated that the enzyme activity is regulated by sterol-induced degradation in addition to transcriptional regulation through sterol-regulatory-element-binding proteins (SREBPs). While 25-hydroxycholesterol induces both HMGCR degradation and SREBP inhibition in a nonselective manner, lanosterol selectively induces HMGCR degradation. Here, to clarify the structural determinants of selectivity for the two activities, we established a luciferase-based assay monitoring HMGCR degradation and used it to profile the structure-activity/selectivity relationships of oxysterols and (oxy)lanosterols. We identified several sterols that selectively induce HMGCR degradation and one sterol, 25-hydroxycholest-4-en-3-one, that selectively inhibits the SREBP pathway. These results should be helpful in designing more potent and selective HMGCR degraders.
Assuntos
Hidroximetilglutaril-CoA Redutases/metabolismo , Lanosterol/metabolismo , Oxisteróis/metabolismo , Células HEK293 , Humanos , Lanosterol/farmacologia , Estrutura Molecular , Oxisteróis/farmacologia , Proteínas de Ligação a Elemento Regulador de Esterol/antagonistas & inibidores , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Relação Estrutura-AtividadeRESUMO
Polyglutamine diseases are a class of neurodegenerative diseases associated with the accumulation of aggregated mutant proteins. We previously developed a class of degradation-inducing agents targeting the ß-sheet-rich structure typical of such aggregates, and we showed that these agents dose-, time-, and proteasome-dependently decrease the intracellular level of mutant huntingtin with an extended polyglutamine tract, which correlates well with the severity of Huntington's disease. Here, we demonstrate that the same agents also deplete other polyglutamine disease-related proteins: mutant ataxin-3 and ataxin-7 in cells from spino-cerebellar ataxia patients, and mutant atrophin-1 in cells from dentatorubral-pallidoluysian atrophy patients. Targeting cross-ß-sheet structure could be an effective design strategy to develop therapeutic agents for multiple neurodegenerative diseases.
Assuntos
Ataxina-3/antagonistas & inibidores , Ataxina-7/antagonistas & inibidores , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Ataxina-3/genética , Ataxina-7/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Humanos , Estrutura Molecular , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Proteínas Repressoras/genética , Relação Estrutura-AtividadeRESUMO
HMG-CoA reductase (HMGCR) is a rate-limiting enzyme in the cholesterol biosynthetic pathway, and its catalytic domain is the well-known target of cholesterol-lowering drugs, statins. HMGCR is subject to layers of negative feedback loops; excess cholesterol inhibits transcription of the gene, and lanosterols and oxysterols accelerate degradation of HMGCR. A class of synthetic small molecules, bisphosphonate esters exemplified by SR12813, has been known to induce accelerated degradation of HMGCR and reduce the serum cholesterol level. Although genetic and biochemical studies revealed that the accelerated degradation requires the membrane domain of HMGCR and Insig, an oxysterol sensor on the endoplasmic reticulum membrane, the direct target of the bisphosphonate esters remains unclear. In this study, we developed a potent photoaffinity probe of the bisphosphonate esters through preliminary structure-activity relationship study and demonstrated binding of the bisphosphonate esters to the HMGCR membrane domain. These results provide an important clue to understand the elusive mechanism of the SR12813-mediated HMGCR degradation and serve as a basis to develop more potent HMGCR degraders that target the non-catalytic, membrane domain of the enzyme.
Assuntos
Difosfonatos/farmacologia , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Células Cultivadas , Difosfonatos/síntese química , Difosfonatos/química , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/síntese química , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Selective estrogen receptor (ER) down-regulators (SERDs) are pure ER antagonists that also induce ER degradation upon binding to the receptor. Although SERDs have been developed for the treatment of ER-positive breast cancers for nearly a decade, their precise mechanism(s) of action and structure-activity relationship are still unclear. Generally, Western blotting is used to examine the effects of SERDs on ER protein levels, but the methodology is low-throughput and not quantitative. Here, we describe a quantitative, high-throughput, luciferase-based assay for the evaluation of SERDs activity. For this purpose, we established stable recombinant HEK-293 cell lines expressing ERα fused with emerald luciferase. We also designed and synthesized new diphenylmethane derivatives as candidate SERDs, and evaluated their SERDs activity using the developed system in order to examine their structure-activity relationship, taking EC50 as a measure of potency, and Emax as a measure of efficacy.
Assuntos
Compostos Benzidrílicos/química , Regulação para Baixo/efeitos dos fármacos , Receptor alfa de Estrogênio/antagonistas & inibidores , Compostos Benzidrílicos/farmacologia , Sítios de Ligação , Ciclofenil/química , Ciclofenil/metabolismo , Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/metabolismo , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Fenóis/química , Fenóis/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by aggregation of mutant huntingtin (mHtt), and removal of mHtt is expected as a potential therapeutic option. We previously reported protein knockdown of Htt by using hybrid small molecules (Htt degraders) consisting of BE04, a ligand of ubiquitin ligase (E3), linked to probes for protein aggregates. Here, in order to examine the effect of changing the ligand, we synthesized a similar Htt degrader utilizing MV1, an antagonist of the inhibitor of apoptosis protein (IAP) family (a subgroup of ubiquitin E3 ligases), which is expected to have a higher affinity and specificity for IAP, as compared with BE04. The MV1-based hybrid successfully induced interaction between Htt aggregates and IAP, and reduced mHtt levels in living cells. Its mode of action was confirmed to be the same as that of the BE04-based hybrid. However, although the affinity of MV1 for IAP is greater than that of BE04, the efficacy of Htt degradation by the MV1-based molecule was lower, suggesting that linker length between the ligand and probe might be an important determinant of efficacy.
Assuntos
Benzotiazóis/farmacologia , Proteína Huntingtina/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Oligopeptídeos/farmacologia , Benzotiazóis/síntese química , Benzotiazóis/química , Fibroblastos/efeitos dos fármacos , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Ligantes , Oligopeptídeos/síntese química , Oligopeptídeos/química , Ligação ProteicaRESUMO
Niemann-Pick disease type C is a fatal, progressive neurodegenerative disease mostly caused by mutations in Nieamnn-Pick type C1 (NPC1), a late endosomal membrane protein that is essential for intracellular cholesterol transport. The most prevalent mutation, I1061T (Ile to Thr), interferes with the protein folding process. Consequently, mutated but intrinsically functional NPC1 proteins are prematurely degraded via proteasome, leading to loss of NPC1 function. Previously, we reported sterol derivatives as pharmacological chaperones for NPC1, and showed that these derivatives can normalize folding-defective phenotypes of I1061T NPC1 mutant by directly binding to, and stabilizing, the protein. Here, we report a series of compounds containing a phenanthridin-6-one scaffold as the first class of non-steroidal pharmacological chaperones for NPC1. We also examined their structure-activity relationships.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Glicoproteínas de Membrana/antagonistas & inibidores , Fenantridinas/farmacologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Relação Dose-Resposta a Droga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Estrutura Molecular , Mutação , Proteína C1 de Niemann-Pick , Fenantridinas/síntese química , Fenantridinas/química , Relação Estrutura-AtividadeRESUMO
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the aggregation of mutant huntingtin (mHtt), and removal of toxic mHtt is expected to be an effective therapeutic approach. We designed two small hybrid molecules (1 and 2) by linking a ligand for ubiquitin ligase (cellular inhibitor of apoptosis protein 1; cIAP1) with probes for mHtt aggregates, anticipating that these compounds would recruit cIAP1 to mHtt and induce selective degradation by the ubiquitin-proteasome system. The synthesized compounds reduced mHtt levels in HD patient fibroblasts and appear to be promising candidates for the development of a treatment for HD.
Assuntos
Descoberta de Drogas , Proteína Huntingtina/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Ligantes , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Niemann-Pick disease type C is a fatal neurodegenerative disease, and its major cause is mutations in NPC1 gene. This gene encodes NPC1 protein, a late endosomal polytopic membrane protein required for intracellular cholesterol trafficking. One prevalent mutation (I1061T) has been shown to cause a folding defect, which results in failure of endosomal localization of the protein, leading to loss-of-function phenotype. We have previously demonstrated that several oxysterols and their derivatives act as pharmacological chaperones; binding of these compounds to NPC1(I1061T) mutant protein corrects the localization/maturation defect of the mutant protein. Here, we disclose detailed structure-activity relationships of oxysterol derivatives as pharmacological chaperones for NPC1(I1061T) mutant.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Glicoproteínas de Membrana/antagonistas & inibidores , Esteróis/farmacologia , Proteínas de Transporte/genética , Relação Dose-Resposta a Droga , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/genética , Estrutura Molecular , Mutação , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Esteróis/síntese química , Esteróis/química , Relação Estrutura-AtividadeRESUMO
Niemann-Pick type C1-like 1 (NPC1L1) is an intestinal cholesterol transporter that is known to be the target of the cholesterol absorption inhibitor ezetimibe. We previously discovered steroidal NPC1L1 ligands by using a novel cell-based assay that employs pharmacological chaperone effect as a readout. Those steroid derivatives bound to a site different from both the sterol-binding domain and the ezetimibe-binding site, implying that they may be a novel class of NPC1L1 inhibitors with a distinct mode of action. As an extension of that work, we aimed here to find non-steroidal NPC1L1 ligands, which may be better candidates for clinical application than steroidal ligands, by using the same assay to screen our focused library of ligands for liver X receptor (LXR), a nuclear receptor that recognizes oxysterols as endogenous ligands. Here we describe identification of a novel class of NPC1L1 ligands with a ring-fused quinolinone scaffold, and an analysis of the structure-activity relationships of their derivatives as NPC1L1 ligands.
Assuntos
Proteínas de Membrana/antagonistas & inibidores , Quinolonas/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Estrutura Molecular , Quinolonas/síntese química , Quinolonas/química , Relação Estrutura-AtividadeRESUMO
A number of hereditary diseases are caused by defective protein trafficking due to a folding defect resulting from point mutations in proteins. Ligands that bind to the folding intermediates of such mutant proteins and rescue their trafficking defects, known as pharmacological chaperones, have promise for the treatment of certain genetic diseases, including Fabry disease, cystic fibrosis, and Niemann-Pick disease type C. Here we show that this pharmacological chaperone effect can be used for ligand screening, that is, binding of candidate ligands can be detected by monitoring the ligand-mediated correction of a localization defect caused by artificially introduced point mutations of the protein of interest. Using this method, we discovered novel steroidal ligands of Niemann-Pick type C1-like 1 (NPC1L1), an intestinal cholesterol transporter that is the target of the cholesterol absorption inhibitor ezetimibe, and conducted structure-activity relationship studies. We also present data indicating that the binding site of the new ligands is distinct from both the N-terminal sterol-binding domain and the ezetimibe-binding site.
Assuntos
Anticolesterolemiantes/química , Proteínas de Membrana/metabolismo , Anticolesterolemiantes/síntese química , Anticolesterolemiantes/farmacologia , Azetidinas/farmacologia , Sítios de Ligação , Transporte Biológico/efeitos dos fármacos , Carbamatos/síntese química , Carbamatos/química , Carbamatos/farmacologia , Ezetimiba , Células HEK293 , Humanos , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Mutação , Esteroides/química , Relação Estrutura-AtividadeRESUMO
TEAD transcription factors are responsible for the transcriptional output of Hippo signaling. TEAD activity is primarily regulated by phosphorylation of its coactivators, YAP and TAZ. In addition, cysteine palmitoylation has recently been shown to regulate TEAD activity. Here, we report lysine long-chain fatty acylation as a posttranslational modification of TEADs. Lysine fatty acylation occurs spontaneously via intramolecular transfer of acyl groups from the proximal acylated cysteine residue. Lysine fatty acylation, like cysteine palmitoylation, contributes to the transcriptional activity of TEADs by enhancing the interaction with YAP and TAZ, but it is more stable than cysteine acylation, suggesting that the lysine fatty-acylated TEAD acts as a "stable active form." Significantly, lysine fatty acylation of TEAD increased upon Hippo signaling activation despite a decrease in cysteine acylation. Our results provide insight into the role of fatty-acyl modifications in the regulation of TEAD activity.