RESUMO
Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients' cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.
Assuntos
Distrofia Miotônica , Animais , Quinases Ciclina-Dependentes , Modelos Animais de Doenças , Humanos , Camundongos , Distrofia Miotônica/tratamento farmacológico , Distrofia Miotônica/genética , RNA , Splicing de RNA/genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
The peptide hormone human relaxin-2 (H2-RLX) has emerged as a potential therapy for cardiovascular and fibrotic diseases, but its short in vivo half-life is an obstacle to long-term administration. The discovery of ML290 demonstrated that it is possible to identify small molecule agonists of the cognate G-protein coupled receptor for H2-RLX (relaxin family peptide receptor-1 (RXFP1)). In our efforts to generate a new medicine for liver fibrosis, we sought to identify improved small molecule functional mimetics of H2-RLX with selective, full agonist or positive allosteric modulator activity against RXFP1. First, we confirmed expression of RXFP1 in human diseased liver. We developed a robust cellular cAMP reporter assay of RXFP1 signaling in HEK293 cells transiently expressing RXFP1. A high-throughput screen did not identify further specific agonists or positive allosteric modulators of RXFP1, affirming the low druggability of this receptor. As an alternative approach, we generated novel ML290 analogues and tested their activity in the HEK293-RXFP1 cAMP assay and the human hepatic cell line LX-2. Differences in activity of compounds on cAMP activation compared with changes in expression of fibrotic markers indicate the need to better understand cell- and tissue-specific signaling mechanisms and their disease-relevant phenotypes in order to enable drug discovery.
Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ativadores de Enzimas/isolamento & purificação , Cirrose Hepática/tratamento farmacológico , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Biópsia , Células Cultivadas , Ativadores de Enzimas/síntese química , Ativadores de Enzimas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Cirrose Hepática/patologiaRESUMO
Circadian rhythms govern a wide variety of physiological and metabolic functions in many organisms, from prokaryotes to humans. We previously reported that silent information regulator 1 (SIRT1), a NAD(+)-dependent deacetylase, contributes to circadian control. In addition, SIRT1 activity is regulated in a cyclic manner in virtue of the circadian oscillation of the coenzyme NAD(+). Here we used specific SIRT1 activator compounds both in vitro and in vivo. We tested a variety of compounds to show that the activation of SIRT1 alters CLOCK:BMAL1-driven transcription in different systems. Activation of SIRT1 induces repression of circadian gene expression and decreases H3 K9/K14 acetylation at corresponding promoters in a time-specific manner. Specific activation of SIRT1 was demonstrated in vivo using liver-specific SIRT1-deficient mice, where the effect of SIRT1 activator compounds was shown to be dependent on SIRT1. Our findings demonstrate that SIRT1 can fine-tune circadian rhythm and pave the way to the development of pharmacological strategies to address a broad range of therapeutic indications.
Assuntos
Ritmo Circadiano/genética , Ativadores de Enzimas/farmacologia , Sirtuína 1/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Linhagem Celular , Cromatina/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Knockout , NAD/metabolismo , Transcrição Gênica/efeitos dos fármacosAssuntos
Baculoviridae/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Técnicas de Transferência de Genes , Animais , Humanos , Canais Iônicos/genética , Proteínas de Transporte de Neurotransmissores/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Esteroides/genética , Proteínas Recombinantes/biossínteseRESUMO
Human BCRP and OATP1B1 have recently been identified as important transporters in the absorption, distribution, and elimination of clinically significant drugs. In this report, we illustrate the use of modified baculoviruses, termed BacMam viruses for the expression of functional BCRP and OATP1B1 in mammalian cells. We show a variety of host cells efficiently transduced to express BCRP including HEK 293, LLC-PK, and U-2 OS, where protein levels on the cell-surface were modulated by titrating different amounts of viral inoculum. In addition, using the BODIPY-prazosin efflux assay and the BacMam reagent we illustrate inhibition of BCRP activity with GF120918 or Fumitremorgin C. Furthermore, we present data demonstrating simultaneous expression of BCRP and OATP1B1 in BacMam transduced mammalian cells by simply adding viral inoculum of each transporter. Thus these results indicate that BacMam mediated gene delivery provides a novel and efficient research tool for the investigation of single or multiple transporters in vitro.
