RESUMEN
The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit 1, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as 2 and 3. Compound 2 was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound 3 modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase.
RESUMEN
Selective inhibition of disease-related proteins underpins the majority of successful drug-target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure. We show that potent repression of PLN expression can be achieved with specificity that approaches single-gene regulation. Moreover, ZFP-driven repression of PLN increases calcium reuptake kinetics and improves contractile function of cardiac muscle both in vitro and in an animal model of heart failure. These results support the development of the PLN repressor as therapy for heart failure, and provide evidence that delivery of engineered ZFP TFs to native organs can drive therapeutically relevant levels of gene repression in vivo. Given the adaptability of designed ZFPs for binding diverse DNA sequences and the ubiquity of potential targets (promoter proximal DNA), our findings suggest that engineered ZFP repressors represent a powerful tool for the therapeutic inhibition of disease-related genes, therefore, offering the potential for therapeutic intervention in heart failure and other poorly treated human diseases.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/terapia , Factores de Transcripción/metabolismo , Dedos de Zinc/fisiología , Adenoviridae/genética , Animales , Western Blotting , Proteínas de Unión al Calcio/genética , Línea Celular , Insuficiencia Cardíaca/genética , Humanos , Cinética , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Dedos de Zinc/genéticaRESUMEN
Advances in understanding the relationship between protein structure and DNA binding specificity have made it possible to engineer zinc finger protein (ZFP) transcription factors to specifically activate or repress virtually any gene. To evaluate the potential clinical utility of this approach for peripheral vascular disease, we investigated the ability of an engineered vascular endothelial growth factor (VEGFa)-activating ZFP (MVZ+426b) to induce angiogenesis and rescue hindlimb ischemia in a murine model. Hindlimb ischemia was surgically induced in advanced-age C57/BL6 mice. Adenovirus (Ad) encoding either MVZ+426b or the fluorescent marker dsRed was delivered to the adducter muscle of the ischemic hindlimb, and the effects on blood flow, limb salvage, and vascularization were assessed. Ad-MVZ+426b induced expression of VEGFa at the mRNA and protein levels and stimulated a significant increase in vessel counts in the ischemic limb. This was accompanied by significantly increased blood flow and limb salvage as measured serially for 4 wk. These data demonstrate that activation of the endogenous VEGFa gene by an engineered ZFP can induce angiogenesis in a clinically relevant model and further document the feasibility of designing ZFPs to therapeutically regulate gene expression in vivo.
Asunto(s)
Regulación de la Expresión Génica/genética , Terapia Genética , Vectores Genéticos/uso terapéutico , Isquemia/terapia , Neovascularización Fisiológica/genética , Factores de Transcripción/fisiología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Dedos de Zinc/fisiología , Adenoviridae/genética , Envejecimiento , Secuencia de Aminoácidos , Animales , Velocidad del Flujo Sanguíneo , Estudios de Factibilidad , Genes Sintéticos , Miembro Posterior/irrigación sanguínea , Flujometría por Láser-Doppler , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Ingeniería de Proteínas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes , Relación Estructura-Actividad , Factores de Transcripción/genética , Factor A de Crecimiento Endotelial Vascular/genética , Dedos de Zinc/genéticaRESUMEN
The cellular heterogeneity and complex circuitry of the central nervous system make it difficult to achieve precise delivery of experimental and therapeutic agents. We report here an in vivo retrograde gene delivery strategy to target mature projection neurons using adeno-associated virus, a vector with low toxicity and the capacity for long-term gene expression. Viral delivery to axon terminal fields in the hippocampus and striatum resulted in viral internalization, retrograde transport, and transgene expression in specific projection neurons in entorhinal cortex and substantia nigra. Retrograde delivery of the anti-apoptotic gene Bcl2l (also known as Bcl-xL) protected entorhinal projection neurons from subsequent damage-induced cell death. Given the broad distribution of neurons affected by neurodegenerative diseases, gene delivery to both the terminal fields and the projection neurons through retrograde infection provides for strategic therapeutic intervention at both levels of the neural circuit. This approach may also facilitate experimental studies of defined neural circuits.