RESUMO
Elastin, a key structural protein essential for the elasticity of the skin and elastogenic tissues, degrades with age. Replenishing elastin holds promise for anti-aging cosmetics and the supplementation of elastic activities of the cardiovascular system. We employed RiboScreenTM, a technology for identifying molecules that enhance the production of specific proteins, to target the production of tropoelastin. We make use of RiboScreenTM in two crucial steps: first, to pinpoint a target ribosomal protein (TRP), which acts as a switch to increase the production of the protein of interest (POI), and second, to identify small molecules that activate this ribosomal protein switch. Using RiboScreenTM, we identified ribosomal protein L40, henceforth eL40, as a TRP switch to boost tropoelastin production. Drug discovery identified a small-molecule hit that binds to eL40. In-cell treatment demonstrated activity of the eL40 ligand and delivered increased tropoelastin production levels in a dose-dependent manner. Thus, we demonstrate that RiboScreenTM can successfully identify a small-molecule hit capable of selectively enhancing tropoelastin production. This compound has the potential to be developed for topical or systemic applications to promote skin rejuvenation and to supplement elastic functionality within the cardiovascular system.
Assuntos
Elastina , Proteínas Ribossômicas , Ribossomos , Tropoelastina , Tropoelastina/metabolismo , Tropoelastina/genética , Humanos , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Elastina/metabolismo , Elastina/genética , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Ligantes , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Severe junctional epidermolysis bullosa is a rare genetic, postpartum lethal skin disease, predominantly caused by nonsense/premature termination codon (PTC) sequence variants in LAMB3 gene. LAMB3 encodes LAMB3, the ß subunit of epidermal-dermal skin anchor laminin 332. Most translational reads of a PTC mRNA deliver truncated, nonfunctional proteins, whereas an endogenous PTC readthrough mechanism produces full-length protein at minimal and insufficient levels. Conventional translational readthrough-inducing drugs amplify endogenous PTC readthrough; however, translational readthrough-inducing drugs are either proteotoxic or nonselective. Ribosome editing is a more selective and less toxic strategy. This technique identified ribosomal protein L35/uL29 (ie, RpL35) and RpL35-ligands repurposable drugs artesunate and atazanavir as molecular tools to increase production levels of full-length LAMB3. To evaluate ligand activity in living cells, we monitored artesunate and atazanavir treatment by dual luciferase reporter assays. Production levels of full-length LAMB3 increased up to 200% upon artesunate treatment, up to 150% upon atazanavir treatment, and up to 170% upon combinatorial treatment of RpL35 ligands at reduced drug dosage, with an unrelated PTC reporter being nonresponsive. Proof of bioactivity of RpL35 ligands in selective increase of full-length LAMB3 provides the basis for an alternative, targeted therapeutic route to replenish LAMB3 in severe junctional epidermolysis bullosa.