RESUMEN
Caspase-3 (Casp-3) is an enzyme that efficiently induces apoptosis, a form of programmed cell death. We report a dendritic molecular glue PCGlue that enables intracellular delivery of Casp-3 and its photoactivation. PCGlue carrying multiple guanidinium (Gu+) ion pendants via photocleavable linkages can tightly adhere to Casp-3 and deliver it into the cytoplasm mainly by direct penetration through the plasma membrane. Casp-3, whose surface is covered by PCGlue, is unable to interact with its cellular substrates and can therefore not induce apoptosis. However, upon exposure to UV or two-photon near-infrared (NIR) light, PCGlue is cleaved off to liberate Casp-3, triggering the apoptotic signaling cascade. This intracellular photoactivation of Casp-3 allows spatiotemporal induction of apoptosis in irradiated cells.
Asunto(s)
Caspasa 3/química , Guanidina/química , Nitrocompuestos/química , Apoptosis , Caspasa 3/metabolismo , Línea Celular Tumoral , Humanos , Rayos Infrarrojos , Estructura Molecular , Procesos Fotoquímicos , Rayos UltravioletaRESUMEN
We developed a dendritic molecular glue PCGlue-NBD that can serve universally to "turn on" protein-protein interactions (PPIs) spatiotemporally. PCGlue-NBD carrying multiple guanidinium ion (Gu+) pendants can adhere strongly to target proteins and cover their surfaces including the PPI interface regions, thereby suppressing PPIs with their receptor proteins. Upon irradiation with UV light, PCGlue-NBD on a target protein is photocleaved at butyrate-substituted nitroveratryloxycarbonyl linkages in the dendrimer framework, so that the multivalency for the adhesion is reduced. Consequently, the guest protein is liberated and becomes eligible for a PPI. We found that hepatocyte growth factor HGF, when mixed with PCGlue-NBD, lost the affinity toward its receptor c-Met. However, upon exposure of the PCGlue-NBD/HGF hybrid to light-emitting diode light (365 nm), the PCGlue-NBD molecules on HGF were photocleaved as described above, so that HGF was liberated and retrieved its intrinsic PPI affinity toward c-Met. The turn-on PPI, thus achieved for HGF and c-Met, leads to cell migration, which can be made spatiotemporally with a millimeter-scale resolution by pointwise irradiation with UV light.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/farmacología , Dendrímeros/farmacología , Guanidinas/farmacología , Factor de Crecimiento de Hepatocito/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-met/metabolismo , 4-Cloro-7-nitrobenzofurazano/síntesis química , 4-Cloro-7-nitrobenzofurazano/efectos de la radiación , Línea Celular Tumoral , Dendrímeros/síntesis química , Dendrímeros/efectos de la radiación , Guanidinas/síntesis química , Guanidinas/efectos de la radiación , Factor de Crecimiento de Hepatocito/química , Humanos , Unión Proteica/efectos de la radiación , Proteínas Proto-Oncogénicas c-met/química , Rayos UltravioletaRESUMEN
The cell nucleus is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is effective for treating various diseases. Here, we demonstrate light-triggered nuclear translocation of guests using caged molecular glue (CagedGlue-R) tags, whose multiple guanidinium ion (Gu+) pendants are protected by an anionic photocleavable group (butyrate-substituted nitroveratryloxycarbonyl; BANVOC). Guests tagged with CagedGlue-R are taken up into living cells via endocytosis and remain in endosomes. However, upon photoirradiation, CagedGlue-R is converted into uncaged molecular glue (UncagedGlue-R) carrying multiple Gu+ pendants, which facilitates the endosomal escape and subsequent nuclear translocation of the guests. This method is promising for site-selective nuclear-targeting drug delivery, since the tagged guests can migrate into the cytoplasm followed by the cell nucleus only when photoirradiated. CagedGlue-R tags can deliver macromolecular guests such as quantum dots (QDs) as well as small-molecule guests. CagedGlue-R tags can be uncaged with not only UV light but also two-photon near-infrared (NIR) light, which can deeply penetrate into tissue.
Asunto(s)
Núcleo Celular/metabolismo , Rayos Ultravioleta , Línea Celular Tumoral , Supervivencia Celular , Endocitosis , Endosomas/metabolismo , Guanidina/química , Humanos , Fotones , Puntos Cuánticos/metabolismoRESUMEN
We developed dendritic caged molecular glues (CagedGlue-R) as tags for nucleus-targeted drug delivery, whose multiple guanidinium ion (Gu+) pendants are protected by an anionic photocleavable unit (butyrate-substituted nitroveratryloxycarbonyl; BANVOC). Negatively charged CagedGlue-R hardly binds to anionic biomolecules because of their electrostatic repulsion. However, upon exposure of CagedGlue-R to UV light or near-infrared (NIR) light, the BANVOC groups of CagedGlue-R are rapidly detached to yield an uncaged molecular glue (UncagedGlue-R) that carries multiple Gu+ pendants. Because Gu+ forms a salt bridge with PO4-, UncagedGlue-R tightly adheres to anionic biomolecules such as DNA and phospholipids in cell membranes by a multivalent salt-bridge formation. When tagged with CagedGlue-R, guests can be taken up into living cells via endocytosis and hide in endosomes. However, when the CagedGlue-R tag is photochemically uncaged to form UncagedGlue-R, the guests escape from the endosome and migrate into the cytoplasm followed by the cell nucleus. We demonstrated that quantum dots (QDs) tagged with CagedGlue-R can be delivered efficiently to cell nuclei eventually by irradiation with light.
Asunto(s)
Adhesivos/metabolismo , Núcleo Celular/metabolismo , Guanidina/metabolismo , Luz , Adhesivos/química , Línea Celular Tumoral , Núcleo Celular/química , Endocitosis , Endosomas/química , Endosomas/metabolismo , Guanidina/química , Humanos , Estructura Molecular , Procesos Fotoquímicos , Puntos Cuánticos/química , Puntos Cuánticos/metabolismoRESUMEN
Molecular adhesion based on multivalent interactions plays essential roles in various biological processes. Hence, "molecular glues" that can adhere to biomolecules may modulate biomolecular functions and therefore can be applied to therapeutics. This tutorial review describes design strategies for developing adhesive motifs for biomolecules based on multivalent interactions. We highlight a guanidinium ion-based salt-bridge as a key interaction for adhesion to biomolecules and discuss the application of molecular glues for manipulation of biomolecular assemblies, drug delivery systems, and modulation of biomolecular functions.
Asunto(s)
Carbohidratos/química , Guanidina/química , Ácidos Nucleicos/química , Péptidos/química , Estructura MolecularRESUMEN
We developed a water-soluble adhesive photoswitch (Gluen-Azo-SA, average n = 5) that selectively binds to a target enzyme and photochemically modulates its enzymatic activity even in cell lysates. Its design strategy features a photochromic azobenzene unit (Azo), which carries on one side an inhibitory motif for the target enzyme and on the other a glue part (Gluen) that utilizes its multiple guanidinium ion (Gu+) pendants for adhering onto the target surface. The target of Gluen-Azo-SA is carbonic anhydrase (CA) because sulfonamide (SA) derivatives, such as SA at the terminus of Gluen-Azo-SA, are known to bind selectively to the CA active site. The SA moiety, upon docking at the CA active site, possibly guides the connecting Gluen part to an oxyanion-rich area in proximity to the active site for adhesion, so that the conjugation between Gluen-Azo-SA and CA is ensured. With this geometry, the photochemical isomerization of the Azo unit likely generates a push-pull motion of SA, resulting in its docking and undocking at the active site of CA with the help of a competing substrate. Consequently, Gluen-Azo-SA can selectively photomodulate the enzymatic action of CA even in cell lysates. Azo-SA without Gluen can likewise suppress the enzymatic activity of CA by docking SA at its active site, but the resulting CA/Azo-SA conjugate, in contrast, does not respond to light.
Asunto(s)
Adhesivos/farmacología , Compuestos Azo/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Guanidina/farmacología , Sulfonamidas/farmacología , Adhesivos/química , Compuestos Azo/química , Sitios de Unión/efectos de los fármacos , Inhibidores de Anhidrasa Carbónica/química , Guanidina/química , Estructura Molecular , Procesos Fotoquímicos , Solubilidad , Sulfonamidas/química , Propiedades de Superficie , Agua/químicaRESUMEN
Water-soluble bioadhesive polymers bearing multiple guanidinium ion (Gu+) pendants at their side-chain termini (Glue n -BA, n = 10 and 29) that were conjugated with benzamidine (BA) as a trypsin inhibitor were developed. The Glue n -BA molecules are supposed to adhere to oxyanionic regions of the trypsin surface, even in buffer, via a multivalent Gu+/oxyanion salt-bridge interaction, such that their BA group properly blocks the substrate-binding site. In fact, Glue10-BA and Glue29-BA exhibited 35- and 200-fold higher affinities for trypsin, respectively, than a BA derivative without the glue moiety (TEG-BA). Most importantly, Glue10-BA inhibited the protease activity of trypsin 13-fold more than TEG-BA. In sharp contrast, m Glue27-BA, which bears 27 Gu+ units along the main chain and has a 5-fold higher affinity than TEG-BA for trypsin, was inferior even to TEG-BA for trypsin inhibition.