RESUMO
Previously we have demonstrated that light can be used to control the release of insulin in diabetic animals, followed by a reduction in blood glucose. This is accomplished using a photoactivated depot (PAD) of insulin injected into the skin, and irradiated by a small external LED light source. In this work for the first time we demonstrate dose-response, showing that we can vary insulin release and commensurate blood glucose reduction by varying the amount of light administered. In addition to demonstrating dose-response, we have shown multi-day depot response, with insulin being released on two different days from the same depot. The material used in these studies was CD-insulin, a form of insulin that has a highly non-polar cyclododecyl group attached, markedly reducing the solubility of the modified material, and allowing it to form a depot upon injection. Upon photolysis, the cyclododecyl group is removed, releasing fully native, soluble insulin. Variable response and multi-day response as demonstrated strongly support the potential utility of the PAD approach for the variable and extended release of therapeutic peptides.
Assuntos
Glicemia , Insulina , Animais , Pele , Solubilidade , FotóliseRESUMO
Design of the next-generation of therapeutics, biosensors, and molecular tools for basic research requires that we bring protein activity under control. Each protein has unique properties, and therefore, it is critical to tailor the current techniques to develop new regulatory methods and regulate new proteins of interest (POIs). This perspective gives an overview of the widely used stimuli and synthetic and natural methods for conditional regulation of proteins.
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Intrinsically disordered proteins (IDPs) and regions (IDRs) form a large part of the eukaryotic proteome. Contrary to the structure-function paradigm, the disordered proteins perform a myriad of functions in vivo. Consequently, they are involved in various disease pathways and are plausible drug targets. Unlike folded proteins, that have a defined structure and well carved out drug-binding pockets that can guide lead molecule selection, the disordered proteins require alternative drug-development methodologies that are based on an acceptable picture of their conformational ensemble. In this review, we discuss various experimental and computational techniques that contribute toward understanding IDP "structure" and describe representative pursuances toward IDP-targeting drug development. We also discuss ideas on developing rational drug design protocols targeting IDPs.
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Biomimetics is a design principle within chemistry, biology, and engineering, but chemistry biomimetic approaches have been generally limited to emulating nature's chemical toolkit while emulation of nature's physical toolkit has remained largely unexplored. To begin to explore this, we designed biophysically mimetic microfluidic reactors with characteristic length scales and shear stresses observed within capillaries. We modeled the effect of shear with molecular dynamics studies and showed that this induces specific normally buried residues to become solvent accessible. We then showed using kinetics experiments that rates of reaction of these specific residues in fact increase in a shear-dependent fashion. We applied our results in the creation of a new microfluidic approach for the multidimensional study of cysteine biomarkers. Finally, we used our approach to establish dissociation of the therapeutic antibody trastuzumab in a reducing environment. Our results have implications for the efficacy of existing therapeutic antibodies in blood plasma as well as suggesting in general that biophysically mimetic chemistry is exploited in biology and should be explored as a research area.
Assuntos
BiomiméticaRESUMO
In this work, we describe methods for synthesizing and incorporating a wide range of photocleavable groups into proteins. These are based on the di-methoxyl nitro phenyl ethyl (DMNPE) group. Using a common ketone starting material, we have modified the DMNPE core with different peptides and small molecules. We describe how these can be incorporated into DMNPE either by solution or solid phase methods. In addition, we show how the ketone group can be effectively converted into a hydrazone group and ultimately into a diazo. The potential pitfall of azine formation is also delineated, as are the strategies for avoiding this side product. We then show how these modified diazo groups can then be reacted with the carboxyl groups of the protein to make the final ester product. Finally, we show how the ultimate product can be purified, and the products identified using 280 and 345nm ratios, as well as ESI-MS characterization. The combined methods should allow the incorporation of many possible photocleavable groups into a range of proteins, and allow the ultimate properties of the modified protein to be subsequently toggled with light.
Assuntos
Compostos Azo/química , Técnicas de Química Sintética/métodos , Nitrobenzenos/química , Proteínas/química , Compostos Azo/síntese química , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Luz , Nitrobenzenos/síntese química , Peptídeos/síntese química , Peptídeos/química , Processos Fotoquímicos , Proteínas/síntese química , Técnicas de Síntese em Fase Sólida/métodosRESUMO
We have previously described the photoactivated depot (PAD) approach for the light-stimulated release of therapeutic proteins such as insulin. The aim of this method is to release insulin from a shallow dermal depot in response to blood glucose information, using transcutaneous irradiation. Our first approach utilized a photocleavable group that linked insulin to an insoluble but injectable polymer bead. The bead conferred insolubility, ensuring that the injected material stayed at the site of injection, until light cleaved the link, and allowed insulin to be absorbed systemically. While this proved to be effective, the use of a polymer to ensure insolubility introduces two major design problems: (1) low concentration of insulin, as a majority of the material is composed of polymer, and (2) upon release of the insulin, the polymer has to be cleared from the system. To address these two problems, in this work, we have pursued "hydrophobic tags", photocleavable small nonpolar molecules that confer insolubility to the modified insulin prior to irradiation without the bulk or need for biodegradation required of polymers. We developed a combined solid- and solution-phase synthetic approach that allowed us to incorporate a range of small nonpolar moieties, including peptides, into the final depot materials. The resulting materials are >90% w/w insulin and have sharply decreased solubilities relative to unmodified insulin (≤1000 × lower). We demonstrated that they can be milled into low micron-sized particles that can be readily injected through a 31G needle. These suspensions can be prepared at an effective concentration of 20 mM insulin, a concentration at which 120 µL contains 7 days of insulin for a typical adult. Finally, upon photolysis, the insoluble particles release soluble, native insulin in a predictable fashion. These combined properties make these new modified insulins nearly ideal as candidates for PAD materials.
Assuntos
Liberação Controlada de Fármacos/efeitos da radiação , Interações Hidrofóbicas e Hidrofílicas/efeitos da radiação , Insulina Regular Humana/química , Insulina Regular Humana/efeitos da radiação , Luminescência , Adulto , Humanos , Injeções , Cinética , Concentração Osmolar , Tamanho da Partícula , Fotólise/efeitos da radiação , Polímeros/administração & dosagem , Polímeros/química , Proteínas Recombinantes/química , Solubilidade , Suspensões/químicaRESUMO
We previously described the photoactivated depot or PAD approach that allows for the light control of therapeutic protein release. This approach relies on the ability to use light to change a protein's solubility. Traditionally this was accomplished by linking the protein to an insoluble but injectable polymer via a light cleaved linker. This allows the injected material to remain at the site of injection, until transcutaneous irradiation breaks the link between polymer and protein, permitting the protein to be absorbed. However, there are multiple problems associated with polymer based approaches: The polymer makes up a majority of the material, making it inefficient. In addition, after protein release, the polymer has to be cleared from the body, a significant design challenge. In this work, we create materials that form photoactivated depots of insulin without the need for polymers, by linking photolysis to an isoelectric point shift, which itself is linked to a solubility shift. Specifically, we linked basic groups to insulin via a light cleaved linker. These shift the normal pI of insulin from 5.4 to approximately 7. The result of this incorporation are materials that are completely soluble in mildly acidic solutions but precipitate upon injection into a pH 7 environment, i.e., the skin. We successfully synthesized four such modified insulins, demonstrating that their pI values were shifted in the expected manner. We then analyzed one of them, P2-insulin, in detail, demonstrating that it behaves as designed: It is soluble in a formulation pH of 4, but precipitates at pH 7.2, its approximate pI value. Upon irradiation, the photocleavable link to insulin is broken, and completely native and soluble insulin is released from the depot in a well behaved, first order fashion. These materials are 90% therapeutic, form completely soluble and injectable formulations in mildly acidic conditions, form insoluble depots at neutral pH, efficiently release soluble protein from these depots when irradiated, and leave behind only small easily absorbed molecules after irradiation. As such they approach ideality for photoactivated depot materials.