Second Generation Photoactivated Insulins

Title form PDF of title page viewed July 15, 2021Dissertation advisor; Simon H. FriedmanVitaIncludes bibliographical references (pages 165-171)Thesis (Ph.D.)--School of Pharmacy and Department of Chemistry. University of Missouri--Kansas City, 2020The photoactivated depot (PAD) is a minimally invasive approach developed for a continuously variable light stimulated release of insulin. In this approach, a protein depot that can last for days is injected under the skin. A light source placed on the skin above the site of injection can trigger protein release into the blood with a transcutaneous irradiation. Since the insulin photorelease is a photochemical reaction, the amount released can be tightly controlled by varying the duration of irradiation. PAD is beneficial for diabetics as it can deliver insulin in a continuously variable fashion and can be automated as an artificial pancreas to avoid dosing errors. A first-generation material was constructed by covalently linking insulin to an insoluble polymer via a photocleavable group. Insolubility is a key requirement of the PAD material to allow retention at the site of injection, prior to irradiation. In vitro and in vivo experiments demonstrated its ability to deliver insulin on exposure to light. However, it needed further improvements due to the use of the polymer. Due to its large size, the material had low insulin density and was injected using a lower gauge needle. The polymer was not eliminated from the injection site after irradiation. Thus, alternative polymer-free approaches were explored to confer insolubility to insulin. In this work, photocleavable small molecules (tags) were explored to lower protein solubility. Firstly, non-polar peptidic and unnatural photocleavable tags were designed that could render insulin less soluble in aqueous conditions. Protein solubility also depends on its net charge. As an alternative approach, insulin solubility was lowered by balancing its charges with tags to be zero at physiological pH. A series of positively charged photocleavable groups were designed to raise the pI of insulin from 5.4 to 7.2. Two ideal materials, one chosen from each category of tags, were tested in diabetic rats. A PAD dose-response relationship was developed for the first time in living systems. These observations can assist in the development of an automated of the delivery system in response to blood glucose (with a continuous glucose monitor) for the diabetics.Introduction -- The tag approach -- The hydrophobic tags -- The charge tags -- Discrete photolysis -- In vivo studies -- Summary, scope, and conclusions -- Appendi

Accelerating Reaction Rates of Biomolecules by Using Shear Stress in Artificial Capillary Systems.

Funder: Frances and Augustus Newman FoundationFunder: Emmanuel College, University of CambridgeFunder: Biotechnology and Biological Sciences Research CouncilFunder: Centre for Misfolding Diseases, University of CambridgeFunder: Wellcome TrustBiomimetics 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

Light Control of Protein Solubility Through Isoelectric Point Modulation

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

Fuzzy drug targets: disordered proteins in the drug-discovery realm

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

Fuzzy Drug Targets: Disordered Proteins in the Drug-Discovery Realm.

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

Strategies for Conditional Regulation of Proteins.

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

Strategies for Conditional Regulation of Proteins.

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