Towards smart insulin development bearing rigid synthetic-diboronate chemistry applying de novo Protein Specific Modification

Insulin – a hormone, a medication, and protein – is one of the most fascinating and influential molecules ever discovered. Scientific understanding of insulin and the ability to adapt it for medical needs cross boundaries of knowledge, technology, and research. For hundreds of millions of people living with diabetes around the world, it is not only a scientific achievement but an urgent necessity – a vital solution that saves lives every single day. The chemical functionalization of insulin has been desired for various applications such as controlled release (e.g. short or long acting), rescue from toxic fibrillation, and searching for alternative administration approaches (e.g. oral medication). In Type-1 diabetes the controlled pancreas mediated secretion of insulin is been diminished, raising the need to apply insulin to the blood circulation on demand and careful control. With the risk of hypoglycemia attempts are being explored continuously to develop chemistry for the rewiring of insulin properties towards gaining an unnatural sensitivity to glucose blood levels, mimicking the failed pancreatic control. Due to their sugars binding capabilities, boronic acids are great candidates for potential insulin built-in glucose molecular sensors. Nevertheless, despite a century of scientific research the synthetic information currently available for multiple and selective insulin modification is inadequate, limiting the possibility to explore the effect of sensitive functional groups implantation into insulin. Moreover, boronic acid compounds were discovered to be less potent towards glucose rather than other sugars (e.g. fructose), limiting its therapeutic potential. Here, we report the development of a de novo Protein Specific Modification (PSM) strategy. The strategy focuses on shifting the paradigm from general-to-specific protein modification protocols. We revealed and utilized a new synthetic kit to perform multiple insulin modification in a precise manner. Utilizing our developed protocol, we were able to modify naked insulin precisely with three different elements. Moreover, we have progressed towards the potential development of smart boronate insulin by the introduction of synthetic rigid diborontae motif with glucose sensitive properties. Also, we have inserted a glucose mimic element to explore potential activity changes upon internal reversible bridging in presence and absence of glucose. Our in-cell activities findings reveal surprised tolerance of two de novo insulin analogues to a fluctuation with sugars levels. We anticipate these findings to pave the way for the further exploration of synthetic diboronate chemistry in the context of insulin modification. In addition, we expect our PSM approach for targeting a solely protein or a member of large protein family to transform the field of protein chemistry, allowing precise tools for more focused aims, with tremendous therapeutic potential and scope. This paradigm shifts from the general to the specific targeted chemistry in peptide and protein science is anticipated to open the door for a future database, we term as; PSM bank, to modify specific protein in the presence of other peptide and proteins, potentially for in vivo spatial selectivity.