A dimer of A62 aptamer agonist for the insulin receptor selectively enhances PI3K-AKT pathway

Abstract

Insulin, secreted by pancreatic beta cells, effectively lowers blood glucose levels and plays a crucial role in glucose homeostasis in the skeletal muscle and adipose tissue. Insulin binding to the insulin receptor (IR) promotes trans-autophosphorylation and initiates downstream signaling, such as the phosphoinositide 3-kinase (PI3K)-AKT pathway for metabolic function and the mitogen-activated protein kinase (MAPK) pathway for mitogenic function. However, disruption of this insulin signaling pathway can lead to insulin resistance, which can result in type 2 diabetes mellitus (T2DM). The modulation of IR activation is a key step in initiating the insulin signaling cascade. Several IR modulators, such as S597 (insulin-mimetic peptides), XMetA (monoclonal antibodies) and A62 aptamers, have been identified to improve blood glucose levels by activating the AKT pathway. Aptamers are single-stranded oligonucleotides and are known as "chemical antibody" because of their ability to bind strongly to target proteins. In addition, aptamer-based modulation of IR activation exhibits significant advantages over protein therapeutics in terms of synthetic modification, size, cost, stability and non-immunogenicity. A recent study has demonstrated that an A62 aptamer, which can bind to IR with high affinity, partially activated the IR signaling pathway. A62 induced the phosphorylation of tyrosine 1150 (Y1150) and selectively activated the AKT pathway. A62 promoted a glucose-lowering effect in diabetic mice and did not stimulated cell proliferation in breast cancer cells. However, one limitation of aptamers is that A62 has a small size of 10 kDa, which make it susceptible to excretion through the kidneys, a process known as renal clearance. To prevent this issue, an A62 dimer (A62D), which is consisted of two A62 monomers (A62M) connected by an 8T linker, has been developed. This A62D has the advantage that by adjusting the length of the linker, the distance between two individual A62M can be modulated to regulate their protein-aptamer interactions. To investigate how A62M and A62D regulate the functions of insulin receptor at protein and transcript levels, Rat-1 cells overexpressing human IR (Rat-1/hIR cells) were stimulated with 50nM insulin, 200nM A62M, and 100nM A62D. Here, I observed that A62M phosphorylates only tyrosine 1150 (Y1150), whereas A62D induces phosphorylation of all tyrosine kinases (Y960, Y1316, Y1322, Y1146, Y1150, Y1150/Y1151) similar to insulin in Rat-1/hIR cells. A62M and A62D selectively activate the PI3K-AKT pathway but not MAPK pathway. Furthermore, changes in the transcript levels stimulated with the compounds (insulin, A62M and A62D) for 1h and 4h differed depending on time. In the 1-hour treated group, insulin induced breast cancer-associated genes alterations through the activation of MAPK pathway, but not A62D. In addition, insulin and A62D commonly involved in the regulation of gene expression related to the PI3K-AKT pathway. Taken together, this preliminary study demonstrated that A62D can induce full activation of IR and selectively stimulate AKT pathway rather than MAPK pathway.