THU048 Determination Of Receptor Binding Affinity Of Growth Hormone Analogues To The Growth Hormone Receptor In A Drug Screening Context

Disclosure: S. Erlendsson: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. A. Kulakova: Employee; Self; Novo Nordisk. S. Poulsen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. P. Thygesen: Employee; Self; Novo Nordisk. Stock Owner; Self; Novo Nordisk. Growth hormone (GH) and GH analogues are characterized by their ability to bind and activate the GH receptor (GHR). Binding affinity and functional activity are pivotal parameters for development and characterization of new GH analogues for treatment of growth disorders. The GHR is a member of the class I cytokine family and obtains a tripartite structure consisting of two extracellular fibronectin type III domains (ECD), a single-pass transmembrane domain (TMD), and a disordered intracellular domain (ICD). These receptors lack intrinsic kinase activity but rely on recruitment of intracellular signalling kinases and regulatory proteins. Activation of the downstream JAK-STAT signalling cascade is initiated by formation of an asymmetric GHR dimer induced by binding of GH to the GHR ECDs via two partially overlapping binding sites designated site I and II. The structural rearrangement in the ECDs propagates through the TMD and results in a separation of the ICDs which leads to activation through cross-phosphorylation of the Janus kinases 2 (JAK2). Here we utilize isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and small angle x-ray scattering (SAXS) to characterize the binding of native GH and GH analogues including somapacitan and pegvisomant to the monomeric GHR as well as an artificial soluble stabilized dimeric GHR. The GH analogues with good binding properties were subsequently tested in vitro for biological activity using a STAT3-luciferase reporter gene assay (RGA) and a proliferation assay with BAF3 cells expressing the human GHR. Both assays were established in two formats enabling testing of both receptor agonists and antagonists. Using SPR we show that the binding of all tested compounds is preserved between the monomeric and dimeric GHR with affinities ranging from near-picomolar to lower micromolar and we are subsequently able to confirm the expected stoichiometry by utilizing a combination of ITC and SAXS. In the RGA and the proliferation assay we demonstrate that a 1:2 stoichiometry is critical for receptor activation regardless of the affinity and therefore we propose high throughput screening of receptor stoichiometry as key in discovery of new growth disorder therapeutics. Presentation: Thursday, June 15, 2023