Topological control of cytokine receptor signaling induces differential effects in hematopoiesis

Abstract

Although tunable signaling by G protein–coupled receptors can be exploited through medicinal chemistry, a comparable pharmacological approach has been lacking for the modulation of signaling through dimeric receptors, such as those for cytokines. We present a strategy to modulate cytokine receptor signaling output by use of a series of designed C2-symmetric cytokine mimetics, based on the designed ankyrin repeat protein (DARPin) scaffold, that can systematically control erythropoietin receptor (EpoR) dimerization orientation and distance between monomers. We sampled a range of EpoR geometries by varying intermonomer angle and distance, corroborated by several ligand-EpoR complex crystal structures. Across the range, we observed full, partial, and biased agonism as well as stage-selective effects on hematopoiesis. This surrogate ligand strategy opens access to pharmacological modulation of therapeutically important cytokine and growth factor receptor systems.

Modular repeat protein sculpting using rigid helical junctions

“…In contrast to traditional approaches to joining domains with flexible linkers and bispecific antibodies, with the flexible hinge between the fragment crystallizable (FC) region and antigenbinding fragment (Fab) (19), our junction library enables precise control over the orientation of the fused domains. This is important for both design of higher-order protein assemblies and the arraying of receptor-binding domains in precise orientations to engage cell surface receptors in predefined geometries (20). Our junction library makes the exploration of these and other applications limited not by the design of the monomers and assemblies, but the creativity of the protein engineers deploying the methods.…”

Erythropoietin regulation of red blood cell production: from bench to bedside and back

“…These findings have potential medical implications. For example, one study showed that different artificial ligands that resulted in different angles and distance between EPOR homodimer subunits generated unique signaling patterns with stage-selective effects on hematopoiesis ( Figure 6) 134 . The other study 73 identified artificial EPOR ligands that can block EPO-independent signaling by the MPN-associated mutation JAK2V617F, which may inform new therapies for MPNs 134 .…”

Tuning TPO-R signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia

“…While all three DBs bound with comparable affinities to TPO-R, their downstream signaling outputs greatly differed and determined cell fate-maintenance of an HSC-like state with DBs AK111 and AK113 versus full maturation to megakaryocytes with AK119. While the structural basis of the differing signaling outputs elicited by the DBs is not resolved here, most likely the DBs that elicited a similar pharmacological profile of full to partial agonism(28,34).Structural analysis of the DB/EPO-R complexes revealed strikingly different EPO-Rdimerization topologies that presumably impacted the orientation of the intracellular signaling machinery. Here we have extended this concept about basic mechanisms of cytokine-receptor signaling, to applications in HSC biology with therapeutic implications.This study, along with others demonstrating the effects of cytokine partial agonism on cytokine pleiotropy and function in systems such as IL-2, interferons, and stem cell factor, ex vivo by diminishing and/or modulating activating downstream signals through TPO-R.…”