Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface

Abstract

Bcr-Abl is a constitutively active kinase that causes chronic myelogenous leukemia. We have shown that a tandem fusion of two designed binding proteins, termed monobodies, directed to the interaction interface between the Src homology 2 (SH2) and kinase domains and to the phosphotyrosine-binding site of the SH2 domain, respectively, inhibits the Bcr-Abl kinase activity. Because the latter monobody inhibits processive phosphorylation by Bcr-Abl and the SH2-kinase interface is occluded in the active kinase, it remained undetermined whether targeting the SH2-kinase interface alone was sufficient for Bcr-Abl inhibition. To address this question, we generated new, higher affinity monobodies with single nanomolar K D values targeting the kinase-binding surface of SH2. Structural and mutagenesis studies revealed the molecular underpinnings of the monobody-SH2 interactions. Importantly, the new monobodies inhibited Bcr-Abl kinase activity in vitro and in cells, and they potently induced cell death in chronic myelogenous leukemia cell lines. This work provides strong evidence for the SH2-kinase interface as a pharmacologically tractable site for allosteric inhibition of Bcr-Abl.

Monobodies and other synthetic binding proteins for expanding protein science

“…The loop library tends to prefer binding into a concave epitope, whereas the side library prefers a flatter surface. For example, in an unbiased library selection experiment against the Abl SH2 domain, that is, a selection that did not involve a step that steer binders to a specific epitope, a dominant Monobody clone from the loop library bound to the concave, peptide‐binding groove, whereas a dominant clone from the side library bound to a flat surface on the opposite side of the SH2 domain . A similar library design has been reported for another FN3‐based system, Centyrin, although no information was available for the epitopes of resulting molecules .…”

“…In contrast, Monobody AS25 derived from the side library bound to a convex surface on the opposite side of the SH2 domain that is used for intramolecular interaction with the kinase domain of Abl [Fig. (C)] . This surface does not have a cleft for peptide binding, and the Monobody does not mimic the binding mode of the kinase domain.…”

“…Recently more potent Monobodies directed to the SH2‐kinase interface have been developed that inhibit BCR‐ABL without the need to be fused with another Monobody [Fig. (A)] …”

“… Schematics showing allosteric regulation using synthetic binding proteins. (A) Monobody binding to the SH2 domain of ABL kinase disrupts the intramolecular, domain‐domain interaction, which leads to kinase inhibition . (B) Monobody binding to a dimerization interface of RAS disrupts the RAS‐RAF hetero‐trimer and inhibits RAS‐mediated activation of RAF .…”

“…Acute production of synthetic binding proteins in cells overcomes these limitations, although we acknowledge that the technology needs further improvements. Recent studies, in addition to those discussed under the section entitled Allostery, illustrate the effectiveness of this approach.…”