Selective CRAF Inhibition Elicits Transactivation.

Charles W Morgan,James Hunt,Ian L Dale,Andrew P Thomas,Jason W Chin

doi:10.1021/jacs.0c11958

Abstract

Discovering molecules that regulate closely related protein isoforms is challenging, and in many cases the consequences of isoform-specific pharmacological regulation remains unknown. RAF isoforms are commonly mutated oncogenes that serve as effector kinases in MAP kinase signaling. BRAF/CRAF heterodimers are believed to be the primary RAF signaling species, and many RAF inhibitors lead to a “paradoxical activation” of RAF kinase activity through transactivation of the CRAF protomer; this leads to resistance mechanisms and secondary tumors. It has been hypothesized that CRAF-selective inhibition might bypass paradoxical activation, but no CRAF-selective inhibitor has been reported and the consequences of pharmacologically inhibiting CRAF have remained unknown. Here, we use bio-orthogonal ligand tethering (BOLT) to selectively target inhibitors to CRAF. Our results suggest that selective CRAF inhibition promotes paradoxical activation and exemplify how BOLT may be used to triage potential targets for drug discovery before any target-selective small molecules are known.

Highlights

Selective regulation of protein isoforms with small molecules remains an outstanding challenge
We demonstrated that bio-orthogonal ligand tethering (BOLT) ligands exhibited similar cellular responses and paradoxical activation to their parent inhibitors; as expected, BOLT ligands showed a decrease in potency (Figure S1)
We demonstrated that kinase-dead CRAF variants, (D486V, S357BCNK), (D486V, Q436BCNK), (A490T, S357BCNK), (A490T, Q436BCNK),[56,57] transactivate associated RAF monomers in the presence of BOLT ligands; the matched BocK controls did not lead to transactivation (Figure S8)

Summary

Introduction

Selective regulation of protein isoforms with small molecules remains an outstanding challenge. Strategies that define the consequence of selective pharmacological regulation for specific isoforms would provide an approach for triaging molecular targets and enable efforts to be focused on developing selective small molecules for the most valuable and validated targets. We previously described a distinct approach for the selective regulation of protein isoforms named biorthogonal ligand tethering (BOLT) In this approach we site- and cotranslationally encode a noncanonical amino acid (ncAA) bearing a bio-orthogonal group (commonly (2S)-2-amino-6((((1R,8S)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl) amino) hexanoic acid (BCNK (Figure 1A)) or Nε-(((2methylcycloprop-2-en-1-yl)methoxy)carbonyl)- L-lysine (CypK)[5,6] into the target protein using genetic code expansion.[7,8] We add a druglike small molecule−tetrazine conjugate to cells. The conjugate reacts with the target protein through a rapid bioorthgonal inverse electron demand Diels−

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Results

Conclusion