Journal of the American Chemical Society
Article
Prevailing models of paradoxical activation center on
inhibitors promoting RAF dimerization and ultimately eliciting
MEK-ERK pathway activation, an outcome that is amplified by
oncogenic RAS mutations.37 The role of RAF dimerization is
central to both physiological and inhibitor induced signal-
ing.23,39 The homo- and heterodimers formed by wild-type
BRAF and CRAF are responsible for phosphorylating MEK.
While mutant BRAFV600E is constitutively active and has a
limited role in dimerization,40 the BRAF-CRAF heterodimer is
believed to be the primary species in both native signaling and
paradoxical activation.39,41,42 Genetic and biochemical results
have repeatedly implicated CRAF as the primary species
responsible for phosphorylating MEK in paradoxical activation
and native signaling.30−32,43−46 Specifically, inhibitor-bound
BRAF is implicated in promoting heterodimerization with
unbound CRAF, causing transactivation of CRAF through an
allosteric mechanism at the protein−protein interface between
protomer kinase domains.30,32
Decades of genetics research have employed both germline
and conditional allele manipulations of RAF isoforms; these
studies have revealed both redundancy and distinct functions
for BRAF and CRAF in different cell types and stages of cancer
progression.47−50 Recent genetic ablation of CRAF suggested
that removal of the protein may afford a therapeutic
benefit.45,51 However, as no well-characterized CRAF-selective
inhibitors have been reported, the consequences of selective
CRAF inhibition have remained unknown. Here, we develop
BOLT to selectively target inhibitors to CRAF. Our results
suggest that selective CRAF inhibition promotes paradoxical
activation.
Figure 1. Designing BOLT ligands and sites of CRAF tethering. (A)
Structures of noncanonical amino acids (ncAA) BocK and BCNK.
ncAA are site-specifically incorporated into amber (UAG) variants of
the CRAF gene. (B) Schematic of tethering between BOLT ligand,
containing tetrazine (blue), linker (green), and pharmacophore (red),
and BCNK containing proteins following an inverse electron demand
Diels−Alder reaction between the BCNK and tetrazine. (C) Chemical
structures of parent pharmacophores and the corresponding BOLT
ligands. RAF pharmacophores, AZ628 (type II), and PLX4720 (type
I) are shown in red. BOLT ligands AZ13-tet and AZ181-tet are
shown; the tetrazine moiety is in blue, the linker in green, and the
pharmacophore in red. (D) Structural superposition of MEK (gray)
and CRAF (blue) kinase structures. A small-molecule inhibitor
(yellow) occupies the ATP binding pocket. Spheres highlight
positions tested for amber suppression expression and tethering,
MEK (red) and CRAF (orange). Figure created using Pymol. PDB:
3ZLS MEK; 3OMV CRAF. (E) Immunoblot of the indicated CRAF
variants showing full length and ncAA-dependent expression. Variants
(small arrow) include C terminal epitope tags, FLAG, and HA
(3xFLAG-HA) to ensure immunoprecipitation and detection of full
length CRAF. Plasmids containing 4x[tRNAPyl] and CRAF-
(S357TAG) or CRAF(Q436TAG) variants were transfected into
HCT116* cells. Cells were grown with indicated ncAA (2 mM BocK,
200 μM BCNK). Lysates were collected after 48 h of expression.
Extended screening and testing of CRAF(YXXXTAG) alleles are
available in Figure S3; XXX indicates the position at which the codon
for a canonical amino acid (Y) is replaced with the amber codon
(TAG).
RESULTS AND DISCUSSION
■
There are over 2 dozen well-characterized small-molecule
inhibitors targeting RAF kinases, with characterization
spanning in vitro, preclinical, and clinical studies.37 Notably,
none are selective to the CRAF isoform in mutant RAS cells.
Drawing upon available RAF-selective pharmacophores, we
designed and synthesized a series of potential BOLT ligands
composed of three chemical moieties: a pharmacophore, a
tion). Pharmacophores were chosen from classic and distinct
RAF inhibitors, PLX4720 (type I, αC-OUT/DFG-IN) and
AZ628 (type II, αC-IN/DFG-OUT). We created AZ13-tet
(containing the AZ628 pharmacophore) and the AZ181-tet
(containing the PLX4720 pharmacophore). We used the
structure of RAF-inhibitor complexes52,53 to design syntheti-
cally accessible BOLT ligands that should not interfere with
pharmacophore binding. We demonstrated that BOLT ligands
exhibited similar cellular responses and paradoxical activation
to their parent inhibitors; as expected, BOLT ligands showed a
activated.23 Three RAF protein kinase (A, B, and C) serve as
effector kinases in the RAS-ERK signaling cascade. These
kinases drive the activating phosphorylation of MEK, which
ultimately results in an ERK-mediated transcriptional re-
sponse.24 Key to the activation of RAF kinases is the RAS-
mediated disruption of their autoinhibited conformation25 and
the formation of homo- and heterodimers.26−28
A great deal of effort has gone into generating RAF
inhibitors. In cells expressing mutant BRAF (e.g., V600E),
inhibitors suppress RAF activity and ERK signaling, while in
cells expressing wild-type BRAF most inhibitors cause an
undesired increase in RAF activity and ERK signaling (so-
called “paradoxical activation”).29−32 Understanding the
specific mechanism of action of RAF inhibitors has been the
focus of intense research efforts and has challenged the
academic and drug discovery communities for nearly 2
decades.33−38
We chose sites for ncAA incorporation in CRAF based on
the structure of RAF, a structural alignment of its kinase
domain with that of MEK, and previous work developing
BOLT on MEK (Figure 1D and Figure S2).20 We encoded
BCNK at sites in both the N and C lobes of CRAF using the
MmPylRS(Y306A, Y384F)54 /MmtRNAPyl
pair, BCNK,
CUA
selected positions showed BCNK-dependent expression of full
length CRAF and were efficiently labeled with a fluorescent
tetrazine conjugate (Figure S3F). We also incorporated BocK
(Figure 1A), an amino acid containing a nonreactive side
chain.
4601
J. Am. Chem. Soc. 2021, 143, 4600−4606