Current insights of BRAF inhibitors in cancer

Perspective Cite This: J. Med. Chem. 2018, 61, 5775−5793

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Current Insights of BRAF Inhibitors in Cancer Miniperspective Bogos Agianian* and Evripidis Gavathiotis*

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Department of Biochemistry and Department of Medicine, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461, United States ABSTRACT: Oncogenic BRAF kinase deregulates the ERK signaling pathway in a large number of human tumors. FDAapproved BRAF inhibitors for BRAFV600E/K tumors have provided impressive clinical responses extending survival of melanoma patients. However, these drugs display paradoxical activation in normal tissue with BRAFWT due to RAF transactivation and priming, acquired drug resistance, and limited clinical effectiveness in non-V600 BRAF-dependent tumors, underscoring the urgent need to develop improved BRAF inhibitors. This review provides an overview of recent structural and biochemical insights into the mechanisms of BRAF regulation by BRAF inhibitors that are linked to their clinical activity, clinical liabilities, and medicinal chemistry properties. The effectiveness and challenges of structurally diverse next generation RAF inhibitors currently in preclinical and clinical development are discussed, along with mechanistic insights for developing more effective RAF inhibitors targeting different oncogenic BRAF conformations.

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malignancies.6,12−14 BRAFV600E mutation promotes constitutive kinase activation through signaling as a monomer, as well as insensitivity to ERK negative feedback mechanisms.15 In addition to V600E, a number of other hyperactivating point mutations at positions 600 (V600K, V600D, V600R), 601 (K601E, K601D, K601R), 469 (G469A, G469V), and others have been reported.6,11,16 Moreover, fusions and frame deletions are also found in certain tumor types, resulting in constitutive RAS-independent dimerization and activation of BRAF and downstream ERK signaling.17,18 BRAF mutational analysis intensified efforts for the development of selective and potent BRAF inhibitors. BRAF inhibitor development targeting oncogenic BRAFV600E has yielded two FDA-approved drugs for advance stage melanoma, 5 (vemurafenib, Zelboraf) and 6 (dabrafenib, Tafinlar), approved in 2011 and 2013, respectively (Figure 6b). These drugs have produced remarkable responses and improved survival of melanoma patients with BRAFV600 mutant tumors, but their acquired resistance limits their effectiveness and most patients relapse within a year.19 Moreover, colorectal and thyroid BRAFV600E tumors are resistant to 5 and 620 as well as 15−30% of non-smallcell lung carcinomas (NSCLC)21 and ∼85% of pancreatic cancer cells.22 The clinical importance of RAF dimerization in melanoma has been demonstrated by numerous observations whereby current FDA-approved inhibitors are failing to produce sustained tumor remission partly because they unsuccessfully target BRAF and CRAF dimers.2−4,23,24 Specifically, a major mechanism of acquired resistance involves signaling via BRAF homodimeric25−28 or BRAF-CRAF heterodimeric23,29 species.

INTRODUCTION The rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein (MEK)/extracellular signalregulated kinase (ERK) signaling pathway controls cell growth, proliferation, and survival.1 The RAF family of protein kinases (ARAF, BRAF, and CRAF) are critical effectors of this pathway.2,3 Recruitment of RAFs on membrane associated RAS upon upstream activation signals2−4 leads to a cascade of downstream events (ERK-signaling) that involve phosphorylation of MEK1 and MEK2 by RAFs, which in turn phosphorylate and activate ERK1 and ERK2.2 Oncogenic mutations of this pathway are observed frequently in many cancers.5,6 RAS is considered a central therapeutic target of the pathway because it is mutated in approximately 30% of human cancers;4,6 however drugging RAS has been notoriously challenging.7 As a result, efforts to inhibit RAF, MEK, and ERK kinases have gained significant momentum.4 A key step in the activation of RAF proteins is the formation of homo- and heterodimers, as RAF monomers are inactive due to autoinhibition by the N-terminal domain (NTD).2,3 BRAF-CRAF heterodimers are major activators of ERK signaling by mutated RAS;8,9 however physiological RAS signals mainly via BRAF dimers.2,3,10 In addition, oncogenic kinase-impaired BRAF mediates CRAF transactivation in a RAS-independent way.11 The first report of an oncogenic BRAF mutant in 20026 generated wide interest in targeting this protein. Over 45 oncogenic mutations have since been described for BRAF, with the V600E mutation accounting for ∼90% of them.3,4 Overall, BRAF mutations are present in about 8% of human tumors.6 BRAF mutants are present in >50% of melanoma patients as well as in colorectal (5−10%), thyroid carcinomas (25−45%), hairycell leukemia (∼100%) and less commonly in ovarian and lung © 2018 American Chemical Society

Received: September 1, 2017 Published: February 20, 2018 5775

DOI: 10.1021/acs.jmedchem.7b01306 J. Med. Chem. 2018, 61, 5775−5793

Journal of Medicinal Chemistry

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Figure 1. BRAF structure. (a) Ribbon representation of a BRAF protomer kinase domain structure bound to inhibitor 18 (PDB code 5VAM). The kinase consists of the N-lobe (salmon) and the C-lobe (blue) linked by a short flexible linker (hinge). The key structural elements for kinase activation, p-loop (yellow),1 αC-helix (magenta), and activation loop (α-loop, green) are highlighted. The regulatory F595 residue of the DFG motif (orange) adopts the DFG-OUT conformation, the αC-helix is in the IN position, and the α-loop is disordered. The floor of the inhibitor binding pocket (gray) is depicted in surface and the bound inhibitor in sticks. (b) Close-up view of BRAF active site illustrating schematically its ribose (pink), adenine (orange), hydrophobic (blue), type I (brown), and type II (green) subpockets (see text for details). The “gatekeeper” residue T529 and the catalytic residues E501 and D594 (DFG motif) are shown. A salt-bridge between catalytic E501 and K483, a hallmark of active kinase,33 is also shown. (c) The BRAF dimer (ribbons) demonstrates the spatial arrangement of protomers. Packing of residues R509 (ball-and-sticks, green) from each protomer, at the dimer interface, is essential for dimerization. αC-helix residue R506 (ball-and-sticks, yellow) can move toward OUT (red arrow) or IN (green arrow) conformations.

studies evaluate the combination of RAF/MEK inhibitors with immunotherapy.32 The above clinical challenges of RAF inhibitor tumor treatment underscore the pressing need to sustain further development of novel RAF small molecule inhibitors that evade resistance mechanisms. In addition, RAF drug design efforts should address recent breakthroughs in our understanding of the structural, biochemical, and cellular basis of sensitivity and

Clinically resistant tumors may also possess splice variants of BRAFV600E which form resistant dimers.27,30 Approved combination therapies of RAF and MEK inhibitors have improved responses in melanoma patients, although resistance to RAF and MEK inhibitor combination therapy is generated by mechanisms similar to the ones observed in RAF inhibitor monotherapy (see below).31 Additionally, promising ongoing 5776



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Figure 2. Inactive and active BRAF conformations. Ribbon representations of three representative inactive BRAF protomers and a monomer BRAF structure. Residues forming the C-spine (brown) and R-spine (white) (see text for details) are shown in Corey−Pauling−Koltun (CPK) representation. DFG residue F595 is part of the R-spine and is colored magenta. Fully assembled R-spine is depicted schematically with a straight red line, while a fully disassembled R-spine is depicted with a dashed red line. (a, b) BRAF-inhibitor inactive protomer structures showing disordered (part a, complex with 1, PDB code 1UWH) or ordered (part b, β3αC-deletion mutant in complex with 10, PDB code 5HID) α-loop. In both structures, the, inactive DFG-OUT position of F595 prevents full alignment of the R-spines. (c) A monomeric BRAF structure (PDB code 4RZW) demonstrates an inactive kinase conformation in which the α-loop is folded into a helical conformation. The AS forms a short helix that stabilizes αC-helix in the inactive OUT position. The R-spine is assembled but kinked (kinked red line), indicative of inactive kinase.33 (d) Protomer of the active BRAF dimer (PDB code 4MNE). The active kinase conformation is illustrated by the αC-IN position, the unfolding and full extension of the α-loop (including the AS) and the full assembly of the R-spine residues into a straight line (red line).

catalytic E501 residue that often pairs with conserved K483 residue, and a glycine−proline rich loop (p-loop), which stabilizes the β-phosphate of ATP (Figure 1a,b). Under physiological kinase activation, F595 of the DFG motif oscillates between an “inactive” DFG-OUT conformation, in which it occupies part of the ATP binding site, and an “active” DFG-IN conformation, where it swings in toward the base of αC-helix. In the absence of inhibitors, the DFG-OUT conformation favors allosteric movement of the αC-helix away from the ATP site (αC-OUT position), whereas DFG-IN enables the reverse movement (αC-IN position). In most BRAF crystal structures, as with other kinases, the α-loop is highly flexible and thus often structurally unresolved. The floor of the BRAF active site is lined mainly with hydrophobic residues, and the site can be subdivided into the adenine (ATP base), ribose (ATP ribose), hydrophobic, type I, and type II (see below) subpockets (Figure 1b). It is widely accepted that the catalytically active kinase conformation necessitates a DFG-IN/αC-IN conformation, a closed interlobe configuration, and an extended α-loop with unfolded AS (Figure 2d).3 The relative spatial orientation of certain hydrophobic residues of the kinase with each other has been shown to be critical for kinase activation. These residues create the so-called C (catalytic) and R (regulator) spines.33 Precise vertical alignment of the R-spine residues, which involves F595 of DFG motif, is

resistance to inhibitors. In this review, we focus on current developments in our understanding of structural and biochemical BRAF inhibitor-driven effects and their clinical implications. We also discuss the medicinal chemistry properties of RAF inhibitors and focus on the most recent, third generation of BRAF inhibitors, which are developed with the objective to bypass resistance mechanisms. Several inhibitors are currently under clinical trials and hold promise to further improve our repertoire against BRAF-dependent tumors.

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STRUCTURAL BASIS OF BRAF PHYSIOLOGICAL AND ABERRANT ACTIVATION RAFs have a typical kinase structure.3 Two domains, the Nterminal and C-terminal lobes, are linked via a flexible hinge which allows relative movement of the lobes with respect to each other. A deep cleft at the interface of the kinase lobes creates the active site pocket where the nucleotide ATP and peptide substrate (or inhibitors) bind (Figure 1a). Several regulatory elements play a key role in kinase activation: the DFG motif, which comprises the Mg-binding (of ATP) site D594, and the regulatory F595 residue, the phospho-acceptor site (activation segment, AS), which is the beginning of the flexible activation loop (α-loop) adjacent to DFG, the αC-helix, which contains the 5777



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Figure 3. Structural mapping of BRAF activating mutations. Structural mapping of common cancer activation point mutations on inactive BRAF monomer (panel a, PDB code 4RZV), an inactive protomer (panel b, PDB code 5HID), and active protomers (panel c, BRAFWT/MEK1 complex structure, PDB code 4MNE; panel d, BRAFV600E, PDB code 3OG7). Most mutations are concentrated at the AS of the α-loop and the p-loop. Mutations shown are the following: p-loop, G464, G466, S467, G469; α-loop, D594, G596, L597, T599, V600, K601; other locations, L485 and deletions in the 486−490 loop. Cα atoms of mutated residues are shown schematically as gold and magenta spheres, respectively, and F595 of DFG motif in sticks (orange). Residue V600 (pink) participates in a cluster of van der Waals contacts with p-loop or αC-helix residues (molecular surface) which stabilize inactive BRAF conformations (see text). In the inactive BRAF monomer (a) the cluster involves L597, L485, and αC-helix residue F498. In an inactive protomer (b) which comprise an in-frame deletion of BRAF17 positioned at the short segment connecting αC-helix to β3-strand of the Nlobe (β3αC), it involves T599 and the p-loop residue S467. In active BRAF structures, release of steric V600 contacts upon MEK complexation (c) or mutation (d) leads to α-loop extension and positions V600 (or E600) within interaction distance to K507 at the base of αC-helix, which adopts the “active” IN position.

essential for productive catalysis. This linear R-spine alignment, in contrast to other inactive orientations (Figure 2a−c), has been demonstrated for a prototype active BRAF protomer seen in a

recently determined structure of dimeric BRAF bound to MEK34 (Figure 2d). Consistent with an active structure, the αC-helix of 5778



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with inhibitors) with folded α-loops, is characterized by interactions of V600 with p-loop residues and a closed, activelike, interlobe conformation, as originally proposed.16 These interactions may involve direct V600 hydrophobic clustering or contacts via other α-loop residues17 (Figure 3b). In contrast, it is currently accepted that V600E mutation induces unfolding of the AS and an extension of the α-loop which relieves the above inhibited kinase states. The active kinase conformation is stabilized, at least partly, by a salt bridge between V600E and K507 of αC-helix, which is implicit in the active BRAFWT-MEK complex34 (Figure 3c) and is explicitly observed in the BRAFV600E-5 cocrystal structure70 (Figure 3d). In most studies, BRAFV600E adopts a dimeric structure, demonstrating the importance of dimerization for BRAF activation; however BRAFV600E can act as an active monomer in cancer cells with low GTP-RAS levels.8,23,32,38 Similar structural mechanisms of activation to BRAFV600E seem to apply to other V600 oncogenic mutants (V600K, V600D, or V600R) which also can act as monomers.26,40 In addition, inframe β3αC-deletions of the loop connecting the αC-helix and the β-sheet of the N-lobe are also oncogenic, with highest prevalence in pancreatic, lung, ovarian, and thyroid carcinomas.17,42 Structural analysis revealed that the αC-helix-IN conformation of these mutants (Figure 3b) is incompatible to compound 5 binding due to steric clashes.17 Besides V600, several other point mutations including BRAFL597V, BRAFK601E, and BRAFG466E are located in the α-loop and the p-loop of BRAF (Figure 3). Several cellular experiments have shown that activation of these non-V600 mutants depends largely on their spontaneous dimerization capacity, which is RASindependent;42 however, it is controversial whether this is also the case for β3αC-deletions.17,42 In a mutually nonexclusive model, interference of certain mutations to inhibitory autophosphorylation of p-loop residue S467 may also play a role in activation.43 Finally, another class of RAS-dependent catalytically impaired or inactive BRAF mutants mainly found in colorectal, melanoma, and lung tumors,44 such as D594N, G466V, S467L, G596R, were recently shown to depend on dimerization.45 The exact mechanism by which all these mutants dimerize is not very clear due to lack of structural information from these BRAF species. Regardless of the spectacular recent progress in BRAF biology, a better arsenal of BRAF crystal structures, including several mutant structures and the full-length protein which includes the inhibitory N-terminal and the RDB, will be required to obtain a more definitive and detailed understanding of the mechanisms involved in BRAF regulation.

this active protomer adopts an IN position which enables catalysis and a fully extended α-loop (and AS). Under nonactivating conditions, cytosolic BRAF adopts a closed and inactive kinase conformation, which is the result of the inhibitory interaction of the N-terminal regulatory domain on the C-terminal kinase domain. 35 As mentioned above, dimerization is essential for BRAF activation. Upon upstream receptor signaling activation, GTP-RAS levels increase on the plasma membrane. This leads to the release of RAF kinase inhibition due to the recruitment of the N-terminal domain to RAS via strong interactions with its RAS-binding domain (RDB).3 A series of subsequent events that involve BRAF kinase domain homo- or hetero- (with CRAF) dimerization and autophosphorylation of each protomer conclude full activation.4,36 In the vast majority of cases, reported BRAF (or CRAF) structures are dimers37 and almost invariably the constituting protomers adopt inactive kinase conformations (Figure 2a−c). The dimerization interface is mediated by the C-terminal of the kinase domain and the αC-helix (Figure 1c). Importantly, R509 residues at the C-terminal ends of each protomer αC-helix in the dimer pack together with strong van der Waals bonds as well as H-bonds to opposing protomers. These interactions are essential to maintain dimerization as demonstrated in a number of mutagenesis studies.8,27,38 It has been suggested that the orientation of another aC-helix residue, R506, which belongs to the conserved RKTR motif at the dimer interface39 (Figure 1c), determines inhibitor-driven interaction with GTP-RAS40 (see below). An inactive inhibitor-bound monomeric BRAF structure was recently revealed.40,41 This structure demonstrates a closed configuration of kinase lobes, the AS is folded as a short helix which stabilized the αC-helix at the OUT position, and the Rspine is kinked as expected for inactive kinase (Figure 2c). In both BRAF active MEK-bound protomer and the inactive monomer structures, F595 adopts a DFG-IN conformation, suggesting that movement of αC-helix and dimerization are not dependent on the positioning of DFG. These two BRAF structures, along with the collection of other BRAF structures (currently 65 records in the PDB), provided key insights on allosteric conformational transitions that affect the activation and dimerization of the kinase and demonstrated the large structural plasticity of RAFs. Despite these advances, the structural basis of activating BRAF cancer point mutations is not fully understood. On the basis of structures of BRAF bound to first generation RAF inhibitor 1 (sorafenib), Wan et al.16 proposed that disruption of hydrophobic interactions of V600 with residues of the p-loop relieves a closed interlobe kinase conformation and promotes activating dimerization. V600 residue is usually disordered in BRAF crystal structures; however, recent structural insights on inactive monomers and protomers as well as active BRAF have shed light on the potential role of V600 in oncogenic activation.17,34,41 So far, two general modes of interaction of V600 within BRAF seem to promote inhibited kinase states: The first is observed in a distinctive inactive monomeric BRAFWT structure.40,41 In this conformation, the AS folds into a short helix that makes hydrophobic contacts with the αC-helix. This arrangement allows V600 to be part of an extended hydrophobic cluster that stabilizes αC-helix to the inactive OUT conformation (Figure 3a). Steric clashes to αC-helix upon V600E mutation may induce movement of αC-helix toward the IN conformation and allosteric activation. A second mode of V600 interaction, observed in a few inactive BRAFWT structures (in complex

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CONFORMATIONAL AND FUNCTIONAL EFFECTS OF BRAF INHIBITORS A large number of structurally diverse RAF inhibitors have been developed.4,46 The recent burst in the number of BRAF-inhibitor structures and intense investigation on their biochemical and cellular effects have enlarged our understanding on their detailed targeting mode and conformational effects on RAF structure. A central observation has been that RAF inhibitors induce allosteric structural rearrangements, which “lock” their target kinases in discrete conformations and resemble inactive or active kinase states of the αC-helix and DFG motif.3,40 These conformations broadly classify inhibitors as type I (αC-helix-IN/DFG-IN), type II (αC-helix-IN/DFG-OUT), or type I1/2 (αC-helix-OUT/ DFG-IN).42,49 Clinical RAF inhibitors 5 and 6 (Figure 6) belong to type I1/2. 5779



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Figure 4. Inhibitor binding to BRAF structure impacts aC-helix conformation. 3D-superposition of protomers from BRAF dimers illustrate allosteric movements of αC-helix upon inhibitor binding. (a) Binding of type I1/2 inhibitor 5 induces the OUT position of αC-helix (magenta) in one protomer, while the αC-IN position is observed in the inhibitor-free second protomer (cyan). PDB code 3OG7. (b) Type II inhibitor 10 binds to both protomers of a BRAF dimer (PDB code 4RZW). αC-helices of both protomers are stabilized in the αC-IN position (within some variation). (c) The position of DFG motif is not priming the conformation of αC-helix. DFG-IN or DFG-OUT conformations are sterically compatible with either IN or OUT αChelix positions. The corresponding inhibitors and the conformations of BRAF-inhibitor crystal structures are noted. β-Sheets are represented in ribbons and helices as cylinders. Some protein parts of the C-lobe are not shown for clarity. Inhibitors and F595 (part c) are shown in sticks and molecular surface representation.

Table 1. Representative BRAF Inhibitors RAF inhibitor first generation 1

class

originator/developer

in vitro IC50/Ki (nM)a

in cellulo IC50/EC50 (viability) (nM)b

in cellulo IC50/EC50 (pERK) (nM)b

stage of development

refs

II

Bayer/Onyx

22c

3900c

4.4

FDA approved

2 3 4 second generation 5 6 7 8

I I I1/2

GlaxoSmithKline Genentech Plexxikon

0.16 0.17 13

370 >500 500

290 >1000 44

preclnical preclinical preclinical

I1/2 I1/2 I1/2f I

Plexxikon/Hoffman La Roche GlaxoSmithKline Array BioPharma Exelixis

31 0.8 0.3 6

310d 43e 4 200g

32 4 3 2000g

58, 124 125 75, 76 126, 127

9 third generation 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

I/IIh

Boehringer Ing.

0.4

0.9

0.5

FDA approved FDA approved phase III phase I (completed) preclinical

II II II II II II II II II II I1/2 I1/2f IIf N/A II

AstraZeneca Eli Lilly Takeda/Millennium Takeda/Millennium ICR/Basilea ICR/Basilea Beigene Beigene Novartis Novartis Plexxikon Plexxikon Ambit Bio./Ignyta Hanmi/Genentech ICR/Basilea

34 5.8 2.2 N/A 40 15 N/A 23 0.4m 0.5 4.2 3.8 14 7 N/A

383i 9.2 66 248j 15k 10 200 200 950m 140n 170 ∼800o 78 118p N/A

15 37 16 ∼70j ∼5 ∼5 157l 64 100m 160n 16 3.5 84 N/A N/A

preclinical phase I preclinical phase I preclinical preclinical preclinical phase I preclinical phase I/II preclinical phase I/II phase I/II phase I phase I

40 86 89 90, 128 51 51 26 98 101 102 58 58, 106 108, 109 111 51, 91

58, 64, 123 48 67, 68 58, 69

47

a

Activity on BRAFV600E mutant except if noted otherwise. bViability/pERK activity in melanoma A375 (BRAFV600E) cells except if noted otherwise. cActivity on BRAFWT. Viability in SK-MEL-30 cells. dSK-MEL-32 cells. eSK-MEL-28 cells. fPredicted. gpERK activity in Jurkat T cells. Viability in SK-MEL-19 cells. hαC-OUT/DFG-OUT. iSK-MEL-239 cells. jViability and pERK activity in synthetic pediatric low-grade astrocytomas (PLGAs) expressing BRAFV600E. kWM266.4(BRAFV600D) cells. lSK-MEL-239 (BRAFV600E) cells. mActivity on BRAFWT. pERK activity and viability in Calu-6 cells. nSK-MEL-28 cells. oHCC364 lung adenocarcinoma cells expressing p61-V600E. pColo-205 cells. 5780



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Table 2. Structural Data and Specificity of BRAF Inhibitors RAF inhibitor first generation 1 2 3 4 second generation 5 6 7 8 9 third generation 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 a

co-structure protein

BRAF conformation

BRAF specificity, monomer (M)/dimer (D)

PDB codes

BRAFWT, BRAFV600E, β3αC-BRAFWT BRAFWT BRAFV600E BRAFWT

αC-IN/DFG-OUT αC-IN/DFG-IN αC-IN/DFG-IN αC-OUT/DFG-IN

M/D M/D M/D M

1UWH, 1UWJ, 5HI2 2FB8 4MNF 3C4C, 4WO5

BRAFWT(R509H), BRAFV600E BRAFWT, BRAFV600E, β3αC-BRAFWT N/A N/A BRAFWT

αC-OUT/DFG-IN αC-OUT/DFG-IN αC-OUT/DFG-INa αC-IN/DFG-INa αC-OUT/DFG-OUTb

M M Ma M/Da M

4RZV, 3OG7 5CSW, 4XV2, 5HIE N/A N/A 5CSX

BRAFWT(R509H), BRAFV600E, β3αC-BRAFWT BRAFV600E BRAFWT N/A N/A N/A N/A BRAFV600E BRAFWT BRAFWT BRAFV600E N/A N/A N/A N/A

αC-IN/DFG-OUT αC-IN/DFG-OUT αC-IN/DFG-OUT αC-IN/DFG-OUTa αC-IN/DFG-OUTa αC-IN/DFG-OUTa αC-IN/DFG-OUTa αC-IN/DFG-OUT αC-IN/DFG-OUT αC-IN/DFG-OUT αC-OUT/DFG-IN αC-OUT/DFG-INa αC-IN/DFG-OUTa N/A N/A

M/D M/D M/D M/Da M/Da M/Da M/Da M/D M/D M/D M Ma M/Da N/A N/A

4RZW, 4G9R, 5HID 5C9C 4KSP N/A N/A N/A N/A 4R5Y 5VAM 5CT7 4XV1 N/A N/A N/A N/A

Predicted. bThe DGF-OUT conformation is induced by crystallization conditions.

Inhibitor binding induces allosteric conformational transition on the structure of protomers in BRAF dimers, which is characterized by the position of αC-helix, either to the IN or to the OUT conformation. A typical type I1/2 example is 5 which binds only one BRAF protomer of the dimer and induces an αCOUT conformation (Figure 4a). The αC-OUT position of compound 5 bound to protomer creates negative allostery to the second protomer by inducing the αC-IN conformation, which thus becomes incompatible with binding 5.42 Thus, BRAF-5 dimers are highly asymmetric. By contrast, typical type II inhibitors, like 10 (AZ-628) (see below), stabilize an IN conformation of αC-helix in the first protomer and do not create large allosteric αC-helix movements in the second protomer, which remains in the αC-IN conformation allowing another inhibitor binding (Figure 4).42 First and second generation RAF inhibitors (see below) belong mostly to I or I1/2 types (Table 1). They are αC-IN or αC-OUT inducers (Table 2) creating asymmetric BRAF dimers (Figure 5). A notable exception is 6, which occupies both protomers due to a brake in negative allostery exemplified by a less profound αCOUT conformation in both protomers.42,47 The position of DFG does not appear to influence negative allostery as both DFG-IN and OUT conformations are observed in BRAF complexes with αC-OUT and αC-IN inhibitor inducers (Figure 4c).

Figure 5. Inhibitors induce asymmetric BRAF dimers. Illustration of a BRAF dimer showing the two protomers in a molecular surface representation (brown, blue). OUT and IN positions of αC-helices of inhibitor bound protomers A from different BRAF dimers in complex with 5 (yellow, PDB code 5OG7), 9 (magenta, PDB code 5CSX), 10 (green, PDB code 4RZW), and 2 (blue, PDB code 2FB8) are shown after structural superposition. F595 residues (in sticks) adopt either the IN (yellow, cyan) or OUT (magenta, green) positions.

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STRUCTURAL EFFECTS OF RAF INHIBITORS ARE ASSOCIATED WITH THEIR PRECLINICAL AND CLINICAL ACTIVITY Several preclinical and clinical studies of RAF inhibitors demonstrated unusual pharmacologic behavior.9,28,48 While the inhibitors suppressed tumors harboring BRAFV600E or other mutants, in tumors expressing BRAF wild type (BRAFWT), they

do not inhibit but instead paradoxically activate RAF activity and downstream ERK signaling (RAF inhibitor paradox or “paradoxical activation”).4,28,48,49 These studies further established that paradoxical activation of full length BRAF requires active RAS and induced BRAF or BRAF-CRAF dimers. The general trend is that type I inhibitors exhibit more paradoxical activation, followed by type II and type I1/2 RAF inhibitors, an observation 5781



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Figure 6. Chemical structures of BRAF inhibitors. First (a), second (b), and third (c) generation BRAF inhibitors. Inhibitor moieties interacting with subpockets of BRAF active site are highlighted in different colors as in Figure 1b. Coloring key of subpockets: ribose (pink), adenine (orange), hydrophobic (blue), type I (brown), and type II (green). Groups exposed to solvent are colored in gray.

with important clinical implications.40 Recently, integration of previous cell-based studies with crystallographic data enabled the link between structural and biochemical properties of RAF inhibitors and their pharmacological effectiveness in tumors.34,40,41,49 It has become apparent that these effects are dictated by distinct inhibitor-driven allosteric mechanisms that prime specific RAF conformations, as well as the cellular context of active RAS.42 The model of negative allostery described above explains the clinical effectiveness of the current FDA approved αC-OUT RAF inhibitors 5 and 6. These inhibitors are selective for tumors that depend on BRAFV600 mutations (RAF monomers); however tumors driven by BRAF dimers are resistant26,27,40,45 due to the intrinsic inability of these inhibitors to bind both protomers in the dimers. Reduction of the affinity of αC-OUT inhibitors for the second protomer allows it to engage in catalysis, which is the basis of upregulation of ERK signaling.4 At the same time, such inhibitors are predicted to be ineffective to all dimeric BRAF oncogenic mutants. The inability of αC-OUT RAF inhibitors to inhibit physiologically activated dimeric BRAF in normal cells provides a wide therapeutic window for such inhibitors. However, prolonged treatment with αC-OUT inhibitors may lead to drug-induced lesions due to activated RAF signaling induced by negative allostery and resultant transactivation of unbound protomers. Such skin lesions include keratocarcinomas and squamous cell carcinomas.18 In contrast, αC-IN inhibitors can target efficiently BRAF dimers by binding both protomers, usually with similar affinity. In addition, these inhibitors can also target monomeric RAF forms, so they could be viewed as equipotent inhibitors of both

monomeric (e.g., BRAFV600 mutants) and dimeric (e.g., nonV600 mutants) BRAF.26,40,45 Despite these favorable properties, the tumor suppression effectiveness of αC-IN inhibitor monotherpy is limited by a narrower therapeutic window and by allosteric RAF priming. The former is a consequence of concurrent inhibition of BRAFWT (which adopts the active αCIN conformation) in healthy tissue.4 The latter phenomenon is based on the intrinsic propensity of αC-IN inhibitors to promote transactivating dimerization of an inhibitor-bound protomer with another monomer, which then leads to the interaction of RAF with GTP-RAS, followed by activating phosphorylation.2,3,40 Ultimate levels of primed RAF signaling would be context dependent, based on how completely a RAF inhibitor could bind to both protomers in the signaling dimer. Some aC-IN inhibitors target RAF homo- and heterodimers;50−52 however allosteric RAF priming again restricts this inhibitory activity. Allosteric priming by inhibitors has been observed with other kinases, for example, with AKT, protein kinase C (PKC), and Janus kinase (JAK),53−55 as has inhibitorinduced dimerization for instance in the cases of inositol requiring enzyme 1α (IRE1α) or JAK2.56,57 Paradoxical activation of ERK signaling is a property of both αC-OUT and αC-IN inhibitors which is based on their propensity to promote RAF transactivating dimerization as well as their overall inhibition mode.40 Both αC-OUT and αCIN inhibitors induce dimerization; however, αC-IN inhibitors do so typically more potently. Effective inhibition of such dimers depends on the ability of inhibitors to target effectively and durably both protomers. Usually, αC-OUT inhibitors promote paradoxical activation at low (submicromolar to nanomolar) 5782



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Figure 7. Recognition of BRAF active site by type I and I1/2 BRAF inhibitors. Close-up on the active site of BRAF showing binding mode of various type I or I1/2 inhibitors: (a) 5 (PDB code 3OG7); (b) 6 (PDB code 5CSW); (c) 9, a mixed type (I/II, Table 1) inhibitor (PDB code 5CSX); (d) 20 (PDB code 4XV1). Active-site subpockets are colored according to Figure 1b. Key H-bonds are shown (see text for details). In all complexes the terminal sulfonamide of the inhibitors is recognized by specific H-bonds. The E501-K483 salt-bridge is broken, and the αC-helix adopts the OUT position.

stabilizing the DGF-IN conformation, e.g., 2 or 10 (Figure 4c), display more pronounced paradoxical activation compared to their DFG-OUT counterparts.40 This observation may be the result of allosteric formation of RAF-MEK complex by these inhibitors, which activates ERK pathway.34,40 In contrast, αCOUT inhibitors like the clinically used 5 and 6 do not promote RAF interaction with MEK. Two nonmutually exclusive hypotheses attempt to explain the mode of action of paradox breakers. The first claims that they modulate dimerization through altered interaction with αC-helix via residue L505 (Figure 7d) as a result of terminal sulfonamide and sulfamide substitutions (Figure 6), which stabilize the αChelix in the dimerization-resistant OUT conformation.58 The other structural hypothesis involves overall conformational changes mediated by hydrogen-bond interactions with the BRAF gatekeeper residue T52959 (Figure 1b). Nonetheless, all studies demonstrate the critical role of RAF dimerization and the requirement of GTP-RAS for paradoxical activation.

concentrations because they fail to inhibit dimers due to negative allostery. In contrast, αC-IN inhibitors promote RAF priming at low concentrations due to enhanced dimerization; however these dimers are often inhibited due to lower negative allostery, which eventually leads to less paradoxical activation. Thus, transactivation, negative allostery, and potency for individual protomers determine final BRAF inhibitor activity.2,40 Structural comparison of various BRAF-inhibitor complexes led to the hypothesis that the position of αC-helix residue R506 (Figure 1c) determines the extent of inhibitor-induced RAF-RAS interaction.40 This model predicts that displacement of the R506 from the OUT position toward IN (Figure 1c, blue arrow) is compatible to RAF priming, whereas stabilization of R506 to the OUT position (Figure 1c, red arrow) is not. Indeed, αC-IN inhibitors 2 (SB-590885) and 3 (GDC-0879) (Figure 6, Table 1) (R506-IN conformation) promote RAF-RAS interaction, while the “paradox breakers” 20 (PLX7904, PB04) (Figures 6 and 7d) and 21 (PLX8394) (Figure 6, Table 1) (R506-OUT conformation) do not promote priming and paradoxical activation.40,58 Paradox breakers are typically αC-OUT inducers. Notably, 6 and to a lesser extent 5 promote RAF-RAS interaction and RAF dimerization, due to displacement of the R506 residue from the OUT position, despite being αC-OUT inhibitors.40 Moreover, among the group of αC-IN RAF inhibitors, those

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PROGRESS IN RAF KINASE INHIBITOR DEVELOPMENT First Generation RAF Kinase Inhibitors. Initial attempts to inhibit the oncogenic effect of CRAF in tumors identified the first reported ATP-competitive BRAF kinase inhibitor, ZM336372,60

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Figure 8. Recognition of BRAF active site by type II BRAF inhibitors. Close-up on the active site of BRAF showing binding mode of various type II inhibitors: (a) 11 (PDB code 5C9C); (b) 12 (PDB code 4KSP); (c) 18 (PDB code 5VAM); (d) 17 (PDB code 4R5Y). Active-site subpockets are colored according to Figure 1b. The αC-helix adopts the IN position, typical for type II inhibitors complexes. The catalytic E501 recognizes linker or ring nitrogens of the inhibitors, and in some cases (see panels b and c) the salt-bridge to K483 is maintained.

melanoma, while 6 was approved for therapy of melanoma tumors driven by BRAFV600E/K. Both 5 and 6 are αC-OUT/ DFG-IN inducers and, as noted above, demonstrate paradoxical activation and allosteric BRAF dimer transactivation. Thus, healthy (BRAFWT) cells can be activated upon treatment with these inhibitors, which may lead to secondary cancers.73 These drugs show remarkable effectiveness in BRAFV600E tumors; however tumors driven by non-V600 mutants are resistant.26,38,40 As mentioned above, central to the development of resistance is induced RAF dimerization and RAF priming. In a crystal structure of 5 with BRAFV600E the inhibitor demonstrated typical type I1/2 binding mode; the azaindole moiety forms two specific H-bonds to the backbone of hinge residues W531 and C532, the difluorophenyl group occupies the hydrophobic pocket, while the sulfonamide inserts into the type I pocket and H-bonds to K483 and the DFG motif (Figure 7a). A similar orientation in the BRAF active site is observed for 6 (Figure 7b) wherein the aminopyrimidyl moiety is docked in the adenine pocket. In contrast to 5, the ribose site is occupied by the butylthiazol group of 6. Another second generation inhibitor that has demonstrated promising preclinical and clinical properties is 7 (LGX818) (encorafenib, Array BioPharma).74−76 These properties included

a benzamide analogue. Work by Bayer/Onyx Pharmaceuticals developed the biarylurea derivative 1 (αC-IN/DFG-OUT inducer), the only first generation RAF inhibitor that received FDA approval for advanced renal cell carcinoma and hepatocellular carcinoma tumors that express BRAFWT.61−63 Preclinical studies using 1 demonstrated weak affinity for mutated BRAFV60064 (Table 1) and broad specificity. Therefore, it is suggested that the clinical effects of 1 arise from multikinase targeting. Development of several other first generation compounds did not produce any clinically approved RAF inhibitors. These included the indol-2-one derivative GW5074,65,66 the triarylimidazole derivative 2 (SB-590885),67 the pyridylpyrazole derivative 3 (GDC-0879),68 and the azaindole derivative 4 (PLX-4720)69 (Figure 6, Tables 1, and 2). With the exception of 1, which is a type II inhibitor, first generation RAF inhibitors belong to type I or I1/2. Second Generation RAF Kinase Inhibitors. The discovery of the key role of BRAFV600E in melanoma6 advanced interest to develop RAF inhibitors against this mutant. These efforts generated second generation inhibitors which include 5,69,70 an azaindole compound, and the pyrimidine derivative 6,71,72 the only such inhibitors to obtain FDA approval (Figure 6, Table 1). 5 was approved for the treatment of BRAFV600E metastatic 5784



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in contrast to αC-OUT inhibitors. Many third generation inhibitors have been produced, and several of them are currently undergoing phase I or phase II clinical trials (Table 1). Most known third generation RAF inhibitors bind BRAF in type II mode (αC-IN/DFG-OUT), whereby additional, compared to type I or type I1/2, chemical groups insert into a hydrophobic pocket (type II pocket) deep in the BRAF active site cleft (Figure 1b). In most cocrystal structures of BRAF with third generation inhibitors, strong hydrogen bonding of the inhibitor to the catalytic residue E501 of αC-helix or backbone NH of D594 of DFG motif is observed (Figure 8) which is facilitated by the movement of αC-helix toward the IN position. Often, the strong salt-bridge between E501 and K483 (Figure 1b), which stabilizes the αC-IN orientation, is not broken. A collection of key third generation BRAF inhibitors (Figure 6, Table 1) is described below. Among the early αC-IN BRAF inhibitors was the quinazolinole derivative 10 (AstraZeneca) (Figure 6). This compound showed reduced phospho-ERK (pERK) and phospho-MEK (pMEK) levels in RAS mutant cells.48 However, despite suppression of ERK signaling, the inhibitor demonstrated pan-RAF and multikinase inhibition profiles, similar to 1, and induced acquired resistance due to elevated CRAF expression in various cell lines.23 10 inhibits equipotently monomeric and dimeric BRAF40 as indicated by crystal structures with BRAF monomers (Figure 4b, BRAFR509H mutation, PDB code 4RZW)40 and dimes (PDB code 4G9R).85 Compound 10 induces the αC-IN/DFG-OUT conformation in these structures (Table 2). The type II binding to BRAF may correlate with the very slow dissociation rate of 10 from CRAF.48 Due to unfavorable pharmacological properties, 10 did not advance to clinical trials. 11 (LY3009120),86 a urea-linked pyridopyrimidine compound developed by Elli Lilly, is a third generation αC-IN/DFGOUT inhibitor which binds to both RAF protomers creating a mostly symmetrical dimer.52 11 therefore inhibits BRAF monomers and dimers with equal affinity.52,87 Interestingly, this inhibitor was also potent versus BRAF deletion mutants that spontaneously form dimers42 An X-ray structure of BRAFV600E in complex to 11 (PDB code 5C9C) shows that the inhibitor adopts a type II binding mode; the pyridopyrimidine moiety binds at the adenine pocket and the urea linker H-bonds to E501 and the base of DFG motif, while the hydrophobic tert-butylethyl group extends into the type II pocket (Figure 8a). 11 displayed antitumor activity in melanoma resistant to 1 and in RASdependent tumors52,86 as well as against pancreatic and thyroid tumors.42 In HCT116 cells harboring BRAFWT, 11 induced minor paradoxical activation of pERK at >100 nM concentrations, due to its pan-RAF activity profile, in particular its low IC50 for CRAF (15 nM).86 In patient-derived xenograft (PDX) studies, significant dose-dependent growth inhibition of tumors bearing BRAFV600E was observed upon oral treatment of rats with 11 (15−30 mg/kg b.i.d.). Similar results were reported for xenograft models harboring BRAF and NRAS/KRAS mutations.86 In pharmacokinetic experiments, 11 displayed initially low bioavailability in rats, dogs, and monkeys, which was significantly increased using solid dispersion technology.86 Currently 11 is under phase I clinical investigation.88 Similar to 11, compounds 12 (TAK632),89 13 (MLN2480/ TAK580), 90 (Takeda/Millennium) and 25 (CCT-3833/ BAL3833)91,92 (ICR/Basilea) are equipotent inhibitors of BRAF dimers and monomers.40,50 They are therefore expected

a low off-rate from BRAF compared to 5 or 6,24 which leads to a clinically longer residence time and a high selectivity for BRAFV600E.75 There is no available BRAF-LGX818 structure; however, the presence of a terminal type I sulfonamide (Figure 6, Table 1), similar to albeit shorter than that in 5 and 6, suggests that 7 is a type I1/2 inhibitor predicted to induce the αC-OUT/ DFG-IN conformation. Similar to other pyrazolylpyrimidine kinase inhibitors, the aminopyrimidine is expected to contact via H-bonds the hinge, whereas the pyrazolyl moiety extends to the ribose pocket. Currently 7 is in phase III clinical trials.77 8 (XL281, BMS-908662; Exelixis), a benzimidazole derivative78,79 (Figure 6, Table 1), is a second generation RAF inhibitor which held initial promise in biochemical and preclinical studies and advanced to phase I clinical trials.80 In this clinical study, out of 30 patients, one with ocular melanoma achieved a partial remission and 12 subjects had stable disease lasting >3 months. 8 was well tolerated up to 150 mg/day.78 However, its clinical development was discontinued. 8 is predicted to be an αC-IN/ DFG-IN inducer, yet structural information on its mode of binding to BRAF is lacking. Lastly, a class of second generation αC-OUT RAF inhibitors has been developed by Boehringer Ingelheim, with most promising member compound 9 (BI882370),47 a pyrolopyridine derivative containing a sulfonamide moiety typical of type I inhibitors (Figure 6). This inhibitor demonstrates remarkable potency in inhibition of kinase activity and specific cancer cell proliferation (Table 1). In biochemical experiments, it shows low selectivity among BRAF isoforms (IC50(BRAFWT) = 0.8 nM, IC50(CRAF) = 0.6 nM, compared to BRAFV600E IC50 value in Table 1).47 9 is effective in melanoma and colorectal xenograft models; treatment of mice with established A375 melanomas (doses of 6.25 or 12.5 mg/kg twice daily), G-361 melanoma (dose 12.5 mg/kg twice daily), or a COLO205 model (25 mg/kg twice daily) resulted in significant shrinkage of tumors.47 At the same time, the toxicologic profile of 9 in male rats provided no evidence of pathologic changes.47 Oddly, the structure of 9 with BRAFWT illustrates a unique αC-OUT/DFG-OUT conformation, indicative of a mixed type I and type II mode. However, this unique DGF-OUT binding mode is most probably an artifact of the crystallization milieu; a glucose solvent molecule occupies the type II pocket (similar to αC-IN inhibitors), sterically prohibiting movement of F595 toward its DFG-IN position (Figure 7c). Nonetheless, 9 shows characteristic type I active site contacts wherein the pyrimidine interacts with the kinase hinge and the pyrolopyridine group occupies the ribose pocket. Notably, binding of 9 partially orders the α-loop and allows residue K601 to H-bond a sulfonamide oxygen (Figure 7c). Second generation inhibitors can target successfully BRAFV600E melanoma tumors; however, in colorectal and thyroid BRAFV600 tumors their response is limited.81,82 This is attributed to upstream RAS and RTK activation83,84 which is maintained by pronounced feedback reactivation mechanisms and enhanced RAF dimerization.40 Third Generation RAF Kinase Inhibitors. The notion that RAF dimerization confers resistance to first and second generation RAF inhibitors26,27 has recently shifted focus of RAF inhibitor design to the development of compounds that stabilize BRAF in the αC-IN conformation to enable binding to both protomers in dimeric structures and are also capable of inhibiting cellular CRAF. The aim of these efforts has been to design small molecules that inhibit equipotently the monomeric and dimeric forms of RAFs or do not permit dimer formation. Such molecules would be able to target tumors resistant to 5 or 6, 5785



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to induce the αC-IN/DFG-OUT conformation and bind to both protomers of BRAF dimer. The benzothiazole derivative 12 (Figure 6) has been effective in multiple cancers harboring RAS mutations,40,50 in vemurafenib-resistant melanomas, and in BRAFV600E colorectal or thyroid tumors.50 12 is a potent inhibitor of BRAFV600E (Table 1); however it also inhibits BRAFWT (IC50 = 8.3 nM) and CRAF (IC50 = 1.4 nM). 12 demonstrated strong antiproliferative activity in HMVII cells (BRAFG469V, NRAS mutant) resistant to 5, which is thought to be mediated by BRAFWT and CRAF.89 In a human melanoma xenograft model (A375, BRAFV600E) in nude rats, 12 exhibited dose-dependent antitumor efficacy without severe body weight reduction over a dose range of 3.9− 24.1 mg/kg. In the same study, the pharmacokinetic profile of 12 showed significant oral absorption (F = 51% at dose 25 mg/kg)89 The X-ray crystal structure of 12 in complex with BRAFWT demonstrated type II binding for the inhibitor. The benzothiozole group makes specific H-bonds to the hinge residues in the adenine site and the phenylacetamide linker interacts with residue E501 and the backbone of residue G592 before the DFG (Figure 8b). As expected for a type II binder, the trifluoromethylphenyl moiety of 12 inserts into the type II hydrophobic pocket. The cyano substitution points into the ribose pocket. 13 (Figure 6), a pyrimidine derivative, has advanced in phase I clinical trials for patients with melanomas and other tumors93,94 as well as in combinatorial trials with small molecule kinase inhibitors, chemotherapy, and therapeutic antibodies.94,95 A recent study using neuronal cells expressing mutated BRAF showed that 13 inhibits both BRAF monomers and dimers and does not elicit strong paradoxical activation.90 In a clinical study with patients with relapsed or refractory tumors, 13 exhibited rapid absorption (median Tmax of 2 h), low fluctuation at steady state (mean peak to trough ratio 2.1), and mean accumulation half-life of 67 h, with acceptable safety profile.94 A BRAF structure with 13 has not been reported; however, based on its chemical structure, 13 is expected to be a type II inhibitor, whereby the pyrimidine group recognizes the kinase hinge and the trifluoromethylpyridine moiety inserts into the type II pocket. 14 (CCT196969) and 15 (CCT241161) are similar pyridopyrazine derivatives (Figure 6). Both compounds are RAF and sarcoma kinase (SRC) inhibitors and display antiproliferative activity in BRAFV600E melanomas and colorectal malignancies as well as in tumors resistant to second generation RAF inhibitors.51,91,96 Structurally, the pyridopyrazine moiety of these compounds is expected to contact the hinge of BRAF and the butylphenylpyrazole end to recognize the type II pocket; however this has not been shown experimentally by X-ray crystallography. These compounds do not drive paradoxical pathway activation and inhibit the growth of tumors resistant to compound 4 (A375 xenograft model), without causing adverse effects to the mice.46 14 and 15 have not yet progressed to clinical trials; however 25, a parent compound of the same chemical series which blocks both CRAF and BRAF, has entered phase I clinical trials for BRAF mutant and BRAF inhibitor-resistant melanomas.92 However, chemical details or the structure of 25 have not been disclosed. On the basis of an original report on new tetrahydronaphthalene RAF inhibitors,97 the Chinese company Beigene developed the naphthyridine compound 16 (BGB659) (Figure 6), another αC-IN/DFG-OUT third generation RAF inhibitor that displays inhibition of both BRAF dimers and monomers.26 Because of the ability of 16 to bind to both protomers of BRAF

dimer, it evades paradoxical activation and inhibits V600 and non-V600-dependent melanomas as well as melanomas resistant to 1.26 A cyclopropabenzofuran derivative compound from the same company, 17 (BGB283) (Figure 6), inhibits BRAF and EGFR and is effective in BRAFV600E colorectal tumors.98 In a recent study, 15 tumor cell lines with mutant BRAF (bearing mutations BRAFV600E, BRAFV600K, BRAF mutants that constitutively dimerize and p61-BRAFV600E) were sensitive to 16 with IC50 values from 100 to 600 nM.26 In subcutaneous murine xenografts studies carrying SK-MEL-239 (BRAF monomer) and SK-MEL-239 C4 (BRAF dimer) melanoma tumors 16 given daily (100 mg/kg) effectively inhibited growth, with no toxicity to other organs.26 BGB-283 has been shown to be a pan-RAF inhibitor, inhibiting BRAFV600E (IC50 = 23 nM, Table 1), BRAFWT (IC50 = 32 nM), CRAF (IC50 = 7 nM), and ARAF (IC50 = 5.6 nM).98 In vivo, 17 was efficient in suppressing tumor growth in xenograft models of HCC827 lung carcinoma (dose 10 mg/kg b.i.d.), COLO205 colorectal cancer harboring the BRAFV600E mutation (dose 30 mg/kg b.i.d.), and human derived (PDX) colorectal cancer. 17 has entered phase I clinical trials.99 A recent BRAF crystal structure with 17 demonstrated typical αC-IN/DFG-OUT, type II inhibitor binding (Figure 8d) with the naphthyridine ring occupying the adenine site, while the benzimidazole group, which makes a specific H-bond to E501, inserts into the type II pocket. RAF inhibitors developed from Novartis, replaced the urea linker of 1 with an aminoimidazole bioisostere group.100 A dipyridine derivative 18 (RAF709)101 and a benzimidazole derivative 19 (RAF265/CHIR-265)102 (Figure 6) are lead compounds of this series. 18 inhibits BRAFWT (IC50 = 0.4 nM, Table 1) and CRAF (IC50 = 0.5 nM) with similar potency. This high potency is attributed to very slow dissociation kinetics (T1/2 > 6.5 h).101 However, moderate inhibition of proliferation of Calu-6 cells was observed for this compound (Table 1). By use of a Calu-6 xenograft nude mouse model, compound 18 was well tolerated on doses up to 200 mg/kg showing a maximum tumor regression of 92%.101 A recent X-ray structure of the structurally diverse 18 with BRAFWT demonstrated specific protein contacts in all available pockets of the active site, with the morpholino group recognizing the hinge at the adenine pocket, the pyridine moiety extending into the ribose site, and the benzamide linker H-bonding to E501 (Figure 8c). Interestingly, 18 induces a partial structuring of the α-loop which positions residue W604 in proximity to the type II pocket, where it creates van der Waals contacts to the terminal trifluoromethylphenyl group of the inhibitor (Figure 8c). This extensive network of contacts probably explains the very low IC50 of kinase inhibition (Table 1) and the slow dissociation kinetics. 19 is a potent inhibitor of BRAFV600E (IC50 of 0.5 nM, see Table 1, in comparison to IC50 of 70 nM and 19 nM for BRAFWT and CRAF, respectively), displaying long half-life in PK studies and pronounced tumor growth inhibition in a A375M xenograft model.102 Phase I clinical trials on subjects with various tumors are ongoing for compound 19.103 The BRAFWT-19 cocrystal structure (PDB code 5CT7) shows that the imidazolopyridine group docks in the adenine pocket and the amine linker of the benzimidazole creates a typical type II inhibitor H-bond with E501. 19 and 18 induce the αC-IN/DFG-OUT conformation. In contrast to the above group of compounds, certain third generation inhibitors are αC-OUT inducers. aC-OUT paradox breakers comprise a distinct group among them. Typical members include azaindole derivatives developed by Plexxikon: 20 (PLX7904/PB04)58 and its analog 21 (PLX8394/PB03)104 5786



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(Figure 6). These molecules are more potent than 5 in targeting BRAFV600E.58 They are type I1/2 inhibitors with terminal sulfonamides, resembling 5 and 6; however the sulfonamide substitutions are bulkier. In contrast to αC-IN paradox breakers, like inhibitors from Beigene (see above), these compounds circumvent paradoxical activation because they do not induce RAF dimers.58 Data suggest that because of their higher potency against BRAF compared to clinically used 5 (see Table 1), these inhibitors are effective in a number of tumors.105,106 However, in vivo, 20 and 5 produced similar antitumor effects in a subcutaneous COLO205 xenograft model (doses 25 mg/kg twice daily) and good plasma exposures.58 The structure of 20 in complex with BRAF (PDB code 4XV1) confirmed stabilization of αC-helix in the OUT conformation;58 however this position is further OUT as opposed to the one observed with 5, a result of van der Waals contacts of the terminal ethylmethylsulfamoyl group with L505 of αC-helix (Figure 7d). Interestingly, L505 is one of the four residues of the kinase R-spine (see above). As a consequence, 20, as most members of this series, binds only one BRAF protomer. This demonstrates that as with all αC-OUT inhibitors, the clinical effectiveness of paradox breakers will be limited by RAF dimerization.40 The binding mode of 20 in the BRAF active site (Figure 7d) is typical for a type I1/2 inhibitor; the azaindole ring serves as the hinge binder and the terminal sulfonamide moiety recognizes the type I pocket, while the cyclopropylpyrimidine extends to the solvent. The preclinical efficiency of compound 21 was tested in vivo against lung cancer tumors with non-V600 mutant BRAF (BRAFG469A) in a xenograft model using H1755 cells insensitive to 5 treatment. 21 suppressed effectively tumor growth in these xenograft tumors with no overt toxicity in mice treated at a dose of 150 mg/kg per day.104 21 is undergoing phase 1 clinical trial.107 A few third generation BRAF inhibitors, for which limited chemical and functional data are reported and crystallographic information is lacking, are undergoing clinical trials. 22 (CEP32496)108,109 (Ambit Bio/Ignyta), a quinazolinyloxydiarylurea BRAF and multikinase inhibitor (Figure 6), is cytotoxic to cell lines harboring BRAFV600E.108 Compound 22 also demonstrated significant oral efficacy in a 14-day human COLO-205 (BRAFV600E) tumor xenograft mouse model. In the same xenograft experiment, 22 dosed at 30 mg/kg exhibited tumor stasis and a 40% incidence of partial tumor regressions.109 22 is under phase I clinical evaluation in patients with solid malignancies.110 23 (HM95573) (Hanmi Pharmaceutical/ Genentech) is currently in an expansion clinical trial in patients with advanced solid tumors harboring mutations in BRAF and RAS.111−113 Lastly, a compound from Novartis, 24 (LXH254), is an equipotent BRAF monomer−dimer inhibitor currently in clinical phase I development.114 The chemical structures of 23 and 24 have not been disclosed. Finally, a number of BRAF structures with additional selective and nonselective BRAF inhibitors are available in the PDB. Most of these inhibitors are derivatives of inhibitors discussed here. Noteworthy among them is a purinylpyridinylamino-based inhibitor crystal structure (PDB code 5FD2) showing both inhibitor-bound BRAFWT dimer and monomer,115 a low paradoxical activating diarylthiazole inhibitor (PDB code 4CQE),116 and a nonhinge binding pyrazolopyrimidine inhibitor (PDB code 3II5).117

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CHALLENGES AND FUTURE PERSPECTIVES

The development of BRAF inhibitors of variable chemical classes has progressed significantly in the past few years. This fast pace has been supported by technologies that allowed prompt crystallization of BRAF kinase domain constructs, which brought an explosive growth in BRAF-inhibitor complex structures. Structure-guided design efforts helped in the design of the second generation FDA-approved inhibitors 5 and 6 (αC-OUT), which displayed remarkable clinical results and improved survival of patients with BRAFV600E melanoma tumors. However, resistance limits their clinical value. Many challenges in the design of RAF inhibitors with desirable clinical properties remain. αC-OUT first and second generation BRAF inhibitors are highly selective for tumors dependent on monomeric BRAF species and therefore display a wide therapeutic window. However, they suffer from negative allostery and paradoxical activation and are ineffective against malignancies driven by dimer BRAF mutants. In an effort to bypass resistance to αC-OUT inhibitors and achieve potent and durable inhibition of ERK signaling, combinatory regimes containing a mix of BRAF inhibitors and inhibitors of downstream MEK kinase have recently been developed, demonstrating superior efficacy over RAF inhibitor monotherapies. These efforts resulted in two clinically approved combinations, 6 with trametinib118 (Novartis/GSK, FDA approved in 2014) and 5 with cobimetinib119 (Roche/Genentech, FDA approved in 2015). These combinations are currently the current standard of care for melanoma patients with BRAFV600E/K mutations. Clinical trials of drug combinations with second generation encorafenib (LGX818, Array BioPharma) are ongoing.120−122 Third generation, αC-IN, RAF inhibitors should overcome resistance due to RAF dimerization by occupying both protomers of RAF dimers and be effective in a broad range of non-BRAFV600 or RAS-dependent tumors. However, they display allosteric priming and possibly a narrow therapeutic window in patients because they may also suppress physiological BRAFWT dimers in healthy tissue, as suggested by preclinical data.40 On the other hand, third generation αC-OUT paradoxical breakers are expected to generate less secondary tumors in normal tissue due to reduced paradoxical ERK activation. Preclinically, they display a wider therapeutic window compared to second generation inhibitors; however their action is refractory to RAF dimerization and they are not predicted to be effective in tumors with non-V600 mutant BRAF. A number of third generation RAF inhibitors are currently in clinical trials (Table 1). Nevertheless, due to their aforementioned preclinical limitations, their eventual clinical use as monotherapies is unclear. Designing αC-OUT RAF inhibitors that avert negative allosteric and paradoxical activation will be challenging; however, adding small doses of αC-IN inhibitors to currently used RAFMEK combinations may be a successful strategy to target both BRAFV600E monomers and non-V600 dimers in the same tumors.40 Alternatively, superior type II, αC-IN, inhibitors that bind and effectively inhibit at the same time both promoters of BRAF dimer and BRAF monomers may be developed in the future. Finally, another avenue in RAF inhibitor design may explore non-ATP competitive inhibitors that allosterically prevent RAF dimerization or suppress priming via suppressing RAS binding to the BRAF N-terminal inhibitory domain or prevent BRAF-CRAF heterodimerization. Peptide BRAF dimerization breakers have been reported;38 however whether this can be achieved with small molecules remains to be determined. 5787



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(4) Samatar, A. A.; Poulikakos, P. I. Targeting RAS-ERK Signalling in Cancer: Promises and Challenges. Nat. Rev. Drug Discovery 2014, 13 (12), 928−942. (5) Roberts, P. J.; Der, C. J. Targeting the Raf-MEK-ERK MitogenActivated Protein Kinase Cascade for the Treatment of Cancer. Oncogene 2007, 26 (22), 3291−3310. (6) Davies, H.; Bignell, G. R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M. J.; Bottomley, W.; Davis, N.; Dicks, E.; Ewing, R.; Floyd, Y.; Gray, K.; Hall, S.; Hawes, R.; Hughes, J.; Kosmidou, V.; Menzies, A.; Mould, C.; Parker, A.; Stevens, C.; Watt, S.; Hooper, S.; Wilson, R.; Jayatilake, H.; Gusterson, B. A.; Cooper, C.; Shipley, J.; Hargrave, D.; Pritchard-Jones, K.; Maitland, N.; ChenevixTrench, G.; Riggins, G. J.; Bigner, D. D.; Palmieri, G.; Cossu, A.; Flanagan, A.; Nicholson, A.; Ho, J. W. C.; Leung, S. Y.; Yuen, S. T.; Weber, B. L.; Seigler, H. F.; Darrow, T. L.; Paterson, H.; Marais, R.; Marshall, C. J.; Wooster, R.; Stratton, M. R.; Futreal, P. A. Mutations of the BRAF Gene in Human Cancer. Nature 2002, 417 (6892), 949−954. (7) Dang, C. V.; Reddy, E. P.; Shokat, K. M.; Soucek, L. Drugging the “undruggable” Cancer Targets. Nat. Rev. Cancer 2017, 17 (8), 502−508. (8) Rajakulendran, T.; Sahmi, M.; Lefrançois, M.; Sicheri, F.; Therrien, M. A Dimerization-Dependent Mechanism Drives RAF Catalytic Activation. Nature 2009, 461 (7263), 542−545. (9) Heidorn, S. J.; Milagre, C.; Whittaker, S.; Nourry, A.; NiculescuDuvas, I.; Dhomen, N.; Hussain, J.; Reis-Filho, J. S.; Springer, C. J.; Pritchard, C.; Marais, R. Kinase-Dead BRAF and Oncogenic RAS Cooperate to Drive Tumor Progression through CRAF. Cell 2010, 140 (2), 209−221. (10) Röring, M.; Herr, R.; Fiala, G. J.; Heilmann, K.; Braun, S.; Eisenhardt, A. E.; Halbach, S.; Capper, D.; von Deimling, A.; Schamel, W. W.; Saunders, D. N.; Brummer, T. Distinct Requirement for an Intact Dimer Interface in Wild-Type, V600E and Kinase-Dead B-Raf Signalling. EMBO J. 2012, 31 (11), 2629−2647. (11) Garnett, M. J.; Rana, S.; Paterson, H.; Barford, D.; Marais, R. Wild-Type and Mutant B-RAF Activate C-RAF through Distinct Mechanisms Involving Heterodimerization. Mol. Cell 2005, 20 (6), 963−969. (12) Roskoski, R. RAF Protein-Serine/Threonine Kinases: Structure and Regulation. Biochem. Biophys. Res. Commun. 2010, 399 (3), 313− 317. (13) De Luca, A.; Maiello, M. R.; D’Alessio, A.; Pergameno, M.; Normanno, N. The RAS/RAF/MEK/ERK and the PI3K/AKT Signalling Pathways: Role in Cancer Pathogenesis and Implications for Therapeutic Approaches. Expert Opin. Ther. Targets 2012, 16 (Suppl. 2), S17−S27. (14) Schubbert, S.; Shannon, K.; Bollag, G. Hyperactive Ras in Developmental Disorders and Cancer. Nat. Rev. Cancer 2007, 7 (4), 295−308. (15) Pratilas, C. A.; Taylor, B. S.; Ye, Q.; Viale, A.; Sander, C.; Solit, D. B.; Rosen, N. (V600E)BRAF Is Associated with Disabled Feedback Inhibition of RAF-MEK Signaling and Elevated Transcriptional Output of the Pathway. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (11), 4519−4524. (16) Wan, P. T. C.; Garnett, M. J.; Roe, S. M.; Lee, S.; NiculescuDuvaz, D.; Good, V. M.; Jones, C. M.; Marshall, C. J.; Springer, C. J.; Barford, D.; Marais, R.; Cancer Genome Project.. Mechanism of Activation of the RAF-ERK Signaling Pathway by Oncogenic Mutations of B-RAF. Cell 2004, 116 (6), 855−867. (17) Foster, S. A.; Whalen, D. M.; Ö zen, A.; Wongchenko, M. J.; Yin, J.; Yen, I.; Schaefer, G.; Mayfield, J. D.; Chmielecki, J.; Stephens, P. J.; Albacker, L. A.; Yan, Y.; Song, K.; Hatzivassiliou, G.; Eigenbrot, C.; Yu, C.; Shaw, A. S.; Manning, G.; Skelton, N. J.; Hymowitz, S. G.; Malek, S. Activation Mechanism of Oncogenic Deletion Mutations in BRAF, EGFR, and HER2. Cancer Cell 2016, 29 (4), 477−493. (18) Holderfield, M.; Deuker, M. M.; McCormick, F.; McMahon, M. Targeting RAF Kinases for Cancer Therapy: BRAF-Mutated Melanoma and Beyond. Nat. Rev. Cancer 2014, 14 (7), 455−467. (19) Zhang, W. BRAF Inhibitors: The Current and the Future. Curr. Opin. Pharmacol. 2015, 23, 68−73. (20) Barras, D. BRAF Mutation in Colorectal Cancer: An Update. Biomarkers Cancer 2015, 7 (Suppl. 1), 9−12.

Hopefully, further insights on allosteric transitions of RAF kinases upon inhibitor binding and chemical intuition will provide the next generation of RAF inhibitors.

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AUTHOR INFORMATION

Corresponding Authors

*B.A.: e-mail, [email protected]; phone, (718) 430-8590. *E.G.: e-mail, [email protected]; phone, (718) 430-3725. ORCID

Evripidis Gavathiotis: 0000-0001-6319-8331 Notes

The authors declare no competing financial interest. Biographies Bogos Agianian, Ph.D., is a Research Assistant Professor at Albert Einstein College of Medicine. He completed his doctoral studies in Biophysics at the European Molecular Biology Laboratory (EMBL) and received his Ph.D. from Heidelberg University. He was a postdoctoral fellow at the Bijvoet Center for Biomolecular Research and a Marie Curie fellow at EMBL. His research focuses on rational small-molecule drug design and structural mechanisms in allosteric regulation of pharmacologically important proteins. Evripidis Gavathiotis, Ph.D., is an Associate Professor of Biochemistry and Medicine at the Albert Einstein College of Medicine. He received his Ph.D. in Chemistry from the University of Nottingham, U.K., for studies on biomolecular structure and molecular recognition. He worked at De Novo Pharmaceuticals, applying in silico drug design to several therapeutic targets. He pursued postdoctoral studies at Rockefeller University and Dana Farber Cancer Institute/Harvard Medical School on chemical and structural biology of apoptosis signaling. His research program focuses on the use of chemical biology, structural biology, and drug discovery to investigate molecular mechanisms of oncogenic signaling pathways regulating cell survival and cell death and the development of new therapeutics.

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ACKNOWLEDGMENTS We thank Dr. Poulikos Poulikakos for stimulating discussions and collaborative work on BRAF inhibitors. We acknowledge the support from the Melanoma Research Alliance and the Irma T. Hirschl Trust.

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ABBREVIATIONS USED α-loop, activation loop; AS, activation segment; CPK, Corey− Pauling−Koltun; C-spine, catalytic spine; ERK, extracellular signal-regulated kinase; IRE1α, inositol requiring enzyme 1α; JAK, Janus kinase; MEK, mitogen-activated protein; NSCLC, non-small-cell lung carcinomas; NTD, N-terminal domain; PDX, patient-derived xenograft; pERK, phospho-ERK; PKC, protein kinase C; p-loop, proline rich loop; pMEK, phospho-MEK; RAF, rapidly accelerated fibrosarcoma; RAS, rat sarcoma; RDB, RASbinding domain; R-spine, regulator spine; SRC, sarcoma kinase

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REFERENCES

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