Current insights of BRAF inhibitors in cancer - Journal of Medicinal

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Perspective

Current insights of BRAF inhibitors in cancer Bogos Agianian, and Evripidis Gavathiotis J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01306 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 2018

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Journal of Medicinal Chemistry

Current insights of BRAF inhibitors in cancer Bogos Agianian* and Evripidis Gavathiotis* Department of Biochemistry, Department of Medicine, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA

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 Perspective 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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Introduction The rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein (MEK)/ extracellular signal–regulated kinase (ERK) signaling pathway controls cell growth, proliferation and survival1. The RAF family of protein kinases (ARAF, BRAF, and CRAF) are critical effectors of this pathway2,3. 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 ERK22. Oncogenic mutations of this pathway are observed frequently in many cancers5,6. RAS is considered a central therapeutic target of the pathway because it is mutated in approximately 30% of human cancers4,6, however drugging RAS has been notoriously challenging7. As a result, efforts to inhibit RAF, MEK and ERK kinases have gained significant momentum4.

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 RAS8,9, however physiological RAS signals mainly via BRAF dimers2,3,10. In addition, oncogenic kinase-impaired BRAF mediates CRAF transactivation in a RAS-independent way11.

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 them3,4. Overall, BRAF mutations are present in about 8% of human tumors6. BRAF mutants are present in >50% of melanoma patients as well as in colorectal (5-10%), thyroid carcinomas (25-45%), hairy-cell leukemia (~100%) and less commonly in ovarian and lung malignancies6,12–14 .

BRAFV600E mutation promotes

constitutive kinase activation through signaling as a monomer, as well as insensitivity to ERK negative feedback mechanisms15. In addition to V600E, a number of other hyper-activating point mutations at positions 600 (V600K, V600D, V600R), 601 (K601E, K601D, K601R), 469 (G469A, G469V) and other have been


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reported6,11,16. 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 signaling17,18.

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 year19. Moreover, colorectal and thyroid BRAFV600E tumors are resistant to 5 and 620 as well as 15–30% of non-small cell lung carcinomas (NSCLC)21 and ~85% of pancreatic cancer cells22. The clinical importance of BRAF 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 dimers2–4,23,24. Specifically, a major mechanism of acquired resistance involves signaling via BRAF homodimeric25–28 or BRAF-CRAF heterodimeric23,29 species. Clinically resistant tumors may also possess splice variants of BRAFV600E which form resistant dimers27,30.

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 studies evaluate the combination of RAF/MEK inhibitors with immunotherapy32.

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


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cellular basis of sensitivity and resistance to inhibitors. In this perspective, 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.

The structural basis of BRAF physiological and aberrant activation RAFs have a typical kinase structure3. Two domains, the N-terminal and C-terminal lobes, are linked via a flexible hinge which allow 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 catalytic E501 residue which 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 towards 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


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below) sub-pockets (Figure 1b).


It is widely accepted that the catalytically active kinase conformation

necessitates a DFG-IN/αC-IN conformation, a closed inter-lobe 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) spines33. Precise vertical alignment of the R-spine residues, which involves F595 of DFG motif, is 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 to an active structure, the αChelix of this active protomer adopts an IN position which enables catalysis and a fully extended α-loop (and AS).

Under non-activating 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 domain35. 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 activation4,36. 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 Cterminal 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


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mutagenesis studies8,27,38. 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 revealed40,41. 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 R-spine 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 is 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. Based on 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 inter-lobe 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 activation17,34,41. 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 structure40,41. 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


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V600E mutation may induce movement of αC-helix towards the IN conformation and allosteric activation. A second mode of V600 interaction, observed in a few inactive BRAFWT structures (in complex with inhibitors) with folded α-loops, is characterized by interactions of V600 with p-loop residues and a closed, active-like, inter-lobe conformation, as originally proposed16. 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) co-crystal 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 levels8,23,32,38.

Similar structural mechanisms of activation to BRAFV600E seem to apply to other V600

oncogenic mutants (V600K, V600D or V600R) which also can act as monomers26,40. In addition, in-frame 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 carcinomas17,42. Structural analysis revealed that the αC- helix-IN conformation of these mutants (Figure 3b) is incompatible to compound 5 binding due to steric clashes17.

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 RAS-independent42, however, it is controversial whether this is also the case for β3αC-deletions17,42. In a mutually non-exclusive model, interference of certain mutations to inhibitory autophosphorylation of ploop residue S467 may also play a role in activation43. Finally, another class of RAS-dependent catalytically


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impaired or inactive BRAF mutants mainly found in colorectal, melanoma and lung tumors44, such as D594N, G466V, S467L, G596R, were recently shown to depend on dimerization45. 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.

Conformational and functional effects of BRAF inhibitors A large number of structurally diverse RAF inhibitors have been developed4,46. 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 motif3,40. These conformations broadly classify inhibitors as type I (αC-helix-IN/DFG-IN), type II (αC-helixIN/DFG-OUT) or type I½ (αC-helix-OUT/DFG-IN)42,49. Clinical RAF inhibitors 5 and 6 (Figure 6) belong to type I½.

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 OUT conformation. A typical type I½ example is 5 which binds only one BRAF protomer of the dimer and induces an αC-OUT conformation (Figure 4a). The αC-OUT position of compound 5 bound to protomer creates negative allostery to the second protomer


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by inducing the αC-IN conformation, which thus becomes incompatible with binding 542. Thus, BRAF-(5) dimers are highly asymmetric. By contrast, typical type II inhibitors, like 10 (AZ-628) (see below), stabilize an ΙΝ conformation of αC-helix in the first protomer and does not create large allosteric αC-helix movements in the second protomer, which remains in an αC-IN conformation allowing another inhibitor binding (Figure 4)42. First and second generation RAF inhibitors (see below) belong mostly to I or I½ types (Table 1). They are αCIN 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 αC-OUT conformation in both protomers42,47. The position of DFG does not appear to influence negative allostery as both DFG-IN or OUT conformation are observed in BRAF complexes with αC-OUT and αC-IN inhibitor inducers (Figure 4c).

Structural effects of RAF inhibitors are associated to their preclinical and clinical activity Several preclinical and clinical studies of RAF inhibitors demonstrated unusual pharmacologic behavior9,28,48. 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 I½ RAF inhibitors, an observation with important clinical implications40. 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 tumors34,40,41,49. It has become apparent that these effects are dictated by distinct inhibitor-driven allosteric mechanisms which prime specific RAF conformations, as well as the cellular context of active RAS42.

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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 αCOUT inhibitors for the second protomer allows it to engage in catalysis, which is the basis of upregulation of ERK signaling4. 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 αCOUT 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 carcinomas18.

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 non-V600 mutants) BRAF26,40,45.

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 αC-IN conformation) in healthy tissue4. 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 phosphorylation2,3,40. Ultimate levels of primed


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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 hetero dimers50–52, 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 inhibitor-induced dimerization for instance in the cases of inositol requiring enzyme 1α (IRE1α) or JAK256,57.

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 mode40. Both αC-OUT and αC-IN 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) 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 activity2,40.

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 interaction40. This model predicts that displacement of the R506 from the OUT position towards 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, αCIN inhibitors 2 (SB-590885) or 3 (GDC-0879) (Figure 6, Table 1) (R506-IN conformation) promote RAF-RAS

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interaction, while the ‘paradox breakers’ 20 (PLX7904, PB04) (Figure 6, 7d) and 21 (PLX8394) (Figure 6, Table 1) (R506-OUT conformation) do not promote priming and paradoxical activation40,58. 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 inhibitors40. Moreover, among the group of αC-IN RAF inhibitors, those stabilizing the DGF-IN conformation, e.g. 2 or 5 (Figure 4c) display more pronounced paradoxical activation compared to their DFG-OUT counterparts40. This observation may be the result of allosteric formation of RAF-MEK complex by these inhibitors, which activates ERK pathway34,40. In contrast, αC-OUT inhibitors like the clinically used 5 and 6 do not promote RAF interaction with MEK.

Two non-mutually 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 αC-helix in the dimerization-resistant OUT conformation58. 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.

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, ZM33637260, 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


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hepatocellular carcinoma tumors that express BRAFWT61–63. 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 multi-kinase targeting. Development of several other first generation compounds did not produce any clinically approved RAF inhibitors. These included the indol-2-one derivative GW507465,66, the triarylimidazole derivative 2 (SB-590885)67, the pyridylpyrazole derivative 3 (GDC-0879)68 and the azaindole derivative 4 (PLX-4720)69 (Figure 6, Table 1, 2). With the exception of 1 which is a type II inhibitor, first generation RAF inhibitors belong to type I or I½,.

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 569,70, an azaindole compound, and the pyrimidine derivative 671,72, the only such inhibitors to obtain FDA approval (Figure 6, Table 1). 5 was approved for the treatment of BRAFV600E metastatic melanoma, while 6 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 cancers73. These drugs show remarkable effectiveness in BRAFV600E tumors, however tumors driven by non-V600 mutants are resistant26,38,40. 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 I½ binding mode; the azaindole moiety forms two specific H-bonds to the backbone of hinge residues Q530 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 butyl-thiazol group of 6.


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Another second generation inhibitor that has demonstrated promising preclinical and clinical properties is 7 (LGX818) (Encorafenib, Array Biopharma)74–76.

These properties included a low off-rate from BRAF

compared to 5 or 624, which leads to a clinically longer residence time and a high selectivity for BRAFV600E75. There is no available BRAF-LGX818 structure, however, the presence of a terminal type I sulfonamide (Figure 6, Table 1), similar albeit shorter that in 5 and 6, suggests that 7 is a type I½ inhibitor predicted to induce the αC-OUT/DFG-IN

conformation.

Similarly

to

other

pyrazolyl-pyrimidine

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 trials77.

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 trials80. 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/day78. 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, compare 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


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twice daily) or a COLO205 model (25 mg/kg twice daily) resulted in significant shrinkage of tumors47. At the same time, the toxicologic profile of 9 in male rats provided no evidence of pathologic changes47. Oddly, the structure of 9 with BRAFWT illustrates a unique αC-OUT/DFG-OUT conformation, indicative of a mixed type I and 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 (similarly to αC-IN inhibitors), sterically prohibiting movement of F595 towards 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 limited81,82. This is attributed to upstream RAS and RTK activation83,84 which is maintained by pronounced feedback reactivation mechanisms and enhanced RAF dimerization40.

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, in contrast to αC-OUT inhibitors. Many third generation inhibitors have been produced and several of them are currently undergoing Phase I or II clinical trials (Table 1). Most known third generation RAF inhibitors bind BRAF in type II mode (αC-

15


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IN/DFG-OUT), whereby additional, compared to type I or type I½, chemical groups insert into a hydrophobic pocket (type II pocket) deep in the BRAF active site cleft (Figure 1b). In most co-crystal structures of BRAF with third generation inhibitors, strong hydrogen bonding of the inhibitor to the catalytic residue E501 of αChelix or backbone NH of D594 of DFG motif is observed (Figure 8) which is facilitated by the movement of αC-helix towards 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 cells48. However, despite suppression of ERK signaling, the inhibitor demonstrated pan-RAF and multi-kinase inhibition profiles, similar to 1, and induced acquired resistance due to elevated CRAF expression in various cell lines23. 10 inhibits equipotently monomeric and dimeric BRAF40 as indicated by crystal structures with BRAF monomers (Figure 4b, BRAFR509H mutation, PDB:4RZW)40 and dimes (PDB: 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 CRAF48. Due to unfavorable pharmacological properties, 10 did not advance to clinical trials.

11 (LY3009120)86, an urea-linked pyridopyrimidine compound developed by Elli Lilly is a third generation αCIN/DFG-OUT inhibitor which binds to both RAF protomers creating a mostly symmetrical dimer52.

11

therefore inhibits BRAF monomers and dimers with equal affinity52,87 . 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: 5C9C) shows that the inhibitor adopts a type II binding mode; the pyridopyrimidine


16

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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 RAS-dependent tumors52,86 as well as against pancreatic and thyroid tumors42. In HCT116 cell 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 patientderived 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 mutations86.

In pharmacokinetic experiments, 11

displayed initially low bioavailability in rats, dogs and monkeys, which was significantly increased using solid dispersion technology86. Currently 11 is under Phase I clinical investigation88.

Similarly to 11, compounds 12 (TAK632)89, 13 (MLN2480/TAK580)90 (Takeda/Millennium) and 25 (CCT3833/BAL3833)91,92 (ICR/Basilea) are equipotent inhibitors of BRAF dimers and monomers40,50. They are therefore expected 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 mutations40,50, in vemurafenib-resistant melanomas and in BRAFV600E colorectal or thyroid tumors50. 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 CRAF89 . 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


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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 phenyl-acetamide 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 antibodies94,95. A recent study using neuronal cells expressing mutated BRAF showed that 13 inhibits both BRAF monomers and dimers and does not elicit strong paradoxical activation90 In a clinical study with patients with relapsed or refractory tumors, 13 exhibited rapid absorption (median Tmax 2 hr), low fluctuation at steady state (mean peak to trough ratio 2.1) and mean accumulation half-life of 67 hr, with acceptable safety profile94. 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 trifluoromethyl-pyridin 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 inhibitors51,91,96. Structurally, the pyridopyrazine moiety of these compounds is expected to contact the hinge of BRAF and the butyl-phenyl-pyrazol 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 mice46. 14 or


18

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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 melanomas92. However, chemical details or the structure of 25 have not been disclosed.

Based on an original report on new tetrahydronaphthalene RAF inhibitors97, 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 monomers26. Because of the ability of 16 to bind to both protomers of BRAF dimer, it evades paradoxical activation and inhibits V600 and non-V600dependent melanomas as well as melanomas resistant to 126. A cyclopropabenzofuran derivative compound from the same company, 17 (BGB283) (Figure 6), inhibits BRAF and EGFR and is effective in BRAFV600E colorectal tumors98 . 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 nM26. 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 organs26. 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 trials99. 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.

19


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RAF inhibitors developed from Novartis, replaced the urea linker of 1 with an aminoimidazole bioisostere group100. 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). Using 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 trifluoromethyl-phenyl 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 0.5 nM, see Table 1, in comparison to IC50 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 model102. Phase I clinical trial on subjects with various tumors are ongoing for compound 19103. The BRAFWT-(19) co-crystal structure (PDB: 5CT7) shows that the imidazolo-pyridine 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. aCOUT paradox breakers comprise a distinct group among them. Typical member include azaindole derivatives developed by Plexxikon; 20 (PLX7904/PB04)58 and its analog 21 (PLX8394/PB03)104 (Figure 6). These


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molecules are more potent than 5 in targeting BRAFV600E58. They are type I½ 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 dimers58 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 tumors105,106. However, in vivo, 20 and 5 produced similar anti-tumour effects in a subcutaneous COLO205 xenograft model (doses 25 mg/kg twice daily) and good plasma exposures58. The structure of 20 in complex with BRAF (PDB: 4XV1) confirmed stabilization of αC-helix in the OUT conformation58, however this position is further OUT as opposed to the one observed with 5, a result of van der Waals contacts of the terminal ethylmethyl-sulfamoyl 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 dimerization40. The binding mode of 20 in the BRAF active site (Figure 7d) is typical for a type I½ inhibitor; the azaindole ring serves as the hinge binder and the terminal sulfonamide moiety recognizes the typeI 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 day104. 21 is undergoing Phase 1 clinical trial107.

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 (CEP-32496)108,109 (Ambit

Bio/Ignyta), a quinazolinyloxy-diaryl-urea BRAF and multi-kinase inhibitor (Figure 6), is cytotoxic to cell


21


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lines harboring BRAFV600E108. 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 regressions109. 22 is under Phase I clinical evaluation in patients with solid malignancies110.

23 (HM95573) (Hanmi Pharmaceutical/Genentech) is

currently in an expansion clinical trial in patients with advanced solid tumors harboring mutations in BRAF and RAS111–113. Lastly, a compound from Novartis, 24 (LXH254), is an equipotent BRAF monomer-dimer inhibitor currently in clinical Phase I development114. The chemical structures of 23 and 24 have not been disclosed.

Finally, a number of BRAF structures with additional selective and non-selective BRAF inhibitors are available in the PDB. Most of these inhibitors are derivatives of inhibitors discussed here. Noteworthy among them is a purinylpyridinyl-amino-based inhibitor crystal structure (PDB: 5FD2) showing both inhibitor-bound BRAFWT dimer and monomer115, a low paradoxical activating diarylthiazole inhibitor (PDB: 4CQE)116 and a non-hinge binding pyrazolo pyrimidine inhibitor (PDB: 3II5)117.

Challenges and future perspectives. The development of BRAF inhibitors of variable chemical classes has progressed significantly the past few years. This fast pace has been supported by technologies which 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.


22

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Many challenges in the design of RAF inhibitor 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 ongoing120–122.

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 data40. 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 which avert negative allosteric and paradoxical activation will be challenging, however, adding small doses of αC-IN

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inhibitors to currently used RAF-MEK combinations may be a successful strategy to target both BRAFV600E monomers and non-V600 dimers in the same tumors40. 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 hetero-dimerization. Peptide BRAF dimerization breakers have been reported38 however whether this can be achieved with small molecules remains to be determined. 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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FIGURES Figure 1. BRAF structure. a. Ribbon representation of a BRAF protomer kinase domain structure bound to inhibitor 18 (PDB: 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 (grey) 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) sub-pockets (see text for details). The ‘gate keeper’ residue T529 and the catalytic residues E501 and D594 (DFG motif) are shown. A salt-bridge between catalytic E501 and R483, a hallmark of active kinase33, 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 towards OUT (red arrow) or IN (green arrow) conformations.

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-PaulingKoltun (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 with a dashed red line. a, b BRAF-inhibitor inactive protomer structures showing disordered (part a, complex with 1, PDB: 1UWH) or ordered (part b, β3αC-deletion mutant in complex with 10, PDB: 5HID) α-loop. In both structures,


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the, inactive DFG-OUT position of F595 prevents full alignment of the R-spines. c, a monomeric BRAF structure (PDB: 4RZW) demonstrates an inactive kinase conformation in which the α-loop is folded into a helical conformation. The AS forms a short helix which stabilizes αC-helix in the inactive OUT position. The R-spine is assembled but kinked (kinked red line), indicative of inactive kinase33. d, a protomer of the active BRAF dimer (PDB ID: 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).

Figure 3. Structural mapping of BRAF activating mutations. Structural mapping of common cancer activation point mutations on inactive BRAF monomer (panel a, PDB: 4RZV), an inactive protomer (panel b, PDB: 5HID) and active protomers (panel c, BRAFWT/MEK1 complex structure, PDB: 4MNE; panel d, BRAFV600E, PDB: 3OG7). Most mutations are concentrated at the AS of the α-loop and the p-loop. Mutations shown: 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 red spheres, respectively. 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 N-lobe (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 αChelix, which adopts the “active” IN position.


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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 I½ 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: 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 αC-helix positions. The corresponding conformations, inhibitors and PDB codes 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.

Figure 5. Inhibitors induce asymmetric BRAF dimers. Illustration of a BRAF dimer showing the two protomers in a molecular surface representation (brown, blue). The position of αC-helices of inhibitor-bound protomers A from different BRAF dimers in complex with 9 (yellow, PDB: 5CSX), 5 (magenta, PDB: 5OG7), 2 (green, PDB: 2FB8) and 10 (cyan, PDB: 4RZW) BRAFαC-helices is shown after structural superposition of αC-helices (blue) from protomers B. Type II inhibitor 10 induces the αC-IN conformation while all other inhibitors induce various degrees of shift towards the αC-OUT conformation. The corresponding F595 residues (in sticks) adopt either the IN (magenta, green) or OUT (yellow, cyan) position.

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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 sub-pockets of BRAF active site are highlighted in different colors as in Figure 1b. Coloring key of sub-pockets: ribose (pink), adenine (orange), hydrophobic (blue), type I (brown) and type II (green). Groups exposed to solvent are colored in gray.

Figure 7. Recognition of BRAF active site by type I and I½ BRAF inhibitors. Close-up on the active site of BRAF showing binding mode of various type I or I½ 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 sub-pockets 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.

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, c) the salt-bridge to K483 is maintained.


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Table 1. Representative BRAF inhibitors RAF Inhibitors

Class

Originator/Developer

In vitro IC50/Ki (nM)*

In cellulo IC50/EC50 (viability) (nM)#

In cellulo IC50/EC50 (pERK) (nM)

Stage of development

Refs

First Generation 1 2 3 4

II I I I½

Bayer/Onyx GlaxoSmithKline Genentech Plexxikon

221 0.16 0.17 13

39001 370 >500 500

4.4 290 >1000 44

FDA approved Preclnical Preclinical Preclinical

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

Second Generation 5

I½

31

3102

32

FDA approved

58,124

6 7 8 9

I½ I½4 I I/II6

Plexxikon/Hoffman La Roche GlaxoSmithKline Array BioPharma Exelixis Boehringer Ing.

0.8 0.3 6 0.4

433 4 2005 0.9

4 3 20005 0.5

FDA approved Phase III Phase I (completed) Preclinical

125 75,76 126,127 47

Third Generation 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

II II II II II II II II II II I½ I½4 II4 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 0.411 0.5 4.2 3.8 14 7 N/A

3837 9.2 66 2488 159 10 200 200 95011 14012 170 ~80013 78 11814 N/A

15 37 16 ~708 ~5 ~5 15710 64 10011 16012 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

*Activity on BRAFV600E mutant, except if noted otherwise. # Viability in melanoma A375 (BRAFV600E) cells, except if noted otherwise. 1

Activity on BRAFWT. Viability in SK-MEL-30 cells. SK-MEL-32 cells. 3 SK-MEL-28 cells. 4 Predicted 5 pERK activity in Jurkat T cells. Viability in SK-MEL-19 cells. 6 αC-OUT/DFG-OUT 7 SK-MEL-239 cells. 8 Viability and pERK activity in synthetic pediatric low-grade astrocytomas (PLGAs) expressing BRAFV600E 9 WM266.4(BRAFV600D) cells. 10 SK-MEL-239 (BRAFV600E) cells. 11 Activity on BRAFWT. pERK activity and viability in Calu-6 cells. 12 SK-MEL-28 cells. 2


29

Journal of Medicinal Chemistry 1 2 31 42 53 64 75 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

13 14

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HCC364 lung Adenocarcinoma cells expressing p61-V600E. Colo-205 cells.


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Table 2. Structural data and specificity of BRAF inhibitors RAF Inhibitors 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

1 2

Co-structure protein

BRAF Conformation

BRAF Specificity Monomer (M)/Dimer (D)

PDB Codes

BRAFWT, BRAFV600E, β3αC-BRAFWT BRAFWT BRAFV600E BRAFWT

αC-IN/DFG-OUT

M/D

1UWH, 1UWJ, 5HI2

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

M/D M/D M

2FB8 4MNF 3C4C, 4WO5

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

αC-OUT/DFG-IN

M

3OG7, 4RZV

αC-OUT/DFG-IN

M

4XV2, 5HIE, 5CSW

αC-OUT/DFG-IN1 αC-IN/DFG-IN1 αC-OUT/DFG-OUT2

M1 M/D1 M

N/A N/A 5CSX

αC-IN/DFG-OUT

M/D

5HID, 4G9R, 4RZW

αC-IN/DFG-OUT αC-IN/DFG-OUT αC-IN/DFG-OUT1 αC-IN/DFG-OUT1 αC-IN/DFG-OUT1 αC-IN/DFG-OUT1 αC-IN/DFG-OUT αC-IN/DFG-OUT αC-IN/DFG-OUT αC-OUT/DFG-IN αC-OUT/DFG-IN1 αC-IN/DFG-OUT1 N/A N/A

M/D M/D M/D1 M/D1 M/D1 M/D1 M/D M/D M/D M M1 M/D1 N/A N/A

5C9C 4KSP N/A N/A N/A N/A 4R5Y 5VAM 5CT7 4XV1 N/A N/A N/A N/A

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

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


31


Page 32 of 65

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.

Correspondence [email protected], Phone: (718) 430-3725 [email protected], Phone: (718) 430-8590

Notes The authors declare no competing financial interests.

Author 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, UK 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


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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.

Abbreviations

α-loop

activation loop

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

RAS-binding domain

R-spine

Regulator-spine


33


SRC

Page 34 of 65

sarcoma kinase

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