Investigation into the Use of Encorafenib to Develop Potential PROTACs Directed against BRAFV600E Protein

BRAF is a serine/threonine kinase frequently mutated in human cancers. BRAFV600E mutated protein is targeted through the use of kinase inhibitors which are approved for the treatment of melanoma; however, their long-term efficacy is hampered by resistance mechanisms. The PROTAC-induced degradation of BRAFV600E has been proposed as an alternative strategy to avoid the onset of resistance. In this study, we designed a series of compounds where the BRAF kinase inhibitor encorafenib was conjugated to pomalidomide through different linkers. The synthesized compounds maintained their ability to inhibit the kinase activity of mutated BRAF with IC50 values in the 40–88 nM range. Selected compounds inhibited BRAFV600E signaling and cellular proliferation of A375 and Colo205 tumor cell lines. Compounds 10 and 11, the most active of the series, were not able to induce degradation of mutated BRAF. Docking and molecular dynamic studies, conducted in comparison with the efficient BRAF degrader P5B, suggest that a different orientation of the linker bearing the pomalidomide substructure, together with a decreased mobility of the solvent-exposed part of the conjugates, could explain this behavior.


Introduction
The RAS/RAF/MEK/ERK signaling pathway controls cellular growth, differentiation, and survival [1,2]. The RAF family of protein kinases (ARAF, BRAF, and CRAF) are critical effectors of this pathway [3], which is normally activated by receptor tyrosine kinase signaling that stimulates binding of GTP to RAS at the plasma membrane [4,5]. RAF proteins are subsequently recruited at the membrane through interaction with the active GTP-bound RAS, where they are activated by dephosphorylation and phosphorylation events [2]. Recruitment of RAFs 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 [6]. Aberrant activation of ERK signaling is a hallmark of many cancers, and is most commonly due to mutations of RAS and BRAF genes [7,8]. 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 [6,9]. While RAF proteins activate ERK signaling as homo-and heterodimers in the presence of active RAS, mutant BRAF V600E can activate ERK signaling independently of RAS as an active monomer [10,11], and promote insensitivity to ERK negative feedback mechanisms [12].
Therapeutic efforts to mitigate BRAF V600E enjoyed initial success with the discovery of small-molecule BRAF kinase inhibitors, with the approval of vemurafenib [18], dabrafenib [19], and encorafenib [20] for the treatment of melanoma patients carrying BRAF V600E mutations. Indeed, acquisition of a BRAF mutation in melanoma cells leads to constitutive signaling through the MAPK pathway, which in turn contributes to immune escape through the recruitment of regulatory T cells, decreased antigen presentation (via downregulation of MHC class I) and the inhibition of IFNγ, IL2, and TNFα release. Inhibition of BRAF in BRAF-mutant melanoma cells reverses these processes and can restore tumor-immune recognition [21].
The clinical benefits of currently approved BRAF kinase inhibitors are greatly restrained by the rapid emergence of acquired resistance. The prevailing explanation for this phenomenon invokes the transactivation model, wherein inhibitor occupation of one protomer within the RAF dimer induces kinase activation of the other protomer. Even when BRAF V600E is bound by an inhibitor and can no longer phosphorylate its substrates, the oncogenic protein can still activate CRAF, another isozyme in the RAF family, through dimerization, to transduce proliferative signals through the ERK pathway [22]. Combination with MEK inhibitors neutralizes the paradoxical activation of the ERK pathway induced by BRAF inhibitors, modestly improving response rate and extending tumor control [23]. However, resistance and tumor recurrence remain inevitable. For the development of more efficacious therapeutics for BRAF-mutant tumors, a blockade of not only BRAF's catalytic activity, but also its non-catalytic functions, is needed.
Hijacking the ubiquitin proteasome system via heterobifunctional small molecule compounds is an emerging pharmaceutical strategy aiming to selectively remove diseasecausing proteins in affected cells. These small molecule degraders, also known as proteolysistargeting chimeras (PROTACs), consist of a chemical tag that binds to a target protein of interest, connected by a linker to a second tag that binds to a cellular E3 ubiquitin ligase. By simultaneously binding to a target and an E3 ligase, the PROTAC stimulates the ubiquitination and subsequent degradation of the target by the proteasome. Targeted degradation of the protein of interest not only compromises the catalytic activities of these proteins but also removes their scaffolding and other non-catalytic functions, providing unique advantages over inhibitors [24,25].
With the aim of inducing BRAF inhibition and degradation, in this study we designed a series of PROTACs built upon the scaffold of the approved drug encorafenib (1, Figure 1). The solvent-exposed terminal group in encorafenib was linked to pomalidomide (2, Figure 1), a widely employed cereblon (CRBN) binder, through spacers endowed with different lengths and flexibility. Two additional molecules containing encorafenib bound to lenalidomide (3, Figure 1), another glutarimide-based CRBN binder, and to the VHL ligand-1 (4R)-3-methyl-L-valyl-4-hydroxy-N-[[4-(4-methyl-5-thiazolyl)phenyl]methyl]-L-prolinamide (4, Figure 1), were also designed. In this study, we report the synthesis and in vitro evaluation of BRAF V600E inhibiti together with the antiproliferative activity in melanoma and colon cancer cell lines A3 and Colo205 of the synthesized compounds. The ability of selected compounds to indu degradation of the target protein is also discussed. Finally, we illustrate the computation studies performed to explain the lack of target degradation in comparison with compou P5B [24], a recently disclosed BRAF V600E degrader.

Chemistry
In order to obtain compounds bearing the BRAF V600E ligand and the CRBN ligand different spatial positions, the PEG-derived linkers were selected to cover a wide ran spanning from three to seventeen atoms (compounds 5-10, Figure 2). To study the pos ble influence of the flexibility in the formation of the ternary complex between the prote of interest (BRAF V600E ) and the ligase, compounds 11-13, bearing a 1,5-disubstituted tr zole ring at different points of the linker, were also synthesized. Finally, in compound the scaffold of pomalidomide was replaced with either lenalidomide (compound 14) VHL ligand-1 (compound 15) to assess the behavior of different ligase-targeting group  In this study, we report the synthesis and in vitro evaluation of BRAF V600E inhibition together with the antiproliferative activity in melanoma and colon cancer cell lines A375 and Colo205 of the synthesized compounds. The ability of selected compounds to induce degradation of the target protein is also discussed. Finally, we illustrate the computational studies performed to explain the lack of target degradation in comparison with compound P5B [24], a recently disclosed BRAF V600E degrader.

Chemistry
In order to obtain compounds bearing the BRAF V600E ligand and the CRBN ligand at different spatial positions, the PEG-derived linkers were selected to cover a wide range, spanning from three to seventeen atoms (compounds 5-10, Figure 2). To study the possible influence of the flexibility in the formation of the ternary complex between the protein of interest (BRAF V600E ) and the ligase, compounds 11-13, bearing a 1,5-disubstituted triazole ring at different points of the linker, were also synthesized. Finally, in compound 8, the scaffold of pomalidomide was replaced with either lenalidomide (compound 14) or VHL ligand-1 (compound 15) to assess the behavior of different ligase-targeting groups.
The potential PROTACs bearing pomalidomide were synthesized following the general strategy, illustrated in Figure 3. According to our approach, in order to insert the desired linkers into the structure of final PROTACs, we needed linker synthons containing a free terminal amino group on one side and a carboxylic acid group on the other side of the linker chain. We envisaged that the amino group could be first inserted into the phthalimide scaffold of pomalidomide through the nucleophilic substitution of the fluorine atom in 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (31); then, the carboxylic group could be used to generate an amide bond with 16 to afford the designed compounds 5-14.
Compound 14 was obtained by conjugation of 16 to lenalidomide (3), as depicted in Scheme 8. In order to synthesize 14, we used a 12-atom linker (49) to join the two selected chemical scaffolds. Linker 49 contains a free carboxyl group at one end and a tert-butyl protected carboxylic acid at the other end. The synthesis of 49 was performed by oxidation of the terminal hydroxyl group in 19 by using a (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO)/(Diacetoxyiodo)benzene BAIB oxidating system [37]. The obtained intermediate was then coupled with 3 using HATU to promote the formation of the amide bond. After chromatographic purification, the intermediate tert-butyl protected carboxylate was deprotected, and the free acid 50 was coupled to 16 to afford the designed 14. Compound 14 was obtained by conjugation of 16 to lenalidomide (3), as dep Scheme 8. In order to synthesize 14, we used a 12-atom linker (49) to join the two s chemical scaffolds. Linker 49 contains a free carboxyl group at one end and a ter protected carboxylic acid at the other end. The synthesis of 49 was performed by ox of the terminal hydroxyl group in 19 by using a (2,2,6,6-Tetramethylpiperidin-1-(TEMPO)/(Diacetoxyiodo)benzene BAIB oxidating system [37]. The obtained inte ate was then coupled with 3 using HATU to promote the formation of the amide After chromatographic purification, the intermediate tert-butyl protected carboxyl deprotected, and the free acid 50 was coupled to 16 to afford the designed 14. Finally, the reference compound 54, lacking the CRBN ligand, was accomplished according to the route reported in Scheme 10. Encorafenib derivative 54, bearing a 13-atom PEG-type linker, was obtained through the reaction of 2-(2-(2-methoxyethoxy)ethoxy)ethan-1-ol (52) with tert-butyl acrylate, followed by hydrolysis of the tert-butyl ester in acidic medium (TFA 10%), to furnish the acid 53. Finally, HATU-mediated formation of the amide bond allowed the coupling with derivative 16. Using this route, it was possible to obtain the desired 54 in 28% overall yield. The same strategy was employed for the synthesis of 15, which bears the VHL-ligand (2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5yl)benzyl)pyrrolidine-2-carboxamide (4) as the terminal VHL-engaging portion (Scheme 9). Finally, the reference compound 54, lacking the CRBN ligand, was accomplished according to the route reported in Scheme 10. Encorafenib derivative 54, bearing a 13-atom PEG-type linker, was obtained through the reaction of 2-(2-(2-methoxyethoxy)ethoxy)ethan-1-ol (52) with tert-butyl acrylate, followed by hydrolysis of the tert-butyl ester in acidic medium (TFA 10%), to furnish the acid 53. Finally, HATU-mediated formation of the amide bond allowed the coupling with derivative 16. Using this route, it was possible to obtain the desired 54 in 28% overall yield.  Finally, the reference compound 54, lacking the CRBN ligand, was accomplished according to the route reported in Scheme 10. Encorafenib derivative 54, bearing a 13-atom PEG-type linker, was obtained through the reaction of 2-(2-(2-methoxyethoxy)ethoxy)ethan-1-ol (52) with tert-butyl acrylate, followed by hydrolysis of the tert-butyl ester in acidic medium (TFA 10%), to furnish the acid 53. Finally, HATU-mediated formation of the amide bond allowed the coupling with derivative 16. Using this route, it was possible to obtain the desired 54 in 28% overall yield.

BRAF V600E Kinase Activity
The synthesized compounds 5-15 and the reference compound 54 were characterized to verify their ability to inhibit the activity of isolated BRAF V600E using a fluorescent assay. The obtained results are reported in Figure 4. All the synthesized compounds maintained the ability to inhibit the kinase activity of mutated BRAF with IC 50 values in the 40-88 nM range, showing no statistical difference in their activity. In the same assay, encorafenib (1) was the most active compound, showing an IC 50 of 21 ± 13 nM. The synthesized conjugates were 2-4-fold less active than the parent compound 1. The examination of the concentration-response curves (Figure 4a-c) shows that all the compounds behave quite similarly toward BRAF V600E inhibition. The length of the PEG-type linker used (compounds 5-10, Figure 4a) does not have an influence on the ability of compounds to inhibit BRAF V600E ; the same applies for compounds containing a triazole ring at different position of the linker (compounds 11-13, Figure 4b). Finally, the comparison between compounds obtained by joining encorafenib (1) with a simple PEG-type linker (compound 54) or the different E3-ligase binders, in particular pomalidomide (compound 11), lenalidomide (compound 14) or VHL-ligand-1 (compound 15), showed that compounds are endowed with similar potencies (Figure 4c). This observation underlines that the encorafenib substructure is still able to bind the target protein when the solvent-exposed moiety is modulated through the introduction of large molecular portions of different chemical composition.

Stability Assays
Glutarimide derivatives pomalidomide (2) and lenalidomide (3) are widely used in the generation of PROTACs, owing to their ability to bind to CRBN. As it is known, their intrinsic hydrolytic instability could limit their biological activity [38]. Therefore, the stability of selected compounds in cell medium (RPMI) and human serum was checked before performing antiproliferative assays in tumor cell lines. Compounds 8 and 10 were chosen as representative examples of the series of synthesized encorafenib conjugates. In our experiments, compounds 8 and 10 showed satisfactory stability with a half-life of 21 h and 19 h in RPMI, and of 24 h and 23 h in human serum, respectively ( Figure 5). We also checked the cell permeability of compound 8, used as a model compound, in Colo205 cells, demonstrating its ability to reach the cytosolic compartment, as expected (Table S1).

Stability Assays
Glutarimide derivatives pomalidomide (2) and lenalidomide (3) are widely used in the generation of PROTACs, owing to their ability to bind to CRBN. As it is known, their intrinsic hydrolytic instability could limit their biological activity [38]. Therefore, the stability of selected compounds in cell medium (RPMI) and human serum was checked before performing antiproliferative assays in tumor cell lines. Compounds 8 and 10 were chosen as representative examples of the series of synthesized encorafenib conjugates. In our experiments, compounds 8 and 10 showed satisfactory stability with a half-life of 21 h and 19 h in RPMI, and of 24 h and 23 h in human serum, respectively ( Figure 5). We also checked the cell permeability of compound 8, used as a model compound, in Colo205 cells, demonstrating its ability to reach the cytosolic compartment, as expected (Table S1).

Antiproliferative Activity
Based on the previous preliminary tests, we sought to investigate the ability of synthesized compounds 5-15 and reference 1 in inhibiting the proliferation of BRAF V600E mutant cancer cells. A first screening was conducted in A375 melanoma cell lines (Table 1, Figure S2). Encorafenib was employed for comparison. We found that all the synthesized compounds were able to inhibit the proliferation of A375 cells, even with a lower potency compared with encorafenib (1).

Antiproliferative Activity
Based on the previous preliminary tests, we sought to investigate the ability of synthesized compounds 5-15 and reference 1 in inhibiting the proliferation of BRAF V600E mutant cancer cells. A first screening was conducted in A375 melanoma cell lines (Table 1, Figure S2). Encorafenib was employed for comparison. We found that all the synthesized compounds were able to inhibit the proliferation of A375 cells, even with a lower potency compared with encorafenib (1). The ability of compounds 5, 10, 11, 15-selected based on their chemical diversity-to inhibit cell proliferation was then tested both in A375 and in Colo205 cancer cell lines. In this new set of experiments, we decided to compare the antiproliferative effects of our compounds with that of a recently published compound, named P5B ( Figure S3), which is highly efficient in inducing BRAF V600E degradation in A375 cells [24]. P5B was obtained by joining the BRAF inhibitor BI 882370 to pomalidomide through a five PEG unit spacer ( Figure S3). In compound P5B, the linker length was of 17 atoms, the same length of the spacer used to generate our compound 10. A375 and Colo205 cells were treated for 72 h with compounds 5, 10, 11, 15, and P5B, and the antiproliferative activity was measured by a luminescent ATP-based assay, which determines the number of viable cells based on their metabolic activity. The concentration-response curves obtained, and IC 50 values are reported in Figure 6. Among the synthesized compounds, 10 and 11 were the most potent in inhibiting the proliferation of BRAF mutant cell lines, with IC 50s in the nanomolar range; meanwhile, compound 15, bearing the VHL ligand-1 at one end, was active only at micromolar concentration. However, P5B was more efficient in inhibiting tumor cell viability in comparison with the other selected compounds. Finally, we investigated the capability of the most antiproliferative compounds in interfering with BRAF pathway, favoring target degradation. To this end, Western blot analysis was conducted 24 h after incubation with 10 and 11 in both A375 and Colo205 cell lines. Compounds 10 and 11 proved able to decrease phosphorylation of MEK and ERK in both A375 and Colo205 cell lines, similarly to the reference compound P5B (Figure  7a,b). This observation was consistent with their ability to inhibit BRAF V600E . Surprisingly, after 24 h treatment of either A375 or Colo205 cells with encorafenib-pomalidomide conjugates 10 and 11 at 100-500 nM, both the synthesized compounds were not able to induce BRAF V600E degradation. On the contrary, P5B efficiently induced BRAF V600E degradation in the same range of concentration in the tested tumor cell lines (Figure 7a,b). When 10 and 11 were tested at higher concentration (up to 10 µM), no degradation of the target protein was in any way observed ( Figure S4). Finally, we investigated the capability of the most antiproliferative compounds in interfering with BRAF pathway, favoring target degradation. To this end, Western blot analysis was conducted 24 h after incubation with 10 and 11 in both A375 and Colo205 cell lines. Compounds 10 and 11 proved able to decrease phosphorylation of MEK and ERK in both A375 and Colo205 cell lines, similarly to the reference compound P5B (Figure 7a,b). This observation was consistent with their ability to inhibit BRAF V600E . Surprisingly, after  24 h treatment of either A375 or Colo205 cells with encorafenib-pomalidomide conjugates 10 and 11 at 100-500 nM, both the synthesized compounds were not able to induce BRAF V600E degradation. On the contrary, P5B efficiently induced BRAF V600E degradation in the same range of concentration in the tested tumor cell lines (Figure 7a,b). When 10 and 11 were tested at higher concentration (up to 10 µM), no degradation of the target protein was in any way observed ( Figure S4). We investigated the possible reasons underlying the failure of our prototype compounds in inducing BRAF V600E degradation. We reasoned that the synthesized compounds: (i) inhibit isolated BRAF V600E kinase activity; (ii) penetrate the cell and inhibit the signaling of BRAF V600E ; (iii) are stable enough in the cell environment to bring about the formation of the ternary complex between the protein target and the E3-ligase. Taking into account the above observations and the structural similarity of compound 10 and P5B, a possible explanation for the lack of target protein degradation could be that the compound, once inside the cell, can bind to the kinase target but is not able to engage the E3-ligase. To investigate this possibility, we performed in-depth molecular modeling studies of compound 10 compared with P5B in BRAF V600E .

Computational Studies Molecular Docking
Docking studies were first performed to investigate the binding mode of warheads BI 882370 and encorafenib in BRAF V600E , as the kinase inhibitor moiety is the main discriminant between P5B and conjugate 10. Both BI 882370 and encorafenib are type I 1 /2 RAF kinase inhibitors, and stabilize a DFG in and αC-helix out conformation of BRAF [3]. While no experimental structure of encorafenib in BRAF is available, two X-ray structures of BI 882370 in BRAF can be found in the PDB (IDs: 5CSX, 6UUO). The structure of BI 882370bound BRAF in PDB ID 5CSX reports an αC-helix out, but also DFG out conformation, with the DFG out being the result of a crystallization artifact, as already discussed elsewhere [24]. In contrast, PDB ID 6UUO shows a DFG in and an αC-helix out conformation of BRAF, but with a low resolution (3.29 Å), and P-loop and activation loop (A-loop) disordered. As a consequence, PDB IDs 5CSX and 6UUO were discarded for modelling BRAF V600E in complex with either P5B or 10, while BRAF in complex with dabrafenib, another type of I 1 /2 BRAF inhibitor (PDB ID: 5CSW, resolution 2.66 Å) was selected (see Methods section). The resulting BRAF V600E model showed a DFG in loop, an αC-helix out and an unfolded A-loop, in accordance with the conformation stabilized by type I 1 /2 RAF kinase inhibitors BI 882370 and encorafenib [3]. After self-docking of dabrafenib in BRAF V600E (RMSD heavy = 0.563 Å), docking of BI 882370 and encorafenib was performed (Figure 8). Docking poses of BI 882370 and encorafenib trace the polar interactions of dabrafenib observed in 5CSW Xray structure, and involve hydrogen bonds between residues Asp594 and Phe595 (DFG segment), Lys483 and the sulfonylamide moiety of the inhibitors. Another key hydrogen bond involves the nitrogen of Cys532 backbone, and the pyrimidinyl substituent of both BI 882370 and encorafenib. Noticeably, two main differences can be spotted between BI 882370 and encorafenib at the docking pose level; (i) the 2-aminopyrimidin-4-yl moiety in encorafenib can establish a typical 1-3 hydrogen bond with the backbone of Cys532 in the hinge region, and (ii) the linker attachment point is oriented differently with respect to the overall binding mode.

Molecular Dynamics
Next, we planned to investigate the stability of P5B and 10, bearing BI 882730 and encorafenib as the kinase ligands, respectively. Three replicas of unbiased molecular dynamics simulations (500 ns each) in explicit solvent were set up (see Methods section) for two systems: (i) BRAF V600E in complex with P5B, and (ii) BRAF V600E in complex with 10, hereafter referred to as BRAF:P5B and BRAF:10, respectively. We monitored the behavior of the P5B and 10 conjugates during simulations, looking for differences at the binary complex level that might explain the lack of degradation activity shown by 10. Intramolecular hydrogen bonds established along the MD production by P5B and 10 were monitored; however, no relevant discrepancies were recorded. Furthermore, the solvent exposure of the P5B and 10 conjugates was evaluated during the simulations as the solvent-accessible surface area (SASA); however, no significant difference was found across replicas ( Figure S5).

Molecular Dynamics
Next, we planned to investigate the stability of P5B and 10, bearing BI 882730 and encorafenib as the kinase ligands, respectively. Three replicas of unbiased molecular dynamics simulations (500 ns each) in explicit solvent were set up (see Methods section) for two systems: (i) BRAF V600E in complex with P5B, and (ii) BRAF V600E in complex with 10, hereafter referred to as BRAF:P5B and BRAF:10, respectively. We monitored the behavior of the P5B and 10 conjugates during simulations, looking for differences at the binary complex level that might explain the lack of degradation activity shown by 10. Intramolecular hydrogen bonds established along the MD production by P5B and 10 were monitored; however, no relevant discrepancies were recorded. Furthermore, the solvent exposure of the P5B and 10 conjugates was evaluated during the simulations as the solventaccessible surface area (SASA); however, no significant difference was found across replicas ( Figure S5).
Through visual inspection of the MD trajectories (see Movies 1-6 in Supporting Info), a different dynamic behavior and interaction pattern of the linker and pomalidomide segment in P5B and 10 was observed. Notably, in P5B, the linker and pomalidomide moiety Through visual inspection of the MD trajectories (see Movies 1-6 in Supporting Info), a different dynamic behavior and interaction pattern of the linker and pomalidomide segment in P5B and 10 was observed. Notably, in P5B, the linker and pomalidomide moiety explored a larger surface of BRAF V600E , while the same segment in 10 was restricted to a narrower area of the binding cavity. Such differences in protein-ligand interactions were characterized by mapping the generic ligand-protein contacts (protein atoms distant 4 Å or less from ligand atoms) along MD replicas, and weighted according to their occupancy, i.e., persistence on the total MD frames (Figure 9). Black arrows in Figure 9 highlight a surface area of the carboxyterminal lobe of BRAF V600E which was accessed by P5B but not by 10 in MD replicas. This area is lined by residues Lys578, Leu613, Ser614, Leu618, Trp619 and Leu654 ( Figure S6). narrower area of the binding cavity. Such differences in protein-ligand interactions were characterized by mapping the generic ligand-protein contacts (protein atoms distant 4 Å or less from ligand atoms) along MD replicas, and weighted according to their occupancy, i.e., persistence on the total MD frames (Figure 9). Black arrows in Figure 9 highlight a surface area of the carboxyterminal lobe of BRAF V600E which was accessed by P5B but not by 10 in MD replicas. This area is lined by residues Lys578, Leu613, Ser614, Leu618, Trp619 and Leu654 ( Figure S6). Figure 9. Occupancy of ligand-protein contacts along MD productions, mapped on the surface of BRAF. Occupancy is defined as the normalized number of MD frames in which atoms of the ligand are at a distance equal or lower than 4 Å from protein atoms. Occupancy is shown as a range of colors spanning from blue (low occupancy) to orange (high occupancy) areas. Black arrows highlight areas of BRAF V600E which were accessed by P5B but not by 10. Protein is represented as surface, ligand is depicted as sticks and labelled. Furthermore, the hydrogen bond pattern of P5B and 10 along MD simulations was analyzed, searching for possible polar contacts that may affect the linker conformation (Table S2 and S3); overall, P5B showed a richer and more variable network of hydrogen bonds with respect to 10. In BRAF:P5B, the linker orientation was stabilized by a persistent hydrogen bond between the 1-oxo-piperidinyl moiety and Asn580 in all replicas, while in BRAF:10 the linker attachment point was constrained in its position by the firm hydrogen bond between the pyrimidinyl ring of 10 and Cys532.
In this section, we analyzed differences between P5B and 10 at the binary complex level that may explain their efficacy or inefficacy as BRAF V600E degraders. By observing MD simulations and analyses, we gathered no data supporting unfavorable interactions between 10 and BRAF V600E that could hinder the recruitment of the E3 ligase CRBN with respect to P5B. A relevant feature emerging from our MD replicas regards the differences in the protein area of BRAF V600E accessed by the linker and pomalidomide segments in P5B and 10 conjugates. Indeed, the linker and pomalidomide moiety in 10 are constrained near Figure 9. Occupancy of ligand-protein contacts along MD productions, mapped on the surface of BRAF. Occupancy is defined as the normalized number of MD frames in which atoms of the ligand are at a distance equal or lower than 4 Å from protein atoms. Occupancy is shown as a range of colors spanning from blue (low occupancy) to orange (high occupancy) areas. Black arrows highlight areas of BRAF V600E which were accessed by P5B but not by 10. Protein is represented as surface, ligand is depicted as sticks and labelled. Furthermore, the hydrogen bond pattern of P5B and 10 along MD simulations was analyzed, searching for possible polar contacts that may affect the linker conformation (Tables S2 and S3); overall, P5B showed a richer and more variable network of hydrogen bonds with respect to 10. In BRAF:P5B, the linker orientation was stabilized by a persistent hydrogen bond between the 1-oxo-piperidinyl moiety and Asn580 in all replicas, while in BRAF:10 the linker attachment point was constrained in its position by the firm hydrogen bond between the pyrimidinyl ring of 10 and Cys532.
In this section, we analyzed differences between P5B and 10 at the binary complex level that may explain their efficacy or inefficacy as BRAF V600E degraders. By observing MD simulations and analyses, we gathered no data supporting unfavorable interactions between 10 and BRAF V600E that could hinder the recruitment of the E3 ligase CRBN with respect to P5B. A relevant feature emerging from our MD replicas regards the differences in the protein area of BRAF V600E accessed by the linker and pomalidomide segments in P5B and 10 conjugates. Indeed, the linker and pomalidomide moiety in 10 are constrained near the binding site of BRAF V600E by the linker attachment point on encorafenib and by the canonical hydrogen bond between the amino-pyrimidinyl moiety and the backbone of Cys532. On the contrary, the linker attachment point in P5B led to more degrees of freedom and could extend to an area of BRAF which is inaccessible to 10. By comparing our results with other BRAF ligands reported in the literature for designing BRAF degraders, we noticed that effective BRAF degraders (BI 882730-, vemurafenib-or PLX-based conjugates) orient the linker attachment point towards one exit vector, while in weakly active or inactive BRAF degraders containing dabrafenib (or encorafenib, in this work) the exit vector for the linker is directed differently [24,25,39,40]. Considering this agreement with BRAF degraders reported by the literature, we speculate that the inactivity of 10, and of other ineffective BRAF degraders exploiting the same exit vector, might not be explained by focusing only on the formation of the binary or ternary complex, but by taking into account several aspects of protein target degradation.
A key factor to be addressed when rationalizing the activity of PROTACs regards the productivity of the ternary complex, i.e., the effective ubiquitination of the target protein.
In this context, it has been largely demonstrated that the formation of an energetically stable ternary complex does not correlate with efficient protein target degradation, while an efficient ubiquitination typically leads to successful protein target degradation [41]. Overall, we may argue that in the case of both P5B and 10, a ternary complex could be formed; however, while P5B might lead to the formation of a productive ternary complex, 10 might lead to an unproductive ternary complex. Furthermore, the productive protein-protein interactions (PPIs) may be formed at the level of the BRAF V600E surface explored by P5B in our MD simulations, which, however, could not be reached by 10. Further work would be required to test such a hypothesis, eventually clarifying the structure of productive BRAF V600E /inhibitor/E3 ligase ternary complexes by coupling different experimental and in silico methods. Indeed, a recent paper by Eron et al. reports that PPIs stabilized by PROTACs can still be very dynamic, and X-ray structures of ternary complexes can also not correspond to a biologically relevant conformation, as they only represent a 'crystallization snapshot' of a highly dynamic interaction [42]. Demonstrating the efficacy of combining in silico methods with experimental data for studying the dynamics of PPIs in PROTACs design, a freshly published work by Dixon et al. was able to eventually predict the structural basis of targeted protein degradation [43]. In this work, the authors showed that productive ternary complexes, modelled in the context of the full Cullin-RING ligase, are characterized by the proximity of lysine residues of the target protein to the E2 ligase.

Chemistry
All the reactions were monitored by thin-layer chromatography (TLC) on Merck 60 F254 (0.25 mm) plates, which were visualized by UV inspection (254 nm) and/or by spraying KMnO 4 (0.5 g in 100 mL 0.1 N NaOH). Na 2 SO 4 was used as drying agent for the organic phases. Flash chromatography (FC) purifications were performed using silica gel Merck with 60 mesh particles. Unless otherwise specified, all reagents were used as received without further purification. Dichloromethane was dried over P 2 O 5 and freshly distilled under nitrogen prior to use. DMF was stored over 3Å molecular sieves. Anhydrous THF was freshly distilled under nitrogen from Na/benzophenone ketyl. 1 H and 13 C-NMR spectra were registered on JEOL ECZR600 spectrometer, at 600 and 151 MHz. Coupling constants (J) are given in Hertz (Hz) and chemical shifts (δ) are given in ppm, calibrated to solvent signal as internal standard. The following abbreviations are used to describe multiplicities: s = singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet and br = broad signal. The following abbreviation is used to identify the exact proton: ArH = aromatic proton. ESI-mass spectra were recorded on a Waters Micromass Quattro Micro equipped with an ESI source. Semi-preparative HPLC purifications were carried out on a Varian Pro-Star 210 chromatograph equipped with a variable wavelength detector (Prostar 325). The chromatography was performed using a 5 µM particle size Hibar LiChrosper C18 endcapped prepacked column (250 × 25 mm), with a flow rate of 20 mL/min; UV detection was performed at 226 and 240 nm. Samples of the mixture were dissolved (ca 0.5%) in mobile phase. The purity of target compounds was checked by RP-HPLC. Analyses were performed on an HP1100 chromatograph system (Agilent Technologies, Palo Alto, CA, USA) equipped with a quaternary pump (G1311A), a membrane degasser (G1379A) and a diode-array detector (DAD) (G1315B) integrated in the HP1100 system. Data analyses were processed by HP ChemStation system (Agilent Technologies). The analytical column was a LiChrospher ® 100 C18-e (250 × 4.6 mm, 5 µm) (Merck KGaA, 64271 Darmstadt, Germany) eluted with acetonitrile/0.1% TFA in a ratio depending on the characteristics of the compound. All compounds were dissolved in the mobile phase at a concentration of about 0.1 mg/mL and injected through a 20 µL loop. HPLC retention times (t R ) were obtained at flow rates of 1.0 mL min −1 , and the column effluent was monitored at 226, 254 and 300 nm, referenced against 800 nm. The purity of the compounds was evaluated as a percentage ratio between the areas of the main peak and of possible impurities at the three wavelengths, and also using DAD purity analysis of the chromatographic peak. The purity of all the target compounds was found to be ≥95% (Table S4 and Figure S7). Compound P5B was a kind gift of Prof. Frank Sicheri (Center for Molecular, Cell and Systems Biology, Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada). Encorafenib (1; 0.350 g; 6.49 mmol) was dissolved in ethanol (10 mL), and KOH 6M aqueous solution (10 mL) was added. The mixture was stirred at 80 • C for 7 h. The reaction mixture was neutralized with HCl 6M to pH 7 and the organic solvent evaporated under reduced pressure. The obtained precipitate was filtered, and the filtrate was extracted with THF (3 × 15 mL). The organic phases and the collected precipitate were combined and purified by flash chromatography using DCM/MeOH 9/1+ 0.1% NH 3 as the eluent. Compound 16 was obtained in 85% yield as a white solid. 1

General procedure for the synthesis of compounds 26-29 and 41.
Phthalimide derivatives 22-25 and 40 were dissolved in EtOH and NH 2 NH 2 ·2H 2 O was added at rt. The reaction mixture was stirred overnight. The solvent was removed and chloroform was added to the residue and stirred for 15 min. The formed precipitate was filtered off. The filtrate was concentrated under reduced pressure and the crude product was purified using flash chromatography (DCM/MeOH 95/5 to 90/10 + 0.1% NH 3 ).

General procedure for the synthesis of compounds 32-35.
Amines 26-29 and 41(1 eq) were dissolved in DMF, DIPEA (2 eq) and 31 (1 eq) were added and the reaction mixture was stirred at 90 • C for 16 h. When the reaction was complete (TLC), the solvent was removed and the obtained residue treated with water (5 mL), extracted with DCM (3 × 20 mL) and the combined organic phases washed with brine (20 mL). After evaporation of the solvent, the protected intermediate was purified by flash chromatography (PE/EtOAc 1/1) to afford the tert-butyl protected acids. The purified protected intermediates were dissolved in a solution of TFA 10% in DCM and stirred overnight at rt. The solvent was evaporated and the precipitate washed several times with DCM. The product was purified by flash chromatography (DCM/MeOH 98/2 to 95/5) to afford the desired acids 32-35.

Tert-butyl 3-(2-(2-bromoethoxy)ethoxy)propanoate (37).
Compound 19 (1.10 g, 4.69 mmol, 1 eq) was dissolved in THF under nitrogen atmosphere. CBr 4 (3.08 g, 9.39 mmol, 2 eq) and PPh 3 (2.46 g, 9.39 mmol, 2 eq) were successively added and the reaction mixture was stirred at rt for 4 h. The formed white precipitate was filtered off, the solvent removed under reduced pressure and the residue purified by flash chromatography (PE/EtoAc 8/2) to afford 37 (1.38 g, 4.64 mmol, 100%)  Compound 37 (0.42 g, 1.41 mmol, 1 eq) was dissolved in DMF (2 mL). NaN 3 (0.46 g, 7.06 mmol, 5 eq) was added, followed by few drops of water in order to allow complete dissolution of the reagents. The reaction mixture was stirred at 70 • C for 16 h. When the reaction was complete (TLC), the mixture was saturated with NaCl and extracted with DCM (3 × 20 mL). The solvent was removed under reduced pressure to give 38 (0.38 g, 1.46 mmol, 85% yield) as a white oil. The obtained product was used in the following steps without further purification. 1  The appropriate amine 41 or 44 (1 eq) was dissolved in DMF (3 mL) then DIPEA (2 eq) and 31 (1 eq) were added. The reaction mixture was stirred for 16 h at 90 • C. When the reaction was complete (TLC), the solvent was removed and the residue dissolved in water and extracted with DCM (3 × 15 mL). The organic phase was washed with brine, dried and evaporated under reduced pressure. The product was isolated by flash chromatography (PE/EtOac 1/1).
HPLC analyses for stability assays were performed with a HP 1200 chromatograph system (Agilent Technologies, Palo Alto, CA, USA) equipped with a quaternary pump (model G1311A), a membrane degasser (G1322A) and a multiple wavelength UV detector (MWD, model G1365D) integrated in the HP1200 system. The samples were eluted on a Aquasil C18 column (200 × 4.6 mm, 5 µm), Thermo (Waltham, MA, USA). The injection volume was 20 µL (Rheodyne, Cotati, CA, USA). The mobile phase consisted of acetonitrile 0.1% TFA (solvent A) and 0.1% TFA (solvent B) at flow rate = 1.0 mL/min in gradient mode: 35% A until 5 min, from 35 to 45% A between 5 and 8 min, 45% A between 8 and 14 min, and from 45 to 35% A between 14 and 16 min.
The column effluent was monitored at 226 and 300 nm, referenced against a 800 nm wavelength. Analytical data were acquired by HP ChemStation system (Agilent Technologies). Quantitation of compounds was obtained by calibration curves (linearity determined in a concentration range of 1-100 µM, r 2 > 0.99).

BRAF V600E Inhibition Assay
Inhibition of B-RAF (V600E) Kinase activity by synthesized compounds was measured with a B-RAF (V600E) Kinase Assay Kit purchased from BPS Bioscience using Kinase-Glo MAX ® (Promega Corporation, Madison, Wisconsin, USA) as detection reagent. In a 96-well plate, the different inhibitor solutions in DMSO were mixed with Buffer, ATP (10 µM) and Raf substrate. The reaction was started by adding B-RAF (V600E) enzyme (0.8 ng/µL), and the mixture was incubated at 30 • C for 45 min. After a 15 min delay to equilibrate plate temperature, 50 µL of Kinase-Glo Max was added in each well, and the luminescence was measured according to supplier's instruction. The results, reported as IC 50 ± S.E.M., were calculated with GrapPad Prism 7 and derive from three different experiments run in duplicate.

Antiproliferative Activity
Cell viability was evaluated by means of CellTiter-Glo Luminescent Assay (Promega), which allows users to measure the ATP in the cell supernatant. The assay was performed according to manufacturer's instructions. In brief, 4.10 3 A375 or Colo205 were seeded into 96-well plates and allowed to adhere at the wall overnight. Cells were treated for 72 h with BRAF inhibitor compounds. Then, 90 µL of the cell culture medium was transferred in a white 96-well plate, to which 10 µL of CellTiter-Glo was added before measurement of luminescence. Luminescence was measured by the SPARK M10 (Tecan, Männedorf, Switzerland) plate reader.

Protein Modelling
BRAF in complex with the second generation BRAF inhibitor dabrafenib (PDBID: 5CSW) was chosen as the template structure for computational studies of P5B and 10. As in PDBID: 5CSW, BRAF contains 15 mutations (in the C-lobe, and distant from the active site) and lacks an ordered A-loop, WT-BRAF sequence (Uniprot ID: P15056), manually modified with the V600E point mutation, was modelled on 5CSW structure using the SWISSMODEL webserver. The resulting BRAF V600E model (hereon, BRAF V600E ) shows DFG in , αC-helix out and an activated unfolded A-loop. The protein was protonated at pH = 7.4 using the H++ webserver [44].

Docking Studies
Ligands were sketched with Moldraw (Molecular Discovery Ltd., Borehamwood, UK) and converted into mol2 format using Open Babel [45]. Docking studies were performed using the GOLD software version 2021.1 (CCDC, Cambridge, UK) [46]. To verify the software's capability of reproducing the co-crystallographic inhibitor binding pose, we extracted dabrafenib from PDBID:5CSW and docked it in BRAF V600E . Centroid of the docking cavity was defined as atoms at 6 Å from the dabrafenib X-ray pose; two relevant water molecules were retained in the binding site and no constraint was applied. The GOLD standard parameters were used, and the ligand was subjected to 50 genetic algorithm runs. Finally, poses were scored with the CHEMPLP function and ranked accordingly. As a good agreement between dabrafenib crystallographic pose in PDBID: 5CSW and dabrafenib docking pose in BRAF V600E (RMSD heavy = 0.563 Å) was observed, BI 882730 and encorafenib warheads were docked using the same parameters and protocol. Top-ranked docking poses of BI 882730 and encorafenib in BRAF V600E were exported for subsequent MD simulations. Importantly, the linker and pomalidomide moieties were not included in the previous docking studies to avoid artifact interactions that could bias the subsequent MD simulation. The linker and pomalidomide segment of P5B was sketched with Moldraw, minimized and converted to mol2 with OpenBabel. It was then attached to the docked warheads, thereby rebuilding P5B and 10 in the binding site and obtaining BRAF:P5B and BRAF:10 systems.

MD Setup, Simulations and Analysis
Before the MD production, P5B and 10 conjugates were parameterized using the DFTbased protocol implemented in BiKi software suite (http://www.bikitech.com/ accessed on 15 July 2022). BRAF:P5B and BRAF:10 systems were embedded in an octahedral box and solvated in water, with addition of 0.15 M NaCl, using AmberTools22, the Amber14SB forcefield in BiKi, and the GROMACS 2022.1 engine for MD simulations. The LINCS algorithm was employed to fix the bond length between hydrogens and heavy atoms at the equilibrium distance. Periodic boundary conditions (PBC) were set, and long range electrostatic effects were adjusted with the Particle-Mesh Ewald (PME) method; a cut-off value of 12 Å was fixed for both electrostatic and van der Waals interactions. Three replicates were set up for each system, as follows. The BRAF:P5B and BRAF:10 complexes were minimized in two steps, using the steepest descendant and conjugate gradient algorithms; subsequently, six thermalization steps in the isothermal-isochoric ensemble (NVT) were performed for heating the system from 0 to 300 K, with a temperature ramp of 50 K every 0.1 ns, and coordinate constraints for protein backbone and ligands during heating from 0 to 200 K. Two final equilibration steps in the isothermal-isobaric canonical (NPT) ensemble were carried out before MD production, the first imposing position restraints to the protein backbone and ligands, and the second with no restraints. Finally, the unbiased MD production was carried out for 500 ns (timestep = 2 fs). Three independent replicas were obtained for BRAF:P5B and three for BRAF:10 (total simulated time = 3 µs). Analyses on trajectories were carried out using GROMACS built-in functions, pictures and movies were generated with PyMOL, and data were plotted using the Matplotlib library.

Conclusions
In this work, we synthesized a series of potential encorafenib-based PROTACs targeting the oncogenic BRAF V600E variant using diverse linkers and E3-ligase moieties. Our heterobifunctional compounds showed inhibition of BRAF V600E mutant, even with a two-tofour-fold decrease in potency with respect to encorafenib, with no differences depending on the nature of the linker or of the E3 ligase ligand. Furthermore, we assessed the antiproliferative activity in A375 and Colo205 cell lines, comparing our conjugates with encorafenib and with P5B, an effective BRAF V600E degrader published by Posternak et al. [24]. Overall, our compounds showed a higher IC 50 with respect to encorafenib; however, compounds 10 and 11 showed an antiproliferative activity comparable to P5B. To assess the protein target degradation efficiency of our best antiproliferative compounds 10 and 11 with respect to P5B, a Western blot analysis was carried out after a 24-h incubation in A375 and Colo205 cell lines. As expected, 10, 11 and P5B decreased the phosphorylation of MEK and ERK, as a consequence of BRAF V600E inhibition. However, in stark contrast with the results shown by the effective P5B BRAF V600E degrader, our compounds 10 and 11 were not able to induce target protein degradation. To generate hypotheses that might explain such discrepant behavior, we ran molecular docking and molecular dynamics simulations, comparing P5B and 10 (bearing the same linker and E3 ligase ligand as P5B). Our in silico results suggest that the linker attachment point on the kinase inhibitor warhead affects the orientation and degrees of freedom of the linker and pomalidomide moieties. As no significant differences in solvent exposure of P5B and 10 were recorded, we hypothesize that a ternary complex can be stabilized by both PROTACs. However, as the BRAF V600E surface areas accessed by the P5B and 10 during MD simulations are different, we hypothesize that the protein-protein interface might also be different, and productive in the case of P5B, while unproductive in the case of 10. Our hypothesis is strengthened by the literature data reporting inactive dabrafenib-based BRAF degraders that present an analogous linker attachment point [24]. Further work would be required to clarify the structure of productive BRAF V600E /E3 ligase interactions, for better rationalizing the inactivity of encorafeniband dabrafenib-based PROTACs and for enhancing the design of novel effective BRAF degraders. In a broader context, the problem of predicting protein degradation efficiency is currently being addressed by a growing body of research, and recent works point out the importance of coupling in silico and experimental methodologies for modelling such a complex and multifactorial process.