Crystal structure of BCL-2 bound to venetoclax

Crystal structures of BCL-2 with ABT-263 and various analogues of venetoclax have been deposited in the PDB and described in the literature (Fig. 1a, b)10. One of those analogues is 4-[4-((4′-chloro-3-[2-(dimethylamino)ethoxy]biphenyl-2-yl)methyl)piperazin-1-yl]−2-(1H-indol-5-yloxy)-N-((3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl)sulfonyl)benzamide, hereafter referred to as compound 1. We obtained crystals of BCL-2 in complex with venetoclax that diffracted to high resolution (1.62 Å) in the space group P2 1 2 1 2 1 with one molecule in the asymmetric unit (Fig. 1a, b, Table 1). The electron density for the drug was well defined (Supplementary Fig. 1a, b) and the binding pose was in general agreement with the published structures of ABT-263 and compound 1, with the 4-chlorophenyl (CP) bound in the BCL-2 P2 pocket, the piperazine bridging the P2 and P4 pockets over residue F104 and the azaindole substitution bound in the BCL-2 P4 pocket. It was possible to model two distinct conformations with the 4–4-dimethylcyclohex-1-ene (4DM) ring flipping at the 4 and 5 positions above the BCL-2 P2 pocket and the benzamide (BA) acyl oxygen adopting two conformations above the BCL-2 P4 pocket (Fig. 1b). An interesting difference was the positioning of the venetoclax 4DM moiety, which was further from the α4 helix and more central over the P2 pocket than the equivalent rings from ABT-263 and 4MAN compounds (Fig. 1c, d, Supplementary Fig. 2, Supplementary Table 1). This small deviation in the positioning of the 4DM was unexpected as the equivalent six membered ring systems from ABT-263 and compound 1 are similar, differing only by the positioning of the gem-dimethyl group in ABT-263 (5 position) and the lack of a methyl and a phenyl ring in compound 1 (Fig. 1a).

Fig. 1 Venetoclax binding to BCL-2. a Chemical structures of venetoclax (Ven) and related compounds ABT-263 and compound 1. Key features are marked, including the benzamide (BA), the 4-chlorophenyl (CP), the partially saturated 4–4-dimethylcyclohex-1-ene (4DM), the 5–5-dimethylcyclohex-1-ene (5DM), the saturated 4′-chlorobiphenyl (BP), the azaindole (AI) and phenyl sulfonyl (PS). b Crystal structures showing binding of the two venetoclax conformers (Ven A and Ven B, orange), and comparison to ABT-263 (green, PDB id 4LVT) and compound 1 (cyan, PDB id 4MAN) binding to the BCL-2 groove. BCL-2 is shown with surface representation (white) and P2 and P4 pockets indicated along with arrows depicting changes between venetoclax alternate conformations for BA and 4DM moieties. c, d overlays of venetoclax with ABT-263 (c) and compound 1 (d) with arrows indicating deviations compared to the venetoclax 4DM above the P2 pocket Full size image

Table 1 Crystal structure data collection and refinement statistics Full size table

Structures of BCL-2 mutants bound to venetoclax

To understand how these BCL-2 mutations compromise drug binding we solved crystal structures of both complexes (Table 1 and Fig. 2). The G101V mutation resides on the BCL-2 α2 helix packing against the α5 helix and is within the BCL-2 BH3 motif. The glycine is a conserved, defining feature of the motif. Adjacent residues A100 and D103 define boundaries of the P4 pocket so the mutation was expected to alter drug binding by changes to this region (Fig. 2a). The BCL-2 G101V:venetoclax complex crystallised in P2 1 spacegroup with two molecules in the asymmetric unit diffracting to 2.2 Å, with well-defined electron density for both copies of the drug (Supplementary Fig. 1). The overall structure of BCL-2 G101V was similar to WT with no significant deviations in α2–α5 core helices or α6-α8 (Fig. 2b, c). There was a minor change in orientation of the α1 helix resulting in a ~1 Å deviation at either end of the helix, but this was far from the drug binding site. The binding pose of venetoclax is conserved between WT and the G101V mutant (Fig. 2b). The P2 pocket volume was maintained at 478 Å3 (480 Å3 for WT) as was the volume of the P4 pocket at 379 Å3 (380 Å3 for WT). Interestingly, the G101V mutation did not alter the positioning of the α2 helix relative to α5 or impact the residues defining the P4 pocket. Instead the additional bulk of the valine sidechain was accommodated by a deviation of the sidechains of Y18 on α1 and E152 on α5 (Fig. 2c). In the BCL-2 G101V:venetoclax structure E152 had a 60º rotamer change relative to WT (mm-40 for WT to tp10 for G101V), placing the sidechain Cγ in van der Waal’s contact with the chlorine atom of the venetoclax chlorophenyl moiety. This conformational change in E152 in the BCL-2 G101V structure causes a small repositioning of the venetoclax 4DM and chlorophenyl moieties in the P2 pocket, moving on average 0.25 Å closer to L137 in the α4 helix (Fig. 2b, Supplementary Fig. 2, Supplementary Table 1), i.e. more similar to the BCL-2 WT complexes with ABT-263 and compound 1. Additionally, we obtained a structure of BCL-2 G101A bound to venetoclax (Table 1), representing a milder introduction of bulk at the G101 position than the valine substitution. The BCL-2 G101A:venetoclax and BCL-2 WT:venetoclax crystals were isomorphous and there were no significant deviations in venetoclax positioning or E152 (Supplementary Fig. 2, 3, Supplementary Table 1). Despite the closer proximity of the azaindole moiety to the mutation site, its orientation in the P4 pocket was conserved between BCL-2 WT and G101V structures. Therefore, the G101V mutation appears to modulate venetoclax affinity more through its interactions with the P2 pocket than the P4 pocket.

Fig. 2 BCL-2 mutations and venetoclax binding. a location of G101V and F104L mutants relative to the BCL-2 groove, α1–8 helices and P2 (orange) and P4 (blue) pockets. Pockets volumes were defined by venetoclax (Ven) chlorophenyl (CP) for P2 pocket and azaindole ring for P4 pocket, see methods for further details. b, c Overlay of BCL-2 WT and BCL-2 G101V bound to venetoclax. d Overlay of BCL-2 WT and BCL-2 F104L bound to venetoclax. e Surface representation of the BCL-2 WT:venetoclax P2 pocket. f Surface representation of the BCL-2 F104L:venetoclax P2 pocket. In a–f Venetoclax is shown in orange for BCL-2 WT and yellow in mutants, BCL-2 WT in green, BCL-2 G101V in pale blue, F104L in purple and surfaces in white. Arrows indicate key residues, dashed arrows indicate residue movements or movement of venetoclax Full size image

The crystals of venetoclax complexed with BCL-2 F104L and BCL-2 WT are isomorphous (Table 1). Well-defined electron density for the drug in the mutant complex structure (Supplementary Fig. 1) suggests two conformations for the 4DM and acyl group of the BA moiety as in WT (Fig. 2d). The side chain of F104 separates the P2 and P4 pockets of BCL-2. The P4 pocket volume was maintained between WT and F104L structures (P4 pocket volume 380 Å3 for both WT and F104L). In contrast the volume of the P2 pocket increased with the F104L mutation as the leucine sidechain occupies a smaller volume than phenylalanine (Fig. 2d–f). In this structure two conformers for F112 on α3 are evident, one like WT and a second occupying some of the volume vacated by the F104L mutation. (Fig. 2c, e and f). The new F112 conformation extends into the P2 pocket, packing against L104. This inserted F112 conformation compensates for the loss of P2 pocket volume in the BCL-2 F104L mutant and is comparable to the BCL-2 WT P2 pocket volume—P2 pocket volumes of 480 Å3 (BCL-2 WT), 475 Å3 (F104L inserted conformation) and 596 Å3 (F104L conserved conformation). The occupancy of F112 refined to 0.48 for the conserved conformation and 0.52 for inserted conformation, indicating that the compensation in P2 pocket volume only occurs 50% of the time and the vacated space is unfavoured. The consequence of the F104L mutation is to alter the packing environment of the chlorophenyl moiety of the drug.

Binding of BH3 peptides and venetoclax to BCL-2 mutants

SPR experiments were performed using a BIMBH3 or BAXBH3 immobilised sensor surface with BCL-2 mutants as the analyte and determining venetoclax affinity by competition experiments17,25, (Fig. 3, Table 2 and Supplementary Fig. 4, 5). We have previously reported BIMBH3 and BAXBH3 affinities for WT, G101V and F104L17, and these were comparable to F104C, with less than 10-fold change relative to WT (Table 2). In contrast, the affinities for venetoclax differed by 25 to ~1500-fold with K I values 0.018, 3.2, 0.46 and 25 nM for WT, G101V, F104L and F104C, respectively (Table 2). This indicates that the BCL-2 mutants maintain tight binding to BH3 domains, allowing their overexpression to prevent apoptosis, whilst selectively reducing the affinity for the drug and thus providing resistance to therapy.

Fig. 3 Steady-state competition SPR with BCL-2 mutants, BIMBH3 and venetoclax or S55746. SPR chip surfaces were immobilised with BIMBH3 peptide and analytes with the indicated BCL-2 mutants pre-combined with either a–e venetoclax (Ven) or f–h S55746 at the indicated concentrations (circles 0 nM, squares 20 nM, triangles point up 40 nM, triangles point down 60 nM). Response at steady-state is plotted against BCL-2 concentration, with fits from a steady-state competition SPR model shown (see methods and Supplementary Figs. 5, 6 for further details), that were used to derive the indicated mean K I values for venetoclax or S55746 binding to BCL-2. Data are representative of at least n = 2 independent experiments Full size image

Table 2 SPR affinity values for BH3 peptides and BCL-2 selective compounds binding to BCL-2 mutants Full size table

The role of E152 in venetoclax affinity

E152 moved into the base of the P2 pocket in the BCL-2 G101V:venetoclax structure (Fig. 2b, c). To test the role of E152 in reducing affinity we generated a BCL-2 G101V/E152A double mutant. Alanine does not have a Cγ or Cδ to impact the base of the P2 pocket and would allow the chlorophenyl to insert unimpeded into the P2 pocket in the G101V mutant. We repeated SPR experiments with the BCL-2 G101V/E152A double mutant and a BCL-2 E152A single mutant (Table 2 and Fig. 3c, d). The E152A single mutant had comparable binding to WT and when combined with G101V as a double mutation restored high affinity venetoclax binding, with WT binding at 18 pM, BCL-2 E152A at 27 pM and BCL-2 G101V E152A at 2 pM (Table 2 and Fig. 3c, d). The BCL-2 G101V/E152A affinity was 10-fold higher than WT, however competition SPR experiments become less accurate as the ligand K I becomes significantly tighter or weaker than the K D for the competing BimBH3 peptide; as such it is unclear whether this increase in affinity is significant. Furthermore, the E152 conformation in a BCL-2 G101A:venetoclax structure matched the WT conformation, not the G101V. The G101A mutant bound to venetoclax with a K I of 110 pM comparable to WT but distinct from G101V (Table 2 and Supplementary Fig. 4). This indicates that E152A mutation rescues high affinity for venetoclax when combined with G101V and confirms the importance of the E152 rotamer change observed in the G101V mutant. Note also that the affinity of BCL-2 G101V/E152A and BCL-2 E152A for BIMBH3 and BAXBH3 was largely unaltered compared to WT (Table 2 and Supplementary Fig. 3).

BCL-2 G101V binding to S55746

S55746 is another BCL-2 selective antagonist that has progressed to the clinic. The recently disclosed crystal structure of BCL-2 WT bound to S55746 revealed binding to the P1, P2 and P3 pockets12, in contrast to venetoclax that binds principally to the P2 and P4 pockets (Fig. 4). We tested the binding of S55746 to both BCL-2 WT and G101V by competition SPR (Table 2, Fig. 3 and Supplementary Fig. 6). S55746 bound to BCL-2 WT with a K I of 0.36 nM and G101V with a 100-fold lower K I of 36 nM, in both cases > 10-fold weaker than venetoclax, K I of 0.018 nM and 3.2 nM for WT and G101V, respectively. This was confirmed in cellular assays using the B-lineage cell line KMS-12-PE (Fig. 4a). BCL-2 WT and G101V were overexpressed in the KMS-12-PE cells and S55746 LC50 concentrations were determined as 0.32 ± 0.15 μM for WT increasing eight-fold to 2.7 ± 0.43 μM with the G101V mutation.

Fig. 4 BCL-2 G101V binding to S55746. a Expression of BCL-2 G101V mutant reduces S55746 sensitivity in the KMS-12-PE B-lineage cell line. WT and G101V mutant BCL-2 proteins were expressed at similar levels in KMS-12-PE cells as demonstrated by the FACS profiles. In vitro sensitivity to S55746 (0–10 μM) was measured after 24 h by a CellTiter-Glo assay. Data are representative of n = 3 independent experiments with mean LC50 values ± SD (1 Standard Deviation) for empty vehicle (EV, black), WT (blue) and G101V (red) indicated. b Overlay of the published BCL-2:S55746 structure (green and magenta, PDB id 6GL8) with the BCL-2 G101V:S55746 structure (light blue) showing consistency in S55746 orientation. c, d Overlay of BCL-2 G101V structures bound to S55746 (light blue) and venetoclax (Ven, orange). c BCL-2 protein surface displayed from the BCL-2 G101V:S55746 structure showing P1–4 pockets. d Cartoon representation with key residues indicated in stick representation, inserted conformation of F112 in the BCL-2 G101V—S55746 and WT conformation from BCL-2 G101V:venetoclax structure are indicated Full size image