Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr 13;9(28):20075-20088.
doi: 10.18632/oncotarget.24744.

S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth

Patrick Casara #  1 , James Davidson #  2 , Audrey Claperon #  3 , Gaëtane Le Toumelin-Braizat  3 , Meike Vogler  4 , Alain Bruno  5 , Maïa Chanrion  3 , Gaëlle Lysiak-Auvity  3 , Thierry Le Diguarher  1 , Jérôme-Benoît Starck  1 , Ijen Chen  2 , Neil Whitehead  2 , Christopher Graham  2 , Natalia Matassova  2 , Pawel Dokurno  2 , Christopher Pedder  2 , Youzhen Wang  6 , Shumei Qiu  6 , Anne-Marie Girard  3 , Emilie Schneider  3 , Fabienne Gravé  3 , Aurélie Studeny  3 , Ghislaine Guasconi  3 , Francesca Rocchetti  3 , Sophie Maïga  7 , Jean-Michel Henlin  1 , Frédéric Colland  3 , Laurence Kraus-Berthier  5 , Steven Le Gouill  7 , Martin J S Dyer  8 , Roderick Hubbard  2 , Mike Wood  2 , Martine Amiot  7 , Gerald M Cohen  9 , John A Hickman  3 , Erick Morris  6 , James Murray  2 , Olivier Geneste  3
Affiliations

S55746 is a novel orally active BCL-2 selective and potent inhibitor that impairs hematological tumor growth

Patrick Casara et al. Oncotarget. .

Abstract

Escape from apoptosis is one of the major hallmarks of cancer cells. The B-cell Lymphoma 2 (BCL-2) gene family encodes pro-apoptotic and anti-apoptotic proteins that are key regulators of the apoptotic process. Overexpression of the pro-survival member BCL-2 is a well-established mechanism contributing to oncogenesis and chemoresistance in several cancers, including lymphoma and leukemia. Thus, BCL-2 has become an attractive target for therapeutic strategy in cancer, as demonstrated by the recent approval of ABT-199 (Venclexta™) in relapsed or refractory Chronic Lymphocytic Leukemia with 17p deletion. Here, we describe a novel orally bioavailable BCL-2 selective and potent inhibitor called S55746 (also known as BCL201). S55746 occupies the hydrophobic groove of BCL-2. Its selectivity profile demonstrates no significant binding to MCL-1, BFL-1 (BCL2A1/A1) and poor affinity for BCL-XL. Accordingly, S55746 has no cytotoxic activity on BCL-XL-dependent cells, such as platelets. In a panel of hematological cell lines, S55746 induces hallmarks of apoptosis including externalization of phosphatidylserine, caspase-3 activation and PARP cleavage. Ex vivo, S55746 induces apoptosis in the low nanomolar range in primary Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma patient samples. Finally, S55746 administered by oral route daily in mice demonstrated robust anti-tumor efficacy in two hematological xenograft models with no weight lost and no change in behavior. Taken together, these data demonstrate that S55746 is a novel, well-tolerated BH3-mimetic targeting selectively and potently the BCL-2 protein.

Keywords: BCL-2; BH3-mimetics; apoptosis; hematological malignancies; inhibitor.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST P Casara and JA Hickman are former employees of Institut de Recherches Servier. M Chanrion, A Claperon, F Colland, O Geneste, AM Girard, F Gravé, G Guasconi, JM Henlin, G Le Toumelin-Braizat, G Lysiak-Auvity, F Rocchetti, E Schneider, JB Starck and A Studeny are full-time employees of Institut de Recherches Servier. T Le Diguarher is a full-time employee of Technology Servier. A Bruno and L Kraus-Berthier are full-time employees of Institut de Recherches Internationales Servier. E Morris, S Qiu and Y Wang are full-time employees of Novartis Institutes for BioMedical Research; E Morris and Y Wang are stock owner of Novartis. S LeGouill has served on advisory board for Servier. Amiot and Cohen's laboratories have received research funds from Servier. I Chen, J Davidson, P Dokurno, C Graham, N Matassova, J Murray, C Pedder, N Whitehead, M Wood are full-time employees of Vernalis Ltd. R Hubbard is a part-time employee of Vernalis Ltd.

Figures

Figure 1
Figure 1. Chemical structure and binding mode of S55746
(A) Chemical structure of S55746. (B) Superposition of S55746 (green sticks) and ABT-199 analogue from PDB 4MAN (orange lines) X-ray crystal structures. Ala149 is highlighted (space-filling spheres) and the hydrogen bond from S55746 phenol to backbone carbonyl of Ala149 is shown (cyan dashed line). The key S1 and S2 binding pockets are shown as translucent yellow surfaces.By comparison with the ABT-199 analogue it can be seen that S55746 forms a compact structure that only accesses pockets S1/S2/S3 and not S4/S5. Image was prepared using Pymol.
Figure 2
Figure 2. S55746 is a potent and selective inhibitor of BCL-2
(A) Expression of BCL-2, BCL-XL, MCL-1, BAX and BAK was assessed by immunoblotting in H146 and RS4;11 cell lines. GAPDH was used as a loading control (left panel). Viability of H146 and RS4;11 cell lines after incubation with increasing concentration of either ABT-263 (open circles), S55746 (open triangles) or ABT-199 (open squares) for 72 h. IC50 indicates concentration at which 50% of inhibition is reached. Mean and individual data of 3 independent experiments are shown (right panel). (B) Co-immunoprecipitation assay of RS4;11 cells treated with different concentrations of S55746 or ABT-263 for 2 h. ‘−’ indicates that cells were treated with DMSO only. Cells were lysed after treatment and immunoprecipitated (IP) with anti-BCL-2 antibody. The cell lysates (input) and immunoprecipitates were analyzed by immunoblotting with BAX or BCL-2 antibodies as indicated. (C) Co-immunoprecipitation assay of HeLa cells transfected with plasmids expressing Flag-tagged BCL-2 or Flag-tagged BCL-XL as indicated. Cells were treated with different concentrations of S55746 or ABT-263 for 2 h. ‘−’ indicates that cells were treated with DMSO only. Cells were lysed after treatment and immunoprecipitated (IP) with anti-Flag antibody. The cell lysates (input) and immunoprecipitates were analyzed by immunoblotting with BAX or Flag antibodies as indicated. Representative blots of three independent experiments are shown.
Figure 3
Figure 3. S55746 induces apoptosis in a BAX-dependent manner
(A) Apoptosis induction in RS4;11 cells treated with S55746 at the indicated concentration for 2 h. Cells were analyzed by flow cytometry for PI and annexin V-FITC labeling. Mean and individual points from two biological replicates are shown. ‘−’ indicates that cells were treated with DMSO only. (B) Expression of PARP and BAX assessed by immunoblotting in RS4;11 cells transduced with shRNA CT (control) or shRNA BAX upon treatment with increasing concentrations of S55746. Actin was used as a loading control. ‘−’ indicates that cells were treated with DMSO only. (C) Viability of RS4;11 cells transduced either with shRNA CT or shRNA BAX treated with increasing concentration of S55746 for 72 h. IC50 indicates concentration at which 50% of inhibition is reached. Mean and individual points from n = 3 biological replicates are shown. (D) FL5.12 cells either expressing BCL-2 (filled circles, thick line) or BCL-XL (open triangles, dashed line) were treated with increasing concentration of either ABT-737 (left panel) or S55746 (right panel) for 24 h upon IL-3 withdrawal. Cells were then analyzed by flow cytometry for PI and annexin V-FITC labeling. Mean ± s.d. of 4 biological replicates are shown. (E) Isolated platelets from 4 healthy donors were treated with increasing concentrations of ABT-737 (squares, thick line) or S55746 (circles, dashed line) for 3 h. CD41-PE positive cells were then gated and analyzed for annexin V-FITC labeling. PS+: Phosphatidylserine positive cells. EC50: half maximal effective concentration.
Figure 4
Figure 4. S55746 induces cell death in a panel of non-Hodgkin lymphoma cell lines
Viability of a panel of Diffuse Large B-Cell (DLBCL, A), Mantle Cell Lymphoma (MCL, B) and Burkitt lymphoma (BL, C) cell lines treated with increasing concentration of S55746 and ABT-199 for 72 h was determined using Cell Titer Glo. IC50 indicates concentration at which 50% of inhibition is reached. Mean and individual points from n = 3 biological replicates are shown. Origins of the cell lines are described in supplementary materials and methods.
Figure 5
Figure 5. S55746 induces cell death in primary CLL and MCL cells
(A) Primary Chronic Lymphocytic Leukemia (CLL) cells freshly isolated from 7 patients were treated for 4 h with increasing concentrations of S55746. Cells were then analyzed by flow cytometry for PI and annexin V-FITC labeling. Individual curves of cells treated with S55746 are shown (top panel). CLL cells isolated from 2 patients were exposed to S55746 (3 nM) for 4 h. Total and cleaved caspase-3, caspase-9 and PARP were detected by immunoblotting using specific antibodies. Tubulin was used as a loading control (lower panel). (B) Primary Mantle Cell Lymphoma (MCL) cells freshly isolated from 8 patients were cultured with increasing concentrations of S55746 for 24 h. Cells were then analyzed by flow cytometry for annexin V-FITC and CD19-APC positive labeling. Individual curves of cells treated with S55746 are demonstrated. PS+: Phosphatidylserine positive cells.
Figure 6
Figure 6. S55746 inhibits xenograft growth in RS4;11 and Toledo models
(A and B) Female SCID mice were inoculated with 1 × 107 RS4;11 cells and randomized 26 days after grafting. After randomization, mice were either not treated, or dosed once orally with vehicle, S55746 (25 mg/kg and 100 mg/kg) and ABT-263 (100 mg/kg). (A) Tumor samples were collected 16 h post dosing and analyzed for caspase-3 activity. Mean and individual data of caspase-3 activity fold increase over untreated group of 3 animals per treatment group are shown. (B) Platelet cells were counted 16 h post dosing. Mean and individual data of platelets counts of 3 animals per treatment group are shown. (C) Female SCID mice were inoculated with 1 × 106 RS4;11 cells and randomized 27 days after grafting. After randomization, mice were either not treated (black circles) or treated orally with S55746 every day for 7 consecutive days at 25 mg/kg (blue reverse triangles), 50 mg/kg (green squares) or 100 mg/kg (purple triangles). Mean tumor volumes with error bars depicting s.e.m. of 8 animals per treatment group are shown. One way ANOVA on day 17, followed by Dunnett post hoc comparison was performed and always compared to vehicle, (* p < 0.05; ***p < 0.001). (D) Female SCID mice were inoculated with 3 × 106 Toledo cells and randomized 24 days after grafting. After randomization, mice were either not treated (black circles) or treated orally five times a week with ABT-199 at 200 mg/kg (inverted red triangles) and S55746 at 200 mg/kg (blue squares) or 300 mg/kg (green triangles) for 3 weeks. Mean tumor volumes with error bars depicting s.e.m. of 6–7 animals per treatment group are shown. One way ANOVA on day 21, followed by Dunnett post hoc comparison was performed and always compared to vehicle, (* p < 0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Green DR, Llambi F. Cell Death Signaling. Cold Spring Harb Perspect Biol. 2015:7. - PMC - PubMed
    1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74. - PubMed
    1. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science. 1984;226:1097–9. - PubMed
    1. Tsujimoto Y, Yunis J, Onorato-Showe L, Erikson J, Nowell PC, Croce CM. Molecular cloning of the chromosomal breakpoint of B-cell lymphomas and leukemias with the t(11;14) chromosome translocation. Science. 1984;224:1403–6. - PubMed
    1. Czabotar PE, Lessene G, Strasser A, Adams JM. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol. 2014;15:49–63. - PubMed