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. 2019 Dec 9;176(23):4491–4509. doi: 10.1111/bph.14809

A non‐covalent inhibitor XMU‐MP‐3 overrides ibrutinib‐resistant BtkC481S mutation in B‐cell malignancies

Fu Gui 1, Jie Jiang 1, Zhixiang He 1, Li Li 1, Yunzhan Li 1, Zhou Deng 1, Yue Lu 1, Xinrui Wu 1, Guyue Chen 1, Jingyi Su 1, Siyang Song 1, Yue‐Ming Zhang 2, Cai‐Hong Yun 2, Xin Huang 3, Ellen Weisberg 4, Jianming Zhang 5,6,✉, Xianming Deng 1,✉
PMCID: PMC6932946  PMID: 31364164

Abstract

Background and Purpose

Bruton's tyrosine kinase (BTK) plays a key role in B‐cell receptor signalling by regulating cell proliferation and survival in various B‐cell malignancies. Covalent low‐MW BTK kinase inhibitors have shown impressive clinical efficacy in B‐cell malignancies. However, the mutant Btk C481S poses a major challenge in the management of B‐cell malignancies by disrupting the formation of the covalent bond between BTK and irreversible inhibitors, such as ibrutinib. The present studies were designed to develop novel BTK inhibitors targeting ibrutinib‐resistant Btk C481S mutation.

Experimental Approach

BTK‐Ba/F3, BTK(C481S)‐Ba/F3 cells, and human malignant B‐cells JeKo‐1, Ramos, and NALM‐6 were used to evaluate cellular potency of BTK inhibitors. The in vitro pharmacological efficacy and compound selectivity were assayed via cell viability, colony formation, and BTK‐mediated signalling. A tumour xenograft model with BTK‐Ba/F3, Ramos and BTK(C481S)‐Ba/F3 cells in Nu/nu BALB/c mice was used to assess in vivo efficacy of XMU‐MP‐3.

Key Results

XMU‐MP‐3 is one of a group of low MW compounds that are potent non‐covalent BTK inhibitors. XMU‐MP‐3 inhibited both BTK and the acquired mutant BTKC481S, in vitro and in vivo. Further computational modelling, site‐directed mutagenesis analysis, and structure–activity relationships studies indicated that XMU‐MP‐3 displayed a typical Type‐II inhibitor binding mode.

Conclusion and Implications

XMU‐MP‐3 directly targets the BTK signalling pathway in B‐cell lymphoma. These findings establish XMU‐MP‐3 as a novel inhibitor of BTK, which could serve as both a tool compound and a lead for further drug development in BTK relevant B‐cell malignancies, especially those with the acquired ibrutinib‐resistant C481S mutation.

What is already known

  • Covalent BTK kinase inhibitors such as ibrutinib have shown impressive clinical efficacy in B‐cell malignancies.

  • Btk C481S mutation poses a major challenge for patients after treatment with covalent BTK kinase inhibitors.

What this study adds

  • The non‐covalent inhibitor XMU‐MP‐3 suppressed BTK kinase activity both in vitro and in vivo.

  • XMU‐MP‐3 also successfully inhibited cells expressing the ibrutinib‐resistant Btk C481S mutation.

What is the clinical significance

  • XMU‐MP‐3 could be a lead for developing BTK‐targeted therapeutic agents, especially for overriding Btk C481S mutation.

Abbreviations

CLL

chronic lymphocytic leukaemia

BTK

Bruton's tyrosine kinase

HTRF

homogeneous time‐resolved fluorescence

MCL

mantle cell lymphoma

MTS

a tetrazolium compound [3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐ (3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt]

STAT

signal transducer and activator of transcription

1. INTRODUCTION

http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1948 (BTK) was initially identified as a defective cytoplasmic, non‐receptor tyrosine kinase in human X‐linked agammaglobulinemia (Qiu & Kung, 2000; Vetrie et al., 1993). BTK is predominantly expressed in B lymphocytes, myeloid cells, and platelets, but not in plasma cells, NK cells, and T lymphocytes (Genevier et al., 1994; Quek, Bolen, & Watson, 1998; Smith et al., 1994). Activation of BTK is crucial for cell proliferation and survival in various B‐cell malignancies (Hendriks, Yuvaraj, & Kil, 2014), such as chronic lymphocytic leukaemia (CLL), acute lymphoblastic leukaemia, mantle cell lymphoma (MCL), diffuse large B‐cell lymphoma, Waldenstroms macroglobunemia, and multiple myeloma (Cinar et al., 2013; Davis et al., 2010; Herman et al., 2011; Uckun, Tibbles, & Vassilev, 2007; G. Yang et al., 2013; Y. Yang et al., 2015). Moreover, the highly restricted expression pattern of BTK in B‐cells and myeloid cells also provides an opportunity to selectively target BTK as an effective therapeutic strategy for B‐cell malignancies.

Several low MW BTK inhibitors have been developed, including reversible ATP‐competitive inhibitors, http://www.guidetoimmunopharmacology.org/GRAC/LigandDisplayForward?ligandId=8066 and http://www.guidetoimmunopharmacology.org/GRAC/LigandDisplayForward?ligandId=8249, and irreversible inhibitors, http://www.guidetoimmunopharmacology.org/GRAC/LigandDisplayForward?ligandId=6912, http://www.guidetoimmunopharmacology.org/GRAC/LigandDisplayForward?ligandId=7837, and QL47 (Di Paolo et al., 2011; Evans et al., 2013; Honigberg et al., 2010; Wu et al., 2014; Xu et al., 2012). Taking advantage of a unique conserved cysteine residue in the ATP‐binding site of the https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=629 family of kinases, the covalent BTK inhibitors have shown impressive clinical activity in B‐cell malignancies. However, acquired resistance has emerged as a challenging therapeutic limitation for these agents. For instance, the cysteine residue 481 in the ATP‐binding site of BTK, which forms the covalent bond between BTK and ibrutinib, is the most commonly mutated BTK residue in the context of acquired resistance (Cheng et al., 2015; Johnson et al., 2016). Recently, PROteolysis TArgeting Chimera (PROTAC)‐mediated BTK degraders derived from BTK inhibitors have been developed to overcome the resistant C481S mutation (Buhimschi et al., 2018; Dobrovolsky et al., 2019; Sun et al., 2018; Zorba et al., 2018). Due to hurdles existing in binding kinetics and stoichiometry, BTK degraders derived from covalent BTK inhibitors showed limited efficacy (Tinworth et al., 2019). Thus, a wide variety of potent non‐covalent inhibitors would be a valuable resource to provide warheads for PROTAC degraders. In response to the challenge of drug resistant Btk C481S mutation, we searched for a new chemical scaffold by combining a high‐throughput compound screen and rational drug design approach.

Here, we report the identification and pharmacological characterization of XMU‐MP‐3, a noncovalent inhibitor with potent BTK inhibitory activity and the ability to effectively overcome the BTK acquired mutation C481S both in vitro and in vivo. We demonstrate that XMU‐MP‐3 exerts its pharmacological activity by inhibiting BTK‐mediated signalling pathways in B‐cell lymphoma. Newly identified analogues in this series are introduced here as excellent tool compounds for the purpose of elucidating the BTK signalling pathway. Additionally, they also represent a novel scaffold that could be optimized to obtain therapeutic agents for the management of B‐cell malignancies with acquired resistance to ibrutinib such as those harbouring the BTKC481S mutation.

2. METHODS

2.1. Cell culture

The cell lines 293T, JeKo‐1, Ramos, NALM‐6, and HeLa were obtained from the American Type Culture Collection (Manassas, VA, USA) and authenticated by short tandem repeat testing. The human MCL cell line JeKo‐1 (RRID:CVCL_1865), human Burkitt's lymphoma cell line Ramos (RRID:CVCL_0597), and human pre‐B acute lymphoblastic leukaemia cell line NALM‐6 (RRID:CVCL_UJ05) were cultured in RPMI medium 1640 (Gibco, Grand Island, NY, USA). The HEK cell line 293T (RRID:CVCL_0063) and the human cervical carcinoma cell line HeLa (RRID:CVCL_0030) were grown in DMEM (Gibco). The murine Pro‐B cell line Ba/F3 (RRID:CVCL_0161) was cultured in RPMI 1640 supplemented with 10% conditioned medium from the WEHI‐3B cells (RRID:CVCL_2239) as a source of https://www.guidetoimmunopharmacology.org/GRAC/LigandDisplayForward?ligandId=4994 (Ymer et al., 1985). All stably transformed Ba/F3 cells were cultured in RPMI 1640 without IL‐3. All culture medium contained 10% FBS (BI, Kibbutz Beit Haemek, Israel), penicillin (100 U·ml−1), and streptomycin (100 μg·ml−1). All cell lines were tested for mycoplasma contamination and were found to be negative.

2.2. Plasmid constructs

The pBABE‐puro plasmid was provided by the Zhou lab (Xiamen University, China). The first 1 kb of Tel was amplified with a Kozak sequence (GCCACC) at the 5′ end and then cloned into the pBABE‐puro vector to create the pBABE‐Tel vector. The full‐length BTK and the TK domain of other 29 human TKs were amplified from the human cDNA library with an His or a Flag tag added to the 3′ end of the TKs and cloned into pBABE‐Tel vectors downstream of the Tel sequence. The mutant BTKs were generated by site‐directed mutagenesis (C481S, T474M, E445M/A/I, and S538A). The cloning primers are listed in Table S1.

2.3. Virus production

Virus was produced as previously described (Zhang et al., 2010) with some slight modifications. Three micrograms of each TK expression vector were separately co‐transfected with 2.5 μg of pEcopac (a packaging plasmid, provided by Dr Zhou, Xiamen University, China) into 60–70% confluent HEK 293T cells maintained in 3 ml of culture medium in a 60‐mm dish (Corning, NY, USA) by the polyethylenimine‐mediated transfection method (Horbinski, Stachowiak, Higgins, & Finnegan, 2001). Transfection media was replaced with fresh media after 10 hr of transfection; 48 hr post‐transfection, the viral supernatant was harvested and centrifuged at 3,000×g for 5 min and filtered with 0.45‐μm membrane (BD, NJ, USA).

2.4. Infection of Ba/F3 cells

For infection of Ba/F3 cells, 1 ml of viral supernatant and 8 μg·ml−1 polybrene were added to 5 million cells per well in six‐well plates. The plate was centrifuged at 600× g for 90 min at 37°C during the spin infection. Next, the virus‐containing medium was removed 24 hr later, and successfully infected cells were selected with the addition of 1 μg·ml−1 puromycin to the medium. After 6 days, cells were transferred into the fresh medium without IL‐3 and puromycin. IL‐3‐independent, transformed cells were maintained in RPMI medium 1640 supplemented with 10% FBS. Expression of BTK and other kinases was examined by immunoblotting and further validated by RT‐PCR of the respective oncogenic fusion kinases.

2.5. Generation of BTK knockdown cell lines

BTK shRNA and control plasmids were obtained from Shanghai GeneChem (Shanghai, China). Lentiviruses were generated by transfecting sub‐confluent HEK293T cells together with the lentiviral vector and packing plasmid by polyethylenimine‐mediated transfection; 48 hr after transfection, viral supernatants were collected and used to infect NALM‐6 cells under puromycin (1 μg·ml−1) selection. The knockdown efficiency was validated by immunoblotting.

2.6. RT‐PCR

For RT‐PCR, stably transformed Ba/F3 cells were harvested. Respectively, RNA was isolated and cDNA was synthesized by using PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) according to the manufacturer's protocol (Takara, Otsu Shiga, Japan). Sequencing analysis confirmed the expression of the correct TKs in individually transformed Ba/F3 cell lines.

2.7. Immunoblotting and immunoprecipitation

Cells were washed in PBS buffer and lysed in lysis buffer (50‐mM Tris–HCL [pH 7.5], 150‐mM NaCl, 1% Triton X‐100, 5% glycerol, and 1‐mM phenylmethylsulfonyl fluoride). Total protein concentrations were measured by bicinchoninic acid assay analysis (Beyotime, China). For immunoblotting, equal amounts of protein were resolved by SDS‐10% polyacrylamide gel, and then electrophoretically transferred to the immobilon‐P PVDF membrane (PALL, MI, USA). Proteins were detected with appropriate primary antibodies and peroxidase‐conjugated secondary antibodies. Immunoreactive protein bands were detected by the enhanced chemiluminescence reagent, according to the manufacturer's instructions (Advansta, CA, USA). For immunoprecipitation, cell lysates were incubated with the appropriate antibody together with protein A/G‐Sepharose beads for 3 hr. The protein‐antibody complexes on the beads were then subjected to immunoblotting. Antibodies against BTK (Cell Signaling Technology Cat# 3533S, RRID:AB_2067811, (C82B8) rabbit IgG, dilution (1:1,000)), phospho‐BTK (Tyr223; Cell Signaling Technology Cat# 5082S, RRID:AB_10561017, rabbit, dilution (1:1,000)), spleen TK (https://www.guidetoimmunopharmacology.org/GRAC/ObjectDisplayForward?objectId=2230; Cell Signaling Technology Cat# 2712S, RRID:AB_10691458, rabbit, dilution (1:1,000)), phospho‐SYK (Tyr525/526; Cell Signaling Technology Cat# 2710, RRID:AB_2197222, (C87C1) rabbit IgG, dilution (1:1,000)), https://www.guidetoimmunopharmacology.org/GRAC/ObjectDisplayForward?objectId=1408 (Cell Signaling Technology Cat# 3872S, RRID:AB_10694495, rabbit, dilution (1:1,000)), phospho‐PLCγ2 (Tyr759; Cell Signaling Technology Cat# 3874S, RRID:AB_2163714, rabbit, dilution (1:1,000)), phospho‐PLCγ2 (Tyr1217; Cell Signaling Technology Cat# 3871S, RRID:AB_2299548, rabbit, dilution (1:1,000)), signal transducer and activator of transcription (https://www.guidetoimmunopharmacology.org/GRAC/FamilyDisplayForward?familyId=9903; Cell Signaling Technology Cat# 9139, RRID:AB_331757, (124H6) mouse IgG2a, dilution (1:1,000)), phospho‐STAT3 (Tyr705; Cell Signaling Technology Cat# 9131, RRID:AB_331586, rabbit, dilution (1:1,000)), STAT5 (Cell Signaling Technology Cat# 9363S, RRID:AB_10693321, rabbit, dilution (1:1,000)), phospho‐STAT5 (Tyr694; Cell Signaling Technology Cat# 9359S, RRID:AB_823649, (C11C5) rabbit IgG, dilution (1:1,000)), https://www.guidetoimmunopharmacology.org/GRAC/ObjectDisplayForward?objectId=1525 (Cell Signaling Technology Cat# 2708, RRID:AB_390722, (49D7) rabbit IgG, dilution (1:1,000)), phospho‐ribosomal protein S6 kinase β‐1 (Thr389; Cell Signaling Technology Cat# 9234, RRID:AB_2269803, (108D2) rabbit IgG, dilution (1:1,000)), https://www.guidetoimmunopharmacology.org/GRAC/FamilyDisplayForward?familyId=5141/2 (Cell Signaling Technology Cat# 9102, RRID:AB_330744, rabbit, dilution (1:1,000)), phospho‐ERK1/2 (Thr202/Tyr204; Cell Signaling Technology Cat# 4370, RRID:AB_2315112, (D13.14.4E) rabbit IgG, dilution (1:1,000)), TFII‐1 (Cell Signaling Technology Cat# 4562S, RRID:AB_10692502, rabbit, dilution (1:1,000)), phospho‐TFII‐1 (Y248; Abcam Cat# ab63344, RRID:AB_1143423, rabbit, dilution (1:1,000)), NF‐κB (Cell Signaling Technology Cat# 3034, RRID:AB_330561, rabbit, dilution (1:1,000)), phospho‐NF‐κB (S536; Cell Signaling Technology Cat# 3033, RRID:AB_331284, (93H1) rabbit IgG, dilution (1:1,000)), https://www.guidetoimmunopharmacology.org/GRAC/ObjectDisplayForward?objectId=1619 (Cell Signaling Technology Cat# 9662S, RRID:AB_10694681, rabbit, dilution (1:1,000)), cleaved‐Caspase 3 (Cell Signaling Technology Cat# 9664S, RRID:AB_2070042, (5A1E) rabbit IgG, dilution (1:1,000)), https://www.guidetoimmunopharmacology.org/GRAC/ObjectDisplayForward?objectId=1623 (Cell Signaling Technology Cat# 12827, RRID:AB_2687912, (D2Q3L) rabbit IgG, dilution (1:1,000)), cleaved‐Caspase 7 (Cell Signaling Technology Cat# 9491, RRID:AB_2068144, rabbit, dilution (1:1,000)), http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=883 (Santa Cruz Biotechnology Cat# sc‐7150, RRID:AB_2160738, rabbit, dilution (1:1,000)), cleaved PARP (Cell Signaling Technology Cat# 5625S, RRID:AB_10699459, (D64E10) rabbit IgG, dilution (1:1,000)), and β‐Actin (Sigma‐Aldrich Cat# A5316, RRID:AB_476743, (AC‐74) mouse IgG, dilution (1:1,000)) were used. Protein expression levels were quantified and normalized based on the band intensity by using ImageJ 1.48v (http://imagej.nih.gov/ij, RRID:SCR_003070).

2.8. MTS proliferation assay

Cells were seeded at a density of 2 × 104 cells per well in 96‐well plates. Compounds were serially diluted in growth medium and then added in triplicate to 96‐well plates and incubated for 48 hr. Cell viability was determined using MTS (Promega, Madison, WI, USA), according to the manufacturer's instructions. Results were analysed, and IC50 values were calculated based on cell viability dose–response curves by using GraphPad Prism 6.0 (https://www.graphpad.com/scientific-software/prism, RRID:SCR_002798) and defined as the concentration of compound needed to reduce cell viability to 50% of the vehicle control (DMSO).

2.9. Kinase assay

The BTK kinase assay was performed using the homogeneous time‐resolved fluorescence (HTRF) KinEASE‐TK kit (Cisbio Bioassays, Codolet, France) following manufacturer instructions. Compounds were diluted with 1X kinase buffer and pre‐incubated with BTK kinase in assay plates for 30 min at room temperature while shaking. The substrate was added followed by ATP to start the reaction in 384‐well plates (NUNC, NY, USA). The reaction was stopped after 60 min by addition of TK‐Antibody‐Cryptate/Sa‐XL665 solution. Readouts were collected after 60 min using POLARstar Omega microplate reader (BMG LABTECH, Ortenbery, Germany). The ratio of emission of 665 and 620 nm was multiplied by a constant value of 10,000 to obtain the “HTRF ratio.” Assays were performed in duplicate for each condition. The reaction in the absence of BTK kinase was carried out as a negative control.

2.10. Cell apoptosis assay

PARP and Caspase 3 cleavages were assessed by immunoblotting. Cell apoptosis was determined using the Annexin‐V‐FLUOS Staining kit (Roche, Mannheim, Germany), according to the manufacturer's instructions. The BD LSRFortessa cell analyser (BD) was used to obtain readouts, and FlowJo 7.6.1 software was used for data analysis (https://www.flowjo.com/solutions/flowjo, RRID:SCR_008520).

2.11. Cell cycle assay

Cells were cultured in RPMI medium 1640 with DMSO or XMU‐MP‐3 for 24 hr before harvesting and washing with cold 1X PBS buffer. The cells were then fixed with cold 70% ethanol (pre‐chilled at −20°C) and incubated at 4°C overnight. On the day of flow cytometry analysis, cells were collected by centrifugation, washed with 1X PBS buffer, and stained in PI staining buffer (50 μg·ml−1 propidium iodide, 10 mg·ml−1 RNase in 1X PBS) for 30 min at room temperature and then analysed by using the EPICS XL flow cytometer (Beckman coulter, FL, USA). Results were analysed by FlowJo 7.6.1 software (https://www.flowjo.com/solutions/flowjo, RRID: SCR_008520).

2.12. Soft agar colony formation assay

The soft agar colony formation assay was performed as described with some modifications (Hellwig et al., 2012); 1 ml of 1.4% low melting point agarose (BBI) combined with 1 ml 2 × growth media (with 20% FBS, Amp/Strep) was used as the bottom matrix in a six‐well plate. The bottom layer was solidified at 4°C. Approximately 3 × 104 cells in 1 ml 2 × growth media were gently combined with 1 ml of 0.6% low melting point agarose, plated on top of the bottom layer and allowed to solidify at 4°C. This cell‐containing layer also contained test compounds at indicated concentrations. The plates were incubated at 37°C with 5% CO2 for 14 days. Soft agar colonies were stained for 1 hr with thiazolyl blue tetrazolium bromide (Sigma) in 1X PBS (1 mg·ml−1, 1 ml per well). The colonies (consisting of at least 50 cells) were counted from scanned images of the plates using the Clono counter software (Niyazi, Niyazi, & Belka, 2007).

2.13. Animals

All animal care and experimental procedures complied with the guidelines from the Institutional Animal Care and Use Committee at Experimental Animal Centre at Xiamen University (acceptance no. XMULAC20120030) and Harvard Medical School. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010; McGrath & Lilley, 2015) and with the recommendations made by the British Journal of Pharmacology. Nu/nu BALB/c mice at 4–6 weeks of age were obtained from Experimental Animal Centre at Xiamen University and acclimated for 1 week in a pathogen‐free enclosure before starting study. Mice were maintained in 12‐hr light/12‐hr dark cycles with free access to food and water.

2.14. Tumour xenograft experiments

Cells were filtered through 70‐μm cell strainers (BD) and then suspended in media without serum. Xenografts were initiated by subcutaneous injection of BTK‐Ba/F3 (1 × 107 cells per 200 μl per mouse, n = 6 per group), BTK(C481S)‐Ba/F3 (5 × 106 cells per 200 μl per mouse, n = 12 per group), or Ramos (1 × 107 cells per 200 μl per mouse, n = 12 per group) into the right flank near the axillary fossa of mice. The tumour volume was monitored once per day using the standard formula: V = length × width2 × 0.5. When tumours were grown to approximately 400–500 mm3, mice (half males and half females) were randomly assigned into each experimental group and treated by tail vein injection with vehicle (10% [w/v] Kolliphor HS 15 [Sigma] in normal saline) or XMU‐MP‐3 formulated in vehicle at indicated doses daily for 14 days. The injection volume was 0.1 ml per 10 g. Mouse body weights were monitored daily. At 6 hr after the final dose, mice were killed and tumour tissues were separated, weighed, and subjected for apoptosis analysis, immunoblotting, immunohistochemistry, TUNEL staining, and routine histopathological examination.

2.15. Immunohistochemistry

The antibody‐based procedures used in this study comply with the recommendations made by the British Journal of Pharmacology. Tumour tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned into 4‐μm thick sections. After dewaxing and rehydration, sections were pretreated with 3% H2O2 (diluted with methanol) for 20 min at room temperature. Antigen retrieval was performed by microwave oven in the medium and heated four times for 2 min each. After blocking with 5% serum for 1 hr, sections were incubated with primary antibodies overnight at 4°C, followed by the Polymer Helper incubation for 20 min and HRP‐conjugated secondary antibody for another 20 min. The colour reaction was developed with 3,3‐diaminobenzidine tetrahydrochloride, and slides were counterstained with haematoxylin. The sections were then dehydrated in ethanol, clarified in xylenes, and mounted with neutral balsam.

2.16. Haematoxylin and eosin staining

Tumour tissues were fixed in 4% paraformaldehyde, embedded in paraffin, cut to 4‐μm thick sections, and applied to slides. Standard staining with haematoxylin and eosin was performed on 4‐μm thick sections from each slide.

2.17. TUNEL staining

TUNEL staining was performed using the DeadEnd Fluorometric TUNEL System (Promega) following manufacturer instructions. Briefly, 4‐μm thick paraformaldehyde‐fixed, paraffin‐embedded tissue sections were deparaffinized and rehydrated. Tissue was fixed with 4% paraformaldehyde and permeabilized with proteinase K solution. Then, the tissue was refixed with paraformaldehyde and equilibrated with equilibration buffer. Following these, sections were incubated with TdT reaction mixture at 37°C for 60 min in a humidified chamber, immersed in 2× SSC for 15 min to stop reaction, mounted, and counterstained with VECTASHIELD + DAPI (Vector Lab, Burlingame, CA, USA) for 15 min at room temperature. Negative controls: sections incubated with rTdT incubation buffer (without rTdT enzyme). Positive controls: sections treated with DNase I to cause DNA fragmentation.

2.18. Molecular docking

The molecular docking procedure was referred to the protocol within LeDock (http://www.lephar.com/software.htm). The protein‐ligand complex crystal structure of https://www.guidetoimmunopharmacology.org/GRAC/ObjectDisplayForward?objectId=1923 kinase (PDB code: 2HIW) was chosen as the template (Okram et al., 2006), which shared the highest sequence similarity (49%) with BTK (residues 402–655, UniProtKB, Q06187). The homology model was generated using SWISS‐MODEL (Biasini et al., 2014). Water molecules were deleted, and the binding site was defined as a cuboid. Docking of the ligand XMU‐MP‐3 to the homology model of BTK and the crystal structure of BTK (PDB ID: 3PJ3 and 3OCS; Di Paolo et al., 2011; Kuglstatter et al., 2011) was performed using LeDock. The initial 3D conformation of compound was optimized in the Avogadro 1.1.1 using UFF force field (Hanwell et al., 2012). Compounds were docked in the defined binding site.

2.19. Data and statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). Calculations were performed using GraphPad Prism 6 (https://www.graphpad.com/scientific-software/prism, RRID: SCR_002798). Data were expressed as mean ± SEM. Two‐tailed unpaired Student's t‐test was used for comparisons between two groups. One‐way or two‐way ANOVA was used for multiple group comparisons, and significant differences between the groups were assessed using the Tukey–Kramer test. Statistically significant level was set at P < .05.

2.20. Materials

XMU‐MP‐3 was synthesized in‐house (details provided in the Supporting Information). CGI‐1746, AVL‐292, and ibrutinib were purchased from MedChem Express (Monmouth Junction, NJ, USA). For biochemical assays and cellular studies, all compounds were dissolved in DMSO to make an initial stock solution (10 mM) and stored at −20°C. The final dosing solution was prepared on the day of use by dilution of the stock solution. For animal treatments, compounds were dissolved in normal saline containing 10% (w/v) Kolliphor HS 15 (Sigma, St Louis, MO). The medicinal chemistry and compounds pharmacological profiling efforts are described in the Supporting Information.

2.21. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Christopoulos, et al., 2017; Alexander, Fabbro, et al., 2017; Alexander, Kelly et al., 2017).

3. RESULTS

3.1. Identification of the BTK inhibitor XMU‐MP‐3

In an effort to identify low MW compounds that can selectively inhibit BTK activity, we carried out high‐throughput screening of an in‐house compound library against many TKs using a Ba/F3 cell‐based differential cytotoxicity assay (Goldstein, Gray, & Zarrinkar, 2008; Melnick et al., 2006). With more than 30,000 chemical entities, our kinase‐focused compound library was designed to target the protein kinase ATP‐binding site (Fan et al., 2016). Ba/F3 cells that depend on IL‐3 for proliferation and survival can become IL‐3‐independent, by transformation with oncogenic kinases. The differential cytotoxicity of kinase inhibitors against oncogenic kinase‐transformed Ba/F3 cells versus parental IL‐3‐dependent Ba/F3 was used to establish the pharmacological activity and selectivity of the compounds tested (Warmuth, Kim, Gu, Xia, & Adrian, 2007). Based on the identified lead scaffold, XMU‐MP‐3 was developed with iterative rounds of medicinal chemistry optimization (Figure 1a). XMU‐MP‐3 inhibited BTK‐transformed Ba/F3 cell proliferation with an IC50 of 11.4 nM, while it showed negligible anti‐proliferative effects on parental wild‐type Ba/F3 cells (IC50 > 10,000 nM). The data indicate an adequately differential cytotoxicity window between parental Ba/F3 cells and BTK‐transformed Ba/F3 cells (Figure 1b). The HTRF‐format biochemical assay further confirmed that XMU‐MP‐3 potently inhibited BTK with an IC50 of 10.7 nM (Figure 1c). In addition, more extensive cellular selectivity profiling against a panel of 30 oncogenic kinase‐transformed Ba/F3 cells demonstrated that XMU‐MP‐3 exhibited the highest potency towards BTK‐transformed cells, suggesting a high degree of selectivity of this compound towards BTK (Figure 1d and Table S2). Taken together, these data suggest that XMU‐MP‐3 is a potent and selective inhibitor of BTK.

Figure 1.

Figure 1

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Characterization of XMU‐MP‐3 as a BTK inhibitor. (a) The chemical structure of XMU‐MP‐3. (b) Dose–response curves of BTK‐transformed and parental Ba/F3 cells (2 × 104 cells per well) exposed to XMU‐MP‐3 for 48 hr. Cell viability was assessed with MTS. Data are presented as mean ± SEM (n = 3). Each concentration point was performed in triplicate. (c) Dose‐inhibition curve of XMU‐MP‐3 on BTK enzymatic activity in the presence of 10‐μM ATP in vitro. Each concentration point was performed in duplicate. (d) The selectivity profiling of XMU‐MP‐3 against a panel of 30 oncogenic protein TK‐transformed Ba/F3 cell lines. Cells (2 × 104 cells per well) were exposed to XMU‐MP‐3 for 48 hr. Cell viability was assessed with MTS. Each concentration point was performed in triplicate. The IC50 values were determined and reported in Table S2. (e) Effects of XMU‐MP‐3 on BTK‐mediated signalling pathway components in BTK‐transformed Ba/F3 cells. Cells were treated with either DMSO or XMU‐MP‐3 for 4 hr. Representative blots from three independent experiments are shown. The relative intensity of each band (p‐BTK Y223 and Y551 normalized to BTK; p‐PLCγ2 Y759 and Y1217 normalized to PLCγ2) is shown under each blot. (f) XMU‐MP‐3 inhibited BTK signalling in a time‐dependent manner. Representative blots from three independent experiments and the data of relative intensity are shown. (g) BTK‐transformed Ba/F3 cells were treated with the indicated dose of XMU‐MP‐3 for 24 hr. Induction of cell apoptosis was assessed by Annexin‐V‐FLUOS/PI double staining. CGI‐1746, AVL‐292, and ibrutinib were used as controls. Representative results from five independent experiments are shown. (h) Annexin‐V‐FLUOS‐positive cells was determined and presented as mean ± SEM (n = 5). * P < .05, significantly different as indicated, NS, not significant; one‐way ANOVA using the Tukey–Kramer test

We further investigated the ability of XMU‐MP‐3 to inhibit BTK activity as well as BTK‐mediated downstream signalling events in BTK‐transformed Ba/F3 cells, in comparison to the known BTK‐selective inhibitors, CGI‐1746 and AVL‐292 (Di Paolo et al., 2011; Evans et al., 2013). CGI‐1746 and AVL‐292 inhibited the growth of stable oncogenic BTK‐transformed Ba/F3 cells with IC50 values of 693 and 12.3 nM respectively (Figures S1 and S2). XMU‐MP‐3 inhibited both the autophosphorylation and trans‐phosphorylation of BTK at residues Y223 and Y551, in a dose‐dependent manner in BTK‐transformed Ba/F3 cells (Figures 1e and S3). After 4 hr of treatment with XMU‐MP‐3, the phosphorylation levels of BTK Y223 and Y551 were reduced significantly at concentrations as low as 100 nM and completely suppressed at the concentration of 1,000 nM. https://www.guidetoimmunopharmacology.org/GRAC/FamilyDisplayForward?familyId=629 kinases are reported to control sustained calcium signalling via phosphorylation of key residues Y759 and Y1217 within the https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1408 SH2‐SH3 linker (Watanabe et al., 2001). XMU‐MP‐3 blocked phosphorylation of PLCγ2 at these sites (Y759 and Y1217; Figure 1e). Furthermore, XMU‐MP‐3 also blocked phosphorylation of https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2994 at Y705, STAT5 at Y694, NF‐κB at S536, and TFII‐1 at Y248, all of which are reported to be downstream effectors of BTK (Figure 1e; Egloff & Desiderio, 2001; Mahajan et al., 2001; Petro, Rahman, Ballard, & Khan, 2000; Uckun et al., 2007). Interestingly, XMU‐MP‐3 also affected the phosphorylation of the https://www.guidetoimmunopharmacology.org/GRAC/ObjectDisplayForward?objectId=2109 signalling pathway mediator, S6K. In comparison, the inhibitory effect of XMU‐MP‐3 against BTK activation was greater than that of CGI‐1746, at 100 nM. We also performed time course studies investigating the ability of XMU‐MP‐3 to inhibit cellular BTK at a concentration of 100 nM. A significant reduction of the phosphorylation of BTK at Y223 was observed as early as after a 1 hr incubation (Figure 1f).

To further understand the anti‐proliferative effect of XMU‐MP‐3 on oncogenic BTK‐transformed Ba/F3 cells, we performed apoptosis analysis. In a 24‐hr endpoint assay, XMU‐MP‐3 significantly induced more apoptosis than that caused by reported BTK inhibitors CGI‐1746 and AVL‐292 (Figure 1g). The percentage of apoptotic BTK‐transformed Ba/F3 cells upon XMU‐MP‐3 treatment was twofold to threefold higher than the treatment of CGI‐1746 and AVL‐292, as assessed by Annexin‐V‐FLUOS/PI staining (Figure 1g,h). Moreover, XMU‐MP‐3 was comparatively ineffective in inducing apoptosis of parental Ba/F3 cells (Figure S4). Taken together, XMU‐MP‐3 is a potent and selective inhibitor of BTK, as assessed by inhibitory effects on BTK kinase activation, BTK‐mediated downstream signalling, and the consequent induction of cellular apoptosis.

3.2. On‐target effects of XMU‐MP‐3 on BTK kinase

To evaluate the selectivity of XMU‐MP‐3, we sought to employ a chemical‐genetic approach by engineering an “inhibitor‐resistant” mutant of BTK. One potential drug‐resistant mutation is the so‐called kinase “gatekeeper” mutation (Azam, Seeliger, Gray, Kuriyan, & Daley, 2008; Gorre et al., 2001; Kobayashi et al., 2005), as it is well known that the size of the gatekeeper residue defines the accessibility of the ATP‐binding pocket in protein kinases (Azam et al., 2008; Gorre et al., 2001; Kobayashi et al., 2005). We generated a gatekeeper mutant BTK(T474M)‐transformed Ba/F3 cell line. The introduction of the gatekeeper mutation, T474M, in BTK leads to enhanced kinase activity which was indicated by increased phosphorylation of both BTKY223 and BTKY551 (Figure 2a). As predicted, XMU‐MP‐3 was considerably less potent (IC50 of 2,815 nM) against proliferation of BTK(T474M)‐transformed Ba/F3 cells, as compared with wild‐type BTK‐transformed Ba/F3 cells (Figures 2b and 1c). This gatekeeper mutant BTK showed resistance to other BTK specific inhibitors as well. AVL‐292 and ibrutinib also showed some anti‐proliferative activity in comparison (Figure 2b). The activity of BTK‐mediated downstream signalling also showed robust resistance to XMU‐MP‐3 inhibition (Figure 2c). Additionally, the ratio of Annexin‐V‐FLUOS‐positive cells further indicated that the gatekeeper mutation T474M caused BTK to be significantly resistant to inhibition by XMU‐MP‐3 (Figure 2d,e). Taken together, these data suggest that inhibition of BTK by XMU‐MP‐3 is “on‐target,” inhibiting the growth and survival of oncogenic BTK‐transformed Ba/F3 cells through suppressing BTK activation and its downstream signalling.

Figure 2.

Figure 2

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XMU‐MP‐3 is “on‐target” to BTK. (a) Establishment of stable BTK and BTK mutant‐transformed Ba/F3 cell lines. Mutations of BTK significantly increased the phosphorylation of BTK. Representative blots from three independent experiments are shown. (b) Dose–response curves of BTK(T474M)‐Ba/F3 cells (2 × 104 cells per well, 48‐hr treatment). The gatekeeper mutant BTK(T474M) significantly elevated cellular IC50 more than 200‐fold. Cell viability was assessed with MTS in triplicate. Data are presented as mean ± SEM (n = 3). (c) Effects of XMU‐MP‐3 on the BTK‐mediated signalling pathway in Ba/F3 cells expressing BTK(T474M) after 4 hr of drug treatment. The gatekeeper mutation, T474M, showed robust resistance to XMU‐MP‐3 treatment. Representative blots from three independent experiments are shown. The relative intensity of each band (p‐BTK Y223 and Y551 normalized to BTK, p‐PLCγ2 Y759 and Y1217 normalized to PLCγ2) is shown under each blot. (d) BTK(T474M)‐Ba/F3 cells were treated with DMSO or escalating doses of XMU‐MP‐3, CGI‐1746, and AVL‐292 for 24 hr. Induction of apoptosis was assayed with Annexin‐V‐FLUOS/PI double staining. Representative results from five independent experiments are shown. (e) The ratio of Annexin‐V‐FLUOS‐positive cells indicates that the gatekeeper mutation BTK(T474M) caused BTK to be significantly resistant to inhibition with XMU‐MP‐3. Data are presented as mean ± SEM (n = 5). * P < .05, significantly different as indicated, NS, not significant; one‐way ANOVA using the Tukey–Kramer test

3.3. The binding mode of XMU‐MP‐3 and structure–activity relationships

We further elucidated the binding mode of XMU‐MP‐3 to BTK by using a molecular modelling approach. Two protein structures were used in our modelling, a reported, incomplete crystal structure of DFG‐out BTK kinase (PDB ID: 3PJ3; Kuglstatter et al., 2011) and a homology model built based on a DFG‐out conformation of ABL (PDB ID: 2HIW; Okram et al., 2006) as template. Both computational modelling results suggest that XMU‐MP‐3 interacts with the hinge region of BTK by forming two hydrogen bonds with Met477 (Figures 3a and S5A). Another two hydrogen bonds were formed by the amide linker of XMU‐MP‐3 with Glu445 and Ser538. The directly attached phenyl ring interacted with Thr474 and the trifluoromethyl phenyl ring displacing the Phe540 (Figures 3a and S5A). Based on our docking model, we further demonstrated the introduction of the mutations E445M/A/I and S538A led to robust resistance to inhibition of XMU‐MP‐3 (Figure 3b). The engineered gatekeeper mutant T474M of BTK was predicted to clash with XMU‐MP‐3, because of steric hindrance caused by the increased residue size. This explains the significant potency drop of XMU‐MP‐3 against BTK(T474M)‐Ba/F3 cells in our previous experiments. No interaction between XMU‐MP‐3 and the residue C481 was registered, as we anticipated. A protein structure of DFG‐in conformation of BTK was also assessed, and no favourable binding poses were found (Figure S5B; Di Paolo et al., 2011). These results suggested that XMU‐MP‐3 binds to the DFG‐out conformation of BTK as a typical “Type‐II” inhibitor (Liu & Gray, 2006).

Figure 3.

Figure 3

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Molecular docking and structure–activity relationships of XMU‐MP‐3 against BTK. (a) The binding mode of XMU‐MP‐3 (left panel) and compound 1e (right panel) to the homology model of BTK kinase domain by using ABL (PDB ID: 2HIW) as the template. BTK kinase is shown in the cartoon representation. Compounds are labelled in colour by atoms. The hydrogen bonds are labelled as dashed lines. The key amino acid residues for the binding are labelled as carbon. The black arrow points to the DFG‐motif. The steric interaction between compound 1e and the residue Phe540 is highlighted by the red arrow. (b) Inhibitory effect of XMU‐MP‐3 on cellular BTK activity in 293T cells expressing mutations of E445M (or E445A, E445I) and S538A showed robust resistance to XMU‐MP‐3. 293T cells with transient expression of BTK, BTK(E445M/A/I), or BTK(S538A) were treated with either DMSO or escalating doses of XMU‐MP‐3 for 4 hr. Representative blots from three independent experiments are shown. The relative intensity of p‐BTK Y223 band normalized to BTK is shown under each blot. (c) Structure–activity relationship for BTK. The N‐methyl substitution (R) was essential to achieve potent cellular inhibitory activity against BTK, which is consistent with the predicted binding mode. Cells were exposed to the compound for 48 hr. Cell viability (IC50 values) was assessed with MTS in triplicate

Next, a structure–activity relationship study revealed that the N‐substituent (R) varying from methyl‐ (1a) to ethyl‐ (1b), cyclopropyl‐ (1c), isopropyl‐ (1d), and cyclopentyl‐ (1e) groups resulted in reduced anti‐proliferative activities against both WT and C481S BTK‐driven Ba/F3 cells (Figure 3c), demonstrating a positive correlation between inhibition of BTK and anti‐proliferative potency against BTK‐positive cells. In conjunction with the molecular docking study, the gradual decrease of the anti‐proliferative activities of these analogues was due to the steric hindrance between N‐substituents and the phenyl group of Phe413 and Phe540 (Figure 3a). Together, these results reveal the key structural features for achieving potent inhibition against BTK.

3.4. XMU‐MP‐3 inhibits malignant B‐cell proliferation

Encouraged by the promising potency and selectivity of XMU‐MP‐3 against BTK‐ and other oncogenic kinase‐transformed BaF3 cells, we next evaluated the anti‐cancer potency of XMU‐MP‐3 in three well‐defined malignant B‐cell lines: JeKo‐1, Ramos, and NALM‐6, in which the BTK expression was detected (Figure S6). XMU‐MP‐3 inhibited the proliferation of JeKo‐1, Ramos, and NALM‐6 cells with IC50 values of 327, 686, and 1,065 nM, respectively (Figure 4a). Interestingly, CGI‐1746 and AVL‐292 were less potent against the proliferation of these cells, which was consistent with the previous report by Wu et al. on Ramos cells (Wu et al., 2014). Furthermore, XMU‐MP‐3 also significantly inhibited colony formation as shown for NALM‐6 cells (Figure 4b). In contrast, XMU‐MP‐3 lost its anti‐proliferative potency against the BTK‐negative cell line, HeLa, with an IC50 of 6674 nM. XMU‐MP‐3 did not effectively block BTK‐relevant signalling events in non‐BTK‐driven cells (Figure S7). Subsequently, we investigated whether the observed anti‐proliferative activity of XMU‐MP‐3 was a consequence of transient arrest of cell division or compound induced apoptosis. In JeKo‐1 cells, XMU‐MP‐3 arrested cell cycle progression at the G2 phase (Figure S8). In addition, XMU‐MP‐3 also induced significant cell apoptosis (Figure 4c,d). Taken together, XMU‐MP‐3 selectively inhibits the proliferation of malignant B‐cells.

Figure 4.

Figure 4

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XMU‐MP‐3 inhibits the growth of malignant B‐cells. (a) Dose–response curves for XMU‐MP‐3 against BTK‐positive B‐cell malignancy cell lines JeKo‐1, Ramos, and NALM‐6 following 48 hr of treatment. Cell viability was assessed with MTS in triplicate. Data are presented as mean ± SEM (n = 3). (b) Anti‐colony formation of XMU‐MP‐3 on NALM‐6. Colony formation were performed in 14‐day soft agar assays from three independent experiments with 3 × 104 cells per well. Data are presented as mean ± SEM (n = 3). (c) XMU‐MP‐3 induced cell apoptosis in malignant B‐cells JeKo‐1, Ramos, and NALM‐6. Induction of cell apoptosis was assessed by Annexin‐V‐FLUOS/PI double staining. CGI‐1746 and AVL‐292 were used as controls. Representative results from five independent experiments are shown. Annexin‐V‐FLUOS‐positive cells was determined and presented as mean ± SEM (n = 5). * P < .05, significantly different as indicated, NS, not significant; one‐way ANOVA using the Tukey–Kramer test. (d) XMU‐MP‐3 induced significant Caspase 3/7 cleavage and PARP activation in JeKo‐1, Ramos, and NALM‐6 cell lines. Representative blots from three independent experiments are shown. The relative intensity of each band normalized to β‐actin is shown under each blot

We further examined whether the anti‐proliferative activity of XMU‐MP‐3 against malignant B‐cells was accompanied by the inhibition of cellular BTK TK activity. In JeKo‐1 cells, the phosphorylation of BTK, at Y551 and Y223, was efficiently blocked by XMU‐MP‐3 (Figure 5a). XMU‐MP‐3 also inhibited phosphorylation of PLCγ2 at Y1217 and Y759, which is the well‐defined effector for BTK signalling (Watanabe et al., 2001). Phosphorylation of other BTK signalling downstream effectors, including STAT3, STAT5, https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=514, and S6K, was also dramatically inhibited by all three BTK inhibitors (Figure 5a). In addition, treatment with XMU‐MP‐3 did not affect the phosphorylation of https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2230&familyId=625&familyType=ENZYME, which is an upstream activator of BTK (Kurosaki & Kurosaki, 1997). Similar results were observed with Ramos and NALM‐6 cells (Figure 5a). To further validate the “on‐target” effect of XMU‐MP‐3, NALM‐6 cells with BTK stably knocked down were established (Figure S9A). BTK depletion significantly inhibited cancer progression as stable knockdown of BTK predominantly decreased colony formation of NALM‐6 cells (Figure S9B, C). Meanwhile, the depletion of BTK by shRNA in NALM‐6 cells also sufficiently attenuated the inhibitory activity of XMU‐MP‐3, compared with that of the sh‐control (Figures 5b and S10). These results demonstrate that the anti‐proliferative activity of XMU‐MP‐3 correlates with the inhibition of BTK activation in malignant B‐cells.

Figure 5.

Figure 5

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XMU‐MP‐3 inhibits cellular BTK activity in B‐cell malignancy cells. (a) Effects of XMU‐MP‐3 on BTK‐mediated signalling pathway in B‐cell malignancy lines JeKo‐1, Ramos, and NALM‐6. Representative blots from three independent experiments are shown. The relative intensity of each band (p‐BTK Y223 and Y551 normalized to BTK, p‐PLCγ2 Y759 and Y1217 normalized to PLCγ2) is shown under each blot. (b) BTK knockdown (1# and 3#) attenuated the anti‐proliferation activity of XMU‐MP‐3 against NALM‐6 cells. Colony formation assays were performed in 14‐day soft agar with the treat of XMU‐MP‐3 on BTK knockdown NALM‐6 cells (#1 and #3) and the control NALM‐6 cells (shCtrl). Representative results are shown as mean ± SEM (n = 3) from three independent experiments

3.5. XMU‐MP‐3 inhibits tumour progression in vivo

Next, we evaluated the in vivo anti‐tumour activity of XMU‐MP‐3 using xenograft models. The pharmacokinetics of XMU‐MP‐3 was determined in SD rat at the dose of 5 mg·kg−1 via intravenous injection, exhibiting moderate pharmacokinetic properties with the t 1/2 of 0.9 hr, clearance (Cl) of 37.6 ml·min−1·kg−1, and volume of distribution (Vd) of 1.34 L·kg−1. Administration of XMU‐MP‐3 led to a significant dose‐dependent inhibition of tumour growth for both BTK‐Ba/F3 and Ramos‐driven tumours without obvious body weight loss or overt signs of toxicity (Figure 6a). Following 14 days of treatment with XMU‐MP‐3, tumour burden was decreased to a significantly lower level in the 50 mg·kg−1 treated group than the vehicle group in both xenograft models (Figure 6b,c). In addition, administration of XMU‐MP‐3 induced tumour cell apoptosis in a dose‐dependent manner, as demonstrated by apoptosis analysis using Annexin‐V‐FLUOS/PI double staining (Figure 6d,e), immunohistochemical staining (Figure 6f), and TUNEL staining (Figure 6g). Furthermore, XMU‐MP‐3 also markedly suppressed the activation of BTK and consequently inhibited the phosphorylation of the downstream effectors PLCγ2, STAT3, and STAT5 in BTK‐transformed Ba/F3‐derived tumours (Figure 6h). Collectively, these results demonstrate that XMU‐MP‐3 displays potent therapeutic activity against BTK‐positive tumours and B‐cell malignancies in vivo.

Figure 6.

Figure 6

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XMU‐MP‐3 substantially suppresses tumour growth in mouse xenograft models. (a) The changes of tumour volume and body weight of stable BTK‐transformed Ba/F3 and Ramos xenograft models in a 14‐day experiment with or without XMU‐MP‐3 treatment. XMU‐MP‐3 significantly reduced the tumour size without affecting animal weights. Data are presented as mean ± SEM (BTK‐Ba/F3, n = 6 per group; Ramos, n = 12 per group).; * P < .05, significantly different from vehicle; two‐way ANOVA with the Tukey–Kramer test ;. (b) Representative photos of BTK‐Ba/F3 and Ramos tumours 14 days after drug treatment. (c) Tumour weights of BTK‐Ba/F3 and Ramos tumours dissected on Day 14 of drug treatments. Data are presented as mean ± SEM (BTK‐Ba/F3, n = 6 per group; Ramos, n = 12 per group).* P < .05 significantly different from vehicle: one‐way ANOVA using the Tukey–Kramer test. (d) Apoptosis analysis of tumours was performed on Day 14 after drug treatment using Annexin‐V‐FLUOS/PI double staining. (e) Annexin‐V‐FLUOS‐positive cells was determined and presented as mean ± SEM (n = 5 per group). * P < .05 significantly different from vehicle: one‐way ANOVA using the Tukey–Kramer test. (f) Immunohistochemical analysis and H&E staining and (g) TUNEL staining indicated that the inhibition of BTK kinase activity by XMU‐MP‐3 induces significant apoptosis in BTK‐transformed Ba/F3 and Ramos tumours. The white scale bar represents 50 μm. (h) Immunoblot analysis of the expression and phosphorylation of BTK in BTK‐transformed Ba/F3 tumours from mice killed 14 days after drug treatment. Representative blots from three animals of each experimental group are shown. The relative intensity of each band (p‐BTK Y223 and Y551 normalized to BTK, p‐PLCγ2 Y759 and Y1217 normalized to PLCγ2) is shown under each blot

3.6. XMU‐MP‐3 inhibits ibrutinib‐resistant BTKC481S mutant

It has been reported that the mutation of BTKC481S poses a major challenge of diminishing the therapeutic efficacy of ibrutinib in the clinical treatment of CLL patients (Cheng et al., 2015; Furman et al., 2014). This is due to the fact that the mutation of the cysteine residue (C481) impairs the ability of ibrutinib or other irreversible inhibitors to form a covalent bond with BTK. Therefore, the BTK C481 mutation significantly decreases the inhibitory effect of irreversible inhibitors. The development and design of XMU‐MP‐3 aimed to override the Cys481 mutation of BTK, while retaining its potency against wild‐type BTK. This idea was tested in a stably transformed BTK(C481S)‐Ba/F3 cell line. Indeed, XMU‐MP‐3 maintained inhibitory potency with an IC50 of 182.3 nM against BTK(C481S)‐Ba/F3 cells (Figure 7a) and with an IC50 of 17.0 nM in biochemical assays (Figure 7b), inducing significant cell apoptosis (Figure 7c,d). To further evaluate the pharmacological properties of XMU‐MP‐3, we transfected human HEK293T cells with WT or C481S BTK. XMU‐MP‐3 maintained its inhibitory potency against C481S BTK and inhibited the autophosphorylation of BTK in a dose‐dependent manner, as well as the phosphorylation of the BTK downstream effector PLCγ2, with similar potency against WT BTK (Figure 7e). The non‐covalent reference inhibitor, CGI‐1746, also blocked BTK phosphorylation with similar potency in WT‐ and C481S BTK‐expressing cells (Figure 7e). In contrast, the covalent reference inhibitor AVL‐292 could effectively inhibit the autophosphorylation of WT BTK but not that of C481S BTK (Figure 7e). Thus, we concluded that XMU‐MP‐3 may serve as a preclinical development candidate for overcoming the recently identified ibrutinib‐resistant mutant C481S BTK in CLL patients (Cheng et al., 2015).

Figure 7.

Figure 7

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XMU‐MP‐3 overcame the ibrutinib‐resistant BTKC481S mutation in cells. (a) Dose–response curves of BTK(C481S)‐transformed Ba/F3 cells (2 × 104 cells per well) exposed to XMU‐MP‐3 for 48 hr. Cell viability was assessed with MTS in triplicate. Data are presented as mean ± SEM (n = 3). (b) Dose‐inhibition curve of XMU‐MP‐3 on BTK(C481S) enzymatic activity in the presence of 10‐μM ATP in vitro. Each concentration point was performed in duplicate. (c) XMU‐MP‐3 induced apoptosis in BTK(C481S)‐Ba/F3 cells. Induction of apoptosis was assayed with Annexin‐V‐FLUOS/PI double staining. Representative results from five independent experiments are shown. (d) Annexin‐V‐FLUOS‐positive cells was determined and presented as mean ± SEM (n = 5). * P < .05 significantly different as indicated: one‐way ANOVA using the Tukey–Kramer test.. (e) Effects of XMU‐MP‐3 on phosphorylation of BTK and the downstream effector PLCγ2 in Ba/F3 cells stably expressed BTK or BTK(C481S). Representative blots from three independent experiments are shown. The relative intensity of each band (p‐BTK Y223 and Y551 normalized to BTK, p‐PLCγ2 Y1217 normalized to PLCγ2) is shown under each blot

3.7. XMU‐MP‐3 suppresses C481S‐driven tumour growth in vivo

In order to determine whether XMU‐MP‐3 still retains the ability to override the C481S BTK mutation in vivo, the anti‐tumour activity of XMU‐MP‐3 was further examined in a xenograft mouse model using BTK(C481S)‐Ba/F3 cells. Administration of XMU‐MP‐3 for 14 days resulted in significant tumour suppression without overt body weight loss or overt signs of toxicity (Figure 8a,b). Significant cancer cell apoptosis induced by the treatment of XMU‐MP‐3 was observed by the assessment with Annexin‐V‐FLUOS/PI staining (Figure 8c), immunohistochemical staining (Figure 8d), and TUNEL staining (Figure 8e). The in vivo therapeutic activity of XMU‐MP‐3 against BTK(C481S)‐driven tumours was consistent with that against WT BTK‐driven tumours. In contrast, the covalent inhibitor ibrutinib showed negligible anti‐tumour activity in the acquired mutant xenograft model of BTK(C481S)‐Ba/F3 (Figure 8a–e). Furthermore, the autophosphorylation of BTKY223 was blocked by XMU‐MP‐3 in BTK(C481S)‐driven tumours, but not by ibrutinib (Figure 8f). Together, these results clearly indicate that the non‐covalent BTK kinase inhibitor XMU‐MP‐3 exhibits significant pharmacological activity against the drug‐resistant C481S mutation of BTK in vivo.

Figure 8.

Figure 8

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XMU‐MP‐3 overrode the ibrutinib‐resistant BTKC481S mutation in vivo. (a) Tumour volumes were significantly reduced by XMU‐MP‐3 compared with both the vehicle and ibrutinib treatment groups in a 14‐day experiment, and body weights were not affected. Data are presented as mean ± SEM (n = 12 per group). * P < .05 significantly different from vehicle: two‐way ANOVA with the Tukey–Kramer test; (b) BTK(C481S)‐driven tumours were harvested on Day 14 after drug treatment. Representative photos and weights of tumours for each experimental group are shown. Data are presented as mean ± SEM (n = 12 per group). * P < .05. significantly different as indicated; one‐way ANOVA using the Tukey–Kramer test; (c) Apoptosis analysis of tumours dissected on Day 14 of dosing using Annexin‐V‐FLUOS/PI double staining. Annexin‐V‐FLUOS‐positive cells was determined and presented as mean ± SEM (n = 12 per group). * P < .05, significantly different as indicated; one‐way ANOVA using the Tukey–Kramer test. (d) Immunohistochemical analysis and H&E staining and (e) TUNEL staining were assayed on BTK(C481S)‐driven tumours from mice killed on Day 14 after drug treatment. The white scale bar represents 50 μm. (f) Immunoblot analysis of expression and phosphorylation of BTK in BTK(C481S)‐Ba/F3 tumours from mice killed 14 days after drug treatment. Representative blots from three animals of each experimental group are shown. The relative intensity of p‐BTK Y223 and Y551 bands normalized to BTK is shown under each blot

4. DISCUSSION

Low MW kinase inhibitors have taken on an increasingly prominent role in the treatment of cancer and other diseases. Because of the highly conserved structure of the ATP‐binding pocket among more than 500 human kinases, development of a kinase‐specific inhibitor is a key challenge in medicinal chemistry. To take advantage of the unique structural features of the TEC kinase family (Hur et al., 2008), medicinal chemists were able to design the compound ibrutinib, which uses an electrophilic acrylamide moiety to react irreversibly with C481 of BTK. By forming a covalent bond with the nucleophilic residue cysteine in the ATP‐binding pocket, this drug design strategy enabled the development of more target‐specific inhibitors of BTK. The covalent BTK inhibitor, ibrutinib has shown impressive clinical activity in patients with B‐cell malignancies, including MCL, CLL, Waldenstroms macroglobunemia, multiple myeloma, and diffuse large B‐cell lymphoma (Advani et al., 2013; Byrd et al., 2013; Wang et al., 2013; Wilson et al., 2015). Unfortunately, acquired resistance to ibrutinib has emerged with disease progression due to the mutation of the key cysteine residue, C481, in BTK. The same challenge certainly would limit the effectiveness of other covalent inhibitors sharing the same covalent binding mode. The first ibrutinib‐resistant BTK mutation reported was C481S (Cheng et al., 2015; Furman et al., 2014). In addition to C481S, other mutations, including C481F/Y/R, T474I/S, L528W, and T316A, have been identified in ibrutinib‐resistant patients (Maddocks et al., 2015; Sharma et al., 2016). Thus, there is an urgent need for development of second generation BTK inhibitors that are able to override drug resistance in mutant BTK‐positive patients. In this study, XMU‐MP‐3 was developed as a selective inhibitor for both wild‐type BTK and the C481 mutant version of BTK. XMU‐MP‐3 showed its pharmacological potency in specifically suppressing the activation and downstream signalling of BTK and BTKC481S mutant in vitro and in vivo. Recently, PROTAC‐mediated BTK kinase degradation has been examined to overcome the ibrutinib‐resistant C481S mutation (Buhimschi et al., 2018; Dobrovolsky et al., 2019; Sun et al., 2018; Zorba et al., 2018). The protein degradation‐based approach is new and could have great potential for overriding BTK resistance, although its clinical efficacy has not been fully confirmed. Even in the usage of PROTAC technology, a reversible warhead is obviously better than irreversible inhibitor in the PROTAC degrader, due to its kinetics and stoichiometry, which has been demonstrated by the work from Tinworth et al. (2019).

Molecular modelling study and site‐directed mutagenesis revealed the unique binding mode of XMU‐MP‐3 with BTK, as the first “Type‐II” BTK inhibitor reported so far. In general, “Type‐II” kinase inhibitors could provide a better selectivity than that of “Type‐I” inhibitors. Interestingly, in comparison with ibrutinib, which is a multitarget inhibitor with nearly equivalent potency against all five TEC family kinases (Honigberg et al., 2010), XMU‐MP‐3 shows a higher selectivity towards BTK. Due to those pharmacological properties, XMU‐MP‐3 is far less potent towards parental Ba/F3 cells than BTK‐transformed Ba/F3 cells. Thus, there was little toxicity observed in mice treated with XMU‐MP‐3, as shown by no change in body weight and no toxicity observed in vital organs. Our data therefore suggest that treatment with XMU‐MP‐3 for targeted therapy of B‐cell malignancies could potentially provide a wider therapeutic window with less adverse side effects. Indeed, the data from mouse xenograft models indicate that XMU‐MP‐3 may serve as a good starting point for drug development overcoming the ibrutinib‐resistant mutant C481S BTK in CLL patients.

In conclusion, we have described XMU‐MP‐3 as a novel, potent and highly selective BTK inhibitor that overrides the resistant BTK mutant C481S. Our findings warrant further optimization and development of XMU‐MP‐3 to potentially yield an improved and effective therapeutic agent for B‐cell malignancies.

AUTHOR CONTRIBUTIONS

X.D. and J.Z. conceived the project. X.D., J.Z., L.L., F.G., and J.J. performed data analysis/statistics. X.D., Z.H., and Z.D. conceived and performed chemical synthesis of XMU‐MP‐3 and its analogues and small‐molecule structure determination. X.D., J.Z., F.G., J.J., and L.L. designed the biology experiments. F.G., J.J., L.L., Y.Li, Y.Lu, X.W., G.C., J.S., and Y.‐M.Z. performed the experiments and acquired data. C.‐H.Y., E.W., and S.S. contributed to analysis and interpretation of data. F.G., J.J., L.L., J.Z., and X.D. co‐wrote the paper. All authors edited the manuscript.

CONFLICT OF INTEREST

X.H. is an employee of Hongyun Biotech Co., Ltd., China. The other authors declare no conflict of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14207, https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14208, and https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14206, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Data S1: Supporting Information

Click here for additional data file. (343.9KB, pdf)

Figure S1, related to Figure 1.The chemical structures of reference inhibitorsCGI‐1746, AVL‐292 and ibrutinib.

Figure S2, related to Figure 1.Antiproliferative activities of CGI‐1746, AVL‐292 and ibrutinib against BTK‐Ba/F3 and WT‐Ba/F3.

Figure S3, related to Figure 1.CGI‐1746, AVL‐292 and ibrutinib inhibited cellular BTK kinase activity in BTK‐Ba/F3 cells.

Figure S4, related to Figure 1.XMU‐MP‐3 did not affect the survival of the parental Ba/F3 cells.

Figure S5, related to Figure 3.Molecular docking of XMU‐MP‐3 with BTK in different binding modes.

Figure S6, related to Figure 4.The expression profile of BTK in human B‐cell malignancy cell lines.

Figure S7, related to Figure 4.No inhibitory effect of XMU‐MP‐3 on BTK‐negative HeLa cells.

Figure S8, related to Figure 4.Quantification of the cell cycle distribution affected by XMU‐MP‐3.

Figure S9, related to Figure 5.BTK knockdown inhibited the proliferation of malignant B cell line NALM‐6.

Figure S10, related to Figure 5.BTK knockdown (1# and 3#) attenuated the potency of XMU‐MP‐3 against cell proliferation in NALM‐6 cells.

Click here for additional data file. (817.1KB, pdf)

Table S1. The list of primer sequences.

Click here for additional data file. (12.9KB, xlsx)

Table S2,related to Figure 1. Cellular antiproliferative IC50s of XMU‐MP‐3 on various oncogenic kinases transformed Ba/F3.

Click here for additional data file. (11.9KB, xlsx)

ACKNOWLEDGEMENTS

We thank Dr D. Zhou for providing the pBABE‐puro vector and Dr J. Han for providing the cDNA of Tel. This work was supported by grants from the Ministry of Science and Technology of the People's Republic of China (Grants 2017YFA0504504 and 2016YFA0502001) and the National Natural Science Foundation of China (Grants 81422045, U1405223, and 81661138005 to X.D. and 81603131 to L.L.), the China's 1000 Young Talents Program to X.D., the Fundamental Research Funds for the Central Universities of China (Grants 20720190101 to X.D. and 20720170067 to L.L.), the Program of Introducing Talents of Discipline to Universities (111 Project, Grant B06016), Shanghai Municipal Education Commission ‐ Gaofeng Clinical Medicine Grant Support, and NSFC MAJOR PROJECTS (Grant 81890994) to J.Z.

Gui F, Jiang J, He Z, et al. A non‐covalent inhibitor XMU‐MP‐3 overrides ibrutinib‐resistant BtkC481S mutation in B‐cell malignancies. Br J Pharmacol. 2019;176:4491–4509. 10.1111/bph.14809

Fu Gui, Jie Jiang and Zhixiang He contributed equally to this work.

Contributor Information

Jianming Zhang, Email: jzuc@shsmu.edu.cn.

Xianming Deng, Email: xmdeng@xmu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1: Supporting Information

Click here for additional data file. (343.9KB, pdf)

Figure S1, related to Figure 1.The chemical structures of reference inhibitorsCGI‐1746, AVL‐292 and ibrutinib.

Figure S2, related to Figure 1.Antiproliferative activities of CGI‐1746, AVL‐292 and ibrutinib against BTK‐Ba/F3 and WT‐Ba/F3.

Figure S3, related to Figure 1.CGI‐1746, AVL‐292 and ibrutinib inhibited cellular BTK kinase activity in BTK‐Ba/F3 cells.

Figure S4, related to Figure 1.XMU‐MP‐3 did not affect the survival of the parental Ba/F3 cells.

Figure S5, related to Figure 3.Molecular docking of XMU‐MP‐3 with BTK in different binding modes.

Figure S6, related to Figure 4.The expression profile of BTK in human B‐cell malignancy cell lines.

Figure S7, related to Figure 4.No inhibitory effect of XMU‐MP‐3 on BTK‐negative HeLa cells.

Figure S8, related to Figure 4.Quantification of the cell cycle distribution affected by XMU‐MP‐3.

Figure S9, related to Figure 5.BTK knockdown inhibited the proliferation of malignant B cell line NALM‐6.

Figure S10, related to Figure 5.BTK knockdown (1# and 3#) attenuated the potency of XMU‐MP‐3 against cell proliferation in NALM‐6 cells.

Click here for additional data file. (817.1KB, pdf)

Table S1. The list of primer sequences.

Click here for additional data file. (12.9KB, xlsx)

Table S2,related to Figure 1. Cellular antiproliferative IC50s of XMU‐MP‐3 on various oncogenic kinases transformed Ba/F3.

Click here for additional data file. (11.9KB, xlsx)

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

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