 |
INTRODUCTION |
Apoptosis is a common mode of eukaryotic cell death that is
triggered by an inducible cascade of biochemical events leading to
activation of endonucleases that cleave the nuclear DNA into oligonucleosome length fragments (1-4). Several of the biochemical events that contribute to apoptotic cell death as well as both positive
and negative regulators of apoptosis have recently been identified
(1-4). Apoptosis plays a pivotal role in the development and
maintenance of a functional immune system by ensuring the timely
self-destruction of autoreactive immature and mature lymphocytes as
well as any emerging target neoplastic cells by cytotoxic T cells
(1-7). Inappropriate apoptosis may contribute to the development as
well as chemotherapy resistance of human leukemias and lymphomas (5-7). Therefore, an improved understanding of the molecular basis of
apoptosis and the pro-apoptotic versus anti-apoptotic regulatory signals may provide further insights into the pathogenesis of human lymphoid malignancies and have important implications for
treatment of leukemias and lymphomas.
Bruton's tyrosine kinase
(BTK),1 a member of the
BTK/Tec family of protein tyrosine kinases (PTKs), is a cytoplasmic PTK
involved in signal transduction pathways regulating growth and
differentiation of B-lineage lymphoid cells (8-10). BTK participates
in signal transduction pathways initiated by the binding of a variety
of extracellular ligands to their cell-surface receptors; following ligation of B cell antigen receptors, BTK activation by the concerted actions of the PTKs Lyn and Syk (9) is required for induction of
phospholipase C-
2 mediated calcium mobilization (9). Mutations in
the human BTK gene are the cause of X-linked
agammaglobulinemia, a male immune deficiency disorder characterized by
a lack of mature, immunoglobulin-producing, peripheral B cells (11,
12). In mice, mutations in the BTK gene have been identified
as the cause of murine X-linked immune deficiency (13).
BTK has an N-terminal region consisting of a 140-residue pleckstrin
homology (PH) domain followed by an 80-residue proline-rich Tec
homology (TH) domain. The PH domain is the site of activation by
phosphatidylinositol phosphates and G-protein 
subunits and inhibition by protein kinase C (14). The remaining portion of BTK
contains Src homology (SH) domains SH3 (49 residues), followed by SH2
(96 residues), and a 250-residue C-terminal SH1 kinase domain. The SH2
domain mediates binding to tyrosine-phosphorylated peptide motifs on
other molecules, and the SH3 domain mediates binding to proline-rich
motifs. BTK is activated by transphosphorylation of Tyr551
in the SH1 domain, followed by autophosphorylation of
Tyr223 in the SH3 domain (9). Phosphorylation of
Tyr223 may function to disrupt an intramolecular TH-SH3
domain interaction, allowing BTK TH domain binding with SH3 domains in
the Src family kinases FYN, LYN, and HCK, and BTK SH3 domain binding
with a proline-rich region of Cbl (9, 15, 16). Mutations in the
catalytic domain, SH2 domain, as well as PH domain of the BTK have been
found to lead to maturational blocks at early stages of B cell ontogeny in human X-linked agammaglobulinemia (17). BTK-deficient mice generated
by introducing PH domain or catalytic domain mutations in embryonic
stem cells showed defective B cell development and function (18). Thus,
different regions of BTK are important for its physiologic functions.
In murine B cells, BTK has been shown to act as an anti-apoptotic
protein upstream of Bcl-xL in the B cell antigen receptor (but not the
CD40 receptor) activation pathway (19). Our recent studies provided
biochemical and genetic evidence that BTK is an inhibitor of the
Fas/APO-1 death-inducing signaling complex in B-lineage lymphoid cells
(20). Furthermore, we found that BTK also prevents ceramide- and
vincristine-induced apoptosis (present study). The fate of
leukemia/lymphoma cells may reside in the balance between the opposing
proapoptotic effects of caspases activated by the death-inducing
signaling complex and an upstream anti-apoptotic regulatory
mechanism involving BTK and/or its substrates (20). Therefore,
inhibitors of BTK are likely to enhance the chemosensitivity of
leukemia/lymphoma cells.
In a systematic effort to design potent inhibitors of BTK as
anti-leukemic agents with apoptosis-promoting properties, we have
constructed a three-dimensional homology model of the BTK kinase
domain. Advanced docking procedures were employed for the rational
design of leflunomide metabolite (LFM) analogs with a high likelihood
to bind favorably to the catalytic site within the kinase domain of
BTK. Here, we first report the identification of the LFM analog
-cyano-
-hydroxy-
-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13) as a potent and specific inhibitor of BTK.
LFM-A13 inhibited recombinant BTK with an IC50
value of 2.5 µM, but it did not affect the enzymatic
activity of other protein tyrosine kinases, including Janus kinases
JAK1 and JAK2, Src family kinase HCK, and receptor family tyrosine
kinases EGF-receptor kinase (EGFR) and insulin receptor kinase (IRK),
at concentrations as high as 278 µM. LFM-A13
enhanced the chemosensitivity of BTK-positive B-lineage leukemia cells
to vincristine and ceramide.
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EXPERIMENTAL PROCEDURES |
Crystal Structures of Leflunomide Metabolite and Its
Analogs--
The leflunomide metabolite (LFM) and two of
its analogs (LFM-A12 and LFM-A13) were
crystallized using various solvents by evaporation or liquid-liquid
diffusion. X-ray data from single crystals were collected using a SMART
CCD area detector (Bruker Analytical X-ray Systems, Madison, WI) with
MoK
radiation (
= 0.7107 Å). The space group was determined
based on systematic absence and intensity statistics. A direct methods solution provided most of the non-hydrogen atoms from the electron density map. Several full-matrix least squares/difference Fourier cycles were performed to locate the remaining non-hydrogen atoms. All
non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were placed in ideal positions and refined as riding atoms with relative isotropic temperature factors. The structure was
refined using full matrix least squares on F2.
Crystal structure calculations were performed using a Silicon Graphics
INDY R4400-SC computer (Silicon Graphics Inc., Mountain View, CA) or a
Pentium computer using the SHELXTL V 5.0 suite of programs (21).
Construction of the Homology Model for the Kinase Domain of
BTK--
We have constructed a homology model of BTK using crystal
structures of homologous kinase domains of protein kinases HCK (22), FGFR (23), IRK (24), and cAMP-dependent protein kinase (25) since no
experimentally determined three-dimensional coordinates have been
reported for the BTK kinase domain. The homology modeling of BTK was
carried out by first obtaining the protein sequence of BTK (Swiss-Prot
accession number Q06187, University of Geneva, Geneva, Switzerland)
from GenBankTM (National Center for Biotechnology
Information, Bethesda, MD). Next, the most reasonable sequence
alignment between the BTK kinase and a coordinate template was
determined. This was done by first superimposing the C-
coordinates
of the kinase domains of HCK, FGFR, IRK, and cAMP-dependent protein
kinase using the InsightII program (26) to provide the best overall
structural comparison. All four sequences were then aligned based on
the superimposition of their structures (amino acid sequences were
aligned together if their C-
positions were spatially related to
each other). The sequence alignment accommodated such features as loops
in a protein that differed from the other protein sequences. The structural superimposition was done using the homology module of the
InsightII (26) program and a Silicon Graphics INDIGO2 computer (Silicon
Graphics Inc., Mountain View, CA). The sequence alignment was manually
adjusted based on the previously mentioned considerations and produced
a sequence variation profile for each superimposed C-
position. The
sequence variation profile served as a basis for the next procedure,
which was sequence alignment of all four proteins with BTK kinase. In
this procedure, the sequence of BTK kinase was read into the program
and manually aligned with the four known kinase proteins based on the
sequence variation profile described previously. Next a set of
three-dimensional coordinates was assigned to the BTK kinase sequence
using the three-dimensional coordinates of HCK as a template, which
employed the Homology module within the InsightII program (26). The
coordinates for a loop region where a sequence insertion occurs
(relative to HCK without the loop) was chosen from a limited number of
possibilities automatically generated by the program and manually
adjusted to a more ideal geometry using the program CHAIN (27).
Finally, the constructed model of BTK was subjected to energy
minimization using the X-plor program (28) so that any steric strain
introduced during the model-building process could be relieved. The
model was screened for unfavorable steric contacts, and if necessary such side chains were remodeled either by using a rotamer library data
base or by manually rotating the respective side chains. The final
homology model of the BTK kinase domain had a root mean square
deviation of 0.01 Å from ideal bond lengths and 2.2° from ideal bond
angles after energy minimization. The homology model of BTK was then
used, in conjunction with model coordinates of LFM and its analogs
(which were later compared with crystal structures), for our modeling
studies of the BTK·inhibitor complexes.
Docking Procedure Using Homology Model of BTK Kinase
Domain--
Modeling of the BTK·LFM analog complexes was done using
the Docking module within the program INSIGHTII (26) and using the Affinity suite of programs for automatically docking a ligand to the
receptor. Energy-minimized coordinates for each LFM molecule were
generated and interactively docked into the ATP-binding site of BTK
based on the position of quercetin in the HCK/quercetin crystal
structure (22). The hydrogen atoms on the kinase domain of BTK were
generated, and potentials were assigned to both receptor and ligand
prior to the start of the docking procedure. The docking method in the
InsightII (26) program used the CVFF force field and a Monte Carlo
search strategy to search for and evaluate docked structures. While the
coordinates for the bulk of the receptor were kept fixed, a defined
region of the binding site was allowed to relax, thereby allowing the
protein to adjust to the binding of different inhibitors. A binding set
was defined within a distance of 5 Å from the inhibitor, allowing
residues within this distance to shift and/or rotate to energetically
favorable positions to accommodate the ligand. An assembly was defined
consisting of the receptor and inhibitor molecule, and docking was
performed using the fixed docking mode. Calculations approximating
hydrophobic and hydrophilic interactions were used to determine the 10 best docking positions of each LFM analog in the BTK catalytic site. The various docked positions of each LFM analog was qualitatively evaluated using Ludi (29, 30) in INSIGHTII (26) which was then used to
estimate a binding constant (Ki) for each compound
in order to rank their relative binding capabilities and predicted
inhibition of BTK. The Ki trends for the LFM analogs
were compared with the trend of the experimentally determined tyrosine
kinase inhibition IC50 values for the compounds, in order
to elucidate the structure-activity relationships determining the
potency of LFM analogs.
Recombinant Baculovirus Construction and Protein
Expression--
Sf21 (IPLB-SF21-AE) cells (20), derived from
the ovarian tissue of the fall armyworm Spodotera
frugiperda, were obtained from Invitrogen and maintained at
26-28 °C in Grace's insect cell medium supplemented with 10%
fetal bovine serum and 1.0% antibiotic/antimycotic (Life Technologies,
Inc.). Stock cells were maintained in suspension at 0.2-1.6 × 106/ml in 600-ml total culture volume in 1-liter Bellco
spinner flasks at 60-90 rpm. Cell viability was maintained at
95-100% as determined by trypan blue dye exclusion.
Recombinant baculovirus containing the murine BTK gene was
constructed as described (20). In brief, the gene encoding BTK was
excised from pBluescript SKII+ vector (Stratagene) by
digestion with BamHI, and this fragment was then ligated
into pFastBac1 (Life Technologies, Inc.). The resulting vector,
pFastBac1-BTK, was then used to generate the recombinant baculovirus by
site-specific transposition in Escherichia coli DH10Bac
cells (Life Technologies, Inc.) which harbor a baculovirus shuttle
vector (bacmid), bMON14272. The resulting recombinant bacmid DNA was
introduced into insect cells by transfection with the standard
liposome-mediated method using Cellfectin reagent (Life Technologies,
Inc.). Four days later, transfection supernatants were harvested for
subsequent plaque purification and analyzed as above. Kinase-dead BTK
was generated as described (20) and cloned into the baculovirus
expression vector as described above for wild-type BTK. Baculovirus
expression vectors for JAK1 and JAK3 kinases were constructed and
introduced into insect cells, as previously reported (31).
Immunoprecipitation of Recombinant Proteins from Insect
Cells--
Sf21 cells were infected with a baculovirus
expression vector for BTK, JAK1, or JAK3 as indicated in the figure
legends. Cells were harvested and lysed (10 mM Tris, pH
7.6, 100 mM NaCl, 1% Nonidet P-40, 10% glycerol, 50 mM NaF, 100 µM
Na3VO4, 50 µg/ml phenylmethylsulfonyl
fluoride, 10 µg/ml aprotonin, 10 µg/ml leupeptin), and the kinases
were immunoprecipitated from the lysates, as reported (20). Antibodies
used for immunoprecipitations from insect cells are as follows:
polyclonal rabbit anti-BTK serum (15), polyclonal rabbit anti-JAK1
(HR-785), catalog number sc-277, rabbit polyclonal IgG affinity
purified, 0.1 mg/ml, Santa Cruz Biotechnology, and polyclonal rabbit
anti-JAK3 (C-21, catalog number sc-513, rabbit polyclonal IgG affinity
purified, 0.2 mg/ml, Santa Cruz Biotechnology). Kinase assays were
performed following a 1-h exposure of the immunoprecipitated tyrosine
kinases to the test compounds, as described in detail elsewhere (15,
32). The immunoprecipitates were subjected to Western blot analysis as
described previously (20).
Cell Lines, Reagents, and Biochemical Assays--
The
establishment and characterization of DT40 lymphoma B cell line as well
as BTK-deficient DT40 and its derivatives reconstituted with wild-type
or mutant human BTK have been previously reported (32). Equal amounts
of BTK protein were detected by Western blot analysis in all of the
BTK-deficient DT40 clones transfected with wild-type or mutated human
BTK genes, but no BTK protein was detectable in the
untransfected BTK-deficient DT40 cells (32). All cell lines derived
from the chicken B cell line DT40 were maintained in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal bovine serum, 1%
heat-inactivated chicken serum, 2 mM glutamine, penicillin,
and streptomycin. Cells were grown at 37 °C in a 5% CO2
water-saturated atmosphere. The BTK positive human B-lineage leukemia
cell lines NALM-6 and ALL-1 were maintained in RPMI 1640 medium
supplemented with 10% heat-inactivated fetal bovine serum (33). COS-7
simian kidney cell line and HepG2 human hepatoma cell line were
obtained from ATCC.
Antibodies directed against BTK, JAK1, JAK3, and HCK have been
described previously (15, 20, 31, 32). Polyclonal antibodies to BTK
were generated by immunization of rabbits with glutathione S-transferase (GST) fusion proteins (Amersham Pharmacia
Biotech) containing the first 150 amino acids of BTK. The monoclonal
anti-Fas antibody (F22120) was obtained from the Transduction
Laboratories, Inc. (Lexington, KY). Immunoprecipitations, immune
complex protein kinase assays, and immunoblotting using the ECL
chemiluminescence detection system (Amersham Pharmacia Biotech) were
conducted as described previously (15, 20, 31, 32). Following
electrophoresis, kinase gels were dried onto Whatman 3M filter paper
and subjected to phosphorimaging on a Molecular Imager (Bio-Rad) as
well as autoradiography on film. Similarly, all chemiluminescent BTK
Western blots were subjected to three-dimensional densitometric
scanning using the Molecular Imager and Imaging Densitometer using the Molecular Analyst/Macintosh version 2.1 software following the specifications of the manufacturer (Bio-Rad). For each drug
concentration, a BTK kinase activity index was determined by
comparing the ratios of the kinase activity in PhosphorImager units
(PIU) and density of the protein bands in densitometric scanning units
(DSU) to those of the base-line sample and using the formula: activity index = [PIU of kinase band/DSU of BTK protein
band]test sample:[PIU of kinase band/DSU of BTK protein
band]baseline control sample. GST-IG
was
sometimes used as an exogenous substrate for BTK immune complex protein
kinase assays, as described (15). Horseradish peroxidase-conjugated
sheep anti-mouse, donkey anti-rabbit secondary antibodies and ECL
reagents were purchased from Amersham Pharmacia Biotech. For insulin
receptor kinase (IRK) assays, HepG2 human hepatoma cells grown to
approximately 80% confluency were washed once with serum-free
Dulbecco's modified Eagle's medium and starved for 3 h at
37 oC in a CO2 incubator. Subsequently, cells
were stimulated with insulin (Lilly, catalog number CP-410;10
units/ml/10 × 106 cells) for 10 min at room
temperature. Following this IRK activation step, cells were washed once
with serum-free medium and lysed in Nonidet P-40 buffer, and IRK was
immunoprecipitated from the lysates with an anti-IR
antibody (Santa
Cruz Biotechnology, catalog number sc-711, polyclonal IgG). Prior to
performing the immune complex kinase assays, the beads were
equilibrated with the kinase buffer (30 mM Hepes, pH 7.4, 30 mM NaCl, 8 mM MgCl2, 4 mM MnCl2).
For HCK kinase assays, we used HCK-transfected COS-7 cells.
The cloning and expression of HCK in COS-7 cells has been described previously (34). The pSV7c-HCK plasmid was transfected into 2 × 106 COS-7 cells using LipofectAMINE (Life Technologies,
Inc.), and the cells were harvested 48 h later. The cells were
lysed in Nonidet P-40 buffer, and HCK was immunoprecipitated from the
whole cell lysates with an anti-HCK antibody.
Apoptosis Assays--
To induce apoptosis, cells were treated
with an agonistic anti-Fas/APO-1 antibody (Bender MedSystems, Lot
number 04/1295) at 0.1 and 0.5 µg/ml final concentrations,
vincristine (vincristine sulfate, USP; Amersham Pharmacia Biotech, NDC
0013-7466-86, lot number VCB019) at 10 and 100 ng/ml final
concentrations, or C2-ceramide (Biomol, lot number M8107)
at 10, 50, and/or 100 µM final concentrations. MC540
binding (as an early marker of apoptosis) and PI permeability (as a
marker of advanced stage apoptosis) were simultaneously measured in
DT40 cells 24 h after exposure to C2-ceramide,
anti-Fas, or vincristine, as described previously (32). Whole cells
were analyzed using a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA). All analyses were done using 488-nm excitation from an
argon laser. MC540 and PI emissions were split with a 600-nm short pass
dichroic mirror, and a 575-nm band pass filter was placed in front of
one photomultiplier tube to measure MC540 emission and a 635-nm band
pass filter was used for PI emission. In order to examine the effects
of the lead BTK inhibitor on ceramide-induced apoptosis in B cell
antigen receptor-ABL positive human ALL cell line ALL-1, cells were
treated for 4 h at 37 °C with 10 µM
C2-ceramide in the presence or absence of the inhibitor
(200 µM LFM-A13). Subsequently, cells were
washed and stained with PI and MC540, and the apoptotic fractions were
determined by multiparameter flow cytometry, as described (32).
To detect apoptotic fragmentation of DNA, DT40 cells were harvested
24 h after exposure to anti-Fas, C2-ceramide, or
vincristine. Similarly, B18.2, NALM-6, and ALL-1 cells were treated
with LFM-A13 (100 µM), vincristine (VCR) (10 ng/ml), C2-ceramide (C2-CER) (10 µM), LFM-A13 (100 µM) + VCR (10 ng/ml), and LFM-A13 (100 µM) + C2-CER (10 µM) for 24 h at 37 °C. DNA
was prepared from Triton X-100 lysates for analysis of fragmentation
(32). In brief, cells were lysed in hypotonic 10 mmol/liter Tris-HCl, pH 7.4, 1 mmol/liter EDTA, 0.2% Triton X-100 detergent and
subsequently centrifuged at 11,000 × g. To detect
apoptosis-associated DNA fragmentation, supernatants were
electrophoresed on a 1.2% agarose gel, and the DNA fragments were
visualized by ultraviolet light after staining with ethidium bromide.
Chemical Synthesis--
All chemicals were purchased from
Aldrich and were used without further purification. Except where noted,
each reaction vessel was secured with a rubber septum, and the reaction
was performed under nitrogen atmosphere. 1H NMR spectrum
was obtained on a Varian Mercury 300 instrument spectrometer (Palo
Alto, CA) at ambient temperature in the solvent specified. Melting
points were determined using a Fisher-Johns melting point apparatus and
are uncorrected. Fourier-Transform-Infrared spectra were recorded on a
Nicolet Protege 460 spectrometer (Madison, WI). Gas chromatography/mass
spectroscopy (MS) was obtained on a HP 6890 GC System (Palo Alto, CA)
equipped with a HP 5973 Mass Selective Detector. EI and CI MS data were
obtained at the University of Minnesota MS Core Facility.
Synthesis of LFM,
LFM-A1-LFM-A14--
Scheme 1 shows the
general synthetic scheme for the preparations of LFM, and
LFM-A1-LFM-A14 (35, 36). Cyanoacetic acid
1 was coupled with the desired substituted-aniline 2 in the presence of diisopropylcarbodiimide to form
3. Compound 3 was treated with NaH and then
acylated with acetyl chloride to afford the final products
LFM and LFM-A1-LFM-A14 (Scheme 1).
General Synthetic Procedures (35,
36)--
1,3-Diisopropylcarbodiimide (1.75 g; 13.9 mmol) was added to
a solution of cyanoacetic acid 1 (1.70 g; 20.0 mmol) and the
desired substituted aniline 2 (12.6 mmol) in tetrahydrofuran (25 ml) at 0 °C. The mixture was stirred for 12 h at room
temperature. The urea precipitate (reaction side product) was removed
by filtration and the filtrate partitioned between ethyl acetate and
0.5 N HCl. The organic layer (a solution of tetrahydrofuran
and ethyl acetate) was sequentially washed with brine twice, dried over
anhydrous Na2SO4, and concentrated by rotary
evaporation. Finally, the crude solid product was recrystallized from
ethyl alcohol to give pure 3. Sodium hydride (0.93 g; 60%
in mineral oil; 23.2 mmol) was added slowly to the solution of
3 in tetrahydrofuran (12 mmol in 40 ml) at 0 °C. After
stirring for 30 min at 0 °C, acetyl chloride (1.04 g; 13.2 mmol) was
added to the reaction mixture. The reaction was continued for another
hour and then was quenched by the addition of acetic acid (2 ml). The
mixture was poured into ice water (100 ml) containing 2.5 ml of
hydrochloric acid to precipitate the crude product, which was collected
by filtration and washed with water. The final pure product was
obtained by recrystallization.
The physical data for
-cyano-
-hydroxy-
-methyl-N-[4-trifluoromethyl)phenyl]propenamide
(LFM) is as follows: mp, 230-233 °C; IR (KBr), 3303, 2218, 1600, and 1555 cm
1; 1H NMR
(Me2SO-d6),
11.01 (s, 1H, NH),
7.75 (d, J = 8.4 Hz, 2H, ArH), 7.64 (d,
J = 8.4 Hz, 2H, ArH), 2.22 (s, 3H, CH3);
GC/MS m/z 270 (M+), 161, 142, 111.
The physical data for
-cyano-
-hydroxy-
-methyl-N-(4-bromophenyl)propenamide
(LFM-A1) is as follows: mp, 213-214 °C; IR (KBr), 3288, 2228, 1615, 1555 cm
1; 1H NMR
(Me2SO-d6),
10.51 (s, 1H, NH),
7.49 (s, 4H, ArH), 2.25 (s, 3H, CH3); MS (EI)
m/z 282 (M+ + 2), 280 (M+), 173, 171.
The physical data for
-cyano-
-hydroxy-
-methyl-N-(4-chlorophenyl)propenamide
(LFM-A2) is as follows: mp, 209-211 °C; IR (KBr), 3298, 2223, 1598, and 1552 cm
1; 1H NMR
(Me2SO-d6),
10.48 (s, 1H, NH),
7.54 (d, J = 8.7 Hz, 2H, ArH), 7.45 (s br, 1H, OH),
7.36 (d, J = 8.7 Hz, 2H, ArH), 2.25 (s, 3H,
CH3); MS (CI) m/z 236 (M+), 129, 127.
The physical data for
-cyano-
-hydroxy-
-methyl-N-(4-fluorophenyl)propenamide
(LFM-A3) is as follows: mp, 165-166 °C; IR (KBr), 3298, 2218, 1610, and 1560 cm
1; 1H NMR
(Me2SO-d6),
10.33 (s, 1H, NH),
7.80 (s br, 1H, OH), 7.53 (m, 2H, ArH), 7.16 (m, 2H, ArH), 2.26 (s, 3H,
CH3); GC/MS m/z 220 (M+), 111.
The physical data for
-cyano-
-hydroxy-
-methyl-N-[2-(trifluoromethyl)phenyl]propenamide
(LFM-A4) is as follows: mp, 61-63 °C; IR (KBr), 3435, 2209, 1619, 1952 and 1548 cm
1; 1H NMR
(Me2SO-d6),
10.99 (s, 1H, NH),
8.03 (d, J = 7.5 Hz, 1H, ArH), 7.67 (d,
J = 7.5 Hz, 1H, ArH), 7.60 (dd, J = 7.5, 7.5 Hz, 1H, ArH), 7.29 (dd, J = 7.5, 7.5 Hz, 1H,
ArH) 5.71 (s br, 1H, OH), 2.20 (s, 3H, CH3); GC/MS
m/z 270 (M+), 161, 141, 114.
The physical data for
-cyano-
-hydroxy-
-methyl-N-(2-bromophenyl)propenamide
(LFM-A5) is as follows: mp, 98-100 °C; IR (KBr), 3351, 2214, 1609, 1585, and 1536 cm
1; 1H NMR
(Me2SO-d6),
10.76 (s, 1H, NH),
8.06 (dd, J = 8.1, 1.5 Hz, 1H, ArH), 7.62 (dd,
J = 8.1, 1.5 Hz, 1H, ArH), 7.33 (m, 1H, ArH), 7.03 (m,
1H, ArH), 6.60 (s br, 1H, OH), 2.22 (s, 3H, CH3); MS (EI)
m/z 282 (M+ + 2), 280 (M+), 173, 171.
The physical data for
-cyano-
-hydroxy-
-methyl-N-(2-chlorophenyl)propenamide
(LFM-A6) is as follows: mp, 93-94 °C; IR (KBr), 3372, 2208, 1644, 1621, and 1587 cm
1; 1H NMR
(Me2SO-d6),
10.96 (s, 1H, NH),
8.16 (d, J = 8.1 Hz, 1H, ArH), 7.46 (dd,
J = 7.5, 1.5 Hz, 1H, ArH), 7.29 (m, 1H, ArH), 7.08 (m,
1H, ArH), 2.22 (s, 3H, CH3); MS (CI) m/z 236 (M+), 129, 127.
The physical data for
-cyano-
-hydroxy-
-methyl-N-(2-fluorophenyl)propenamide
(LFM-A7) is as follows: mp, 118-119 °C; IR (KBr), 3409, 2212, 1613, 1591, and 1532 cm
1; 1H NMR
(Me2SO-d6),
10.70 (s, 1H, NH),
7.91 (m, 1H, ArH), 7.23 (m, 1H, ArH), 7.13 (m, 2H, ArH), 7.10 (s br,
1H, OH), 2.22 (s, 3H, CH3); GC/MS m/z
[M]+, 220.
The physical data for
-cyano-
-hydroxy-
-methyl-N-[3-(trifluoromethyl)phenyl]propenamide
(LFM-A8) is as follows: mp, 182-184 °C; IR (KBr), 3303, 2221, 1619, and 1572 cm
1; 1H NMR
(Me2SO-d6),
10.79 (s, 1H, NH),
8.05 (s br, 1H, OH) 8.04 (s, 1H, ArH), 7.75 (d, J = 8.1 Hz, 1H, ArH), 7.53 (dd, J = 8.1, 7.5 Hz, 1H, ArH), 7.42 (d, J = 7.5 Hz, 1H, ArH), 2.24 (s, 3H, CH3); GC/MS m/z 270 (M+), 161.
The physical data for
-cyano-
-hydroxy-
-methyl-N-(3-bromophenyl)propenamide
(LFM-A9) is as follows: mp, 184-185 °C; IR (KBr), 3303, 2228, 1610, 1595, and 1550 cm
1; 1H NMR
(Me2SO-d6),
10.56 (s, 1H, NH),
7.89 (m, 1H, ArH), 7.47 (m, 1H, ArH), 7.28 (m, 2H, ArH), 6.37 (s br,
1H, OH), 2.26 (s, 3H, CH3); MS (EI) m/z 282 (M+ + H, 81Br), 280 (M+ + H,
79Br), 173, 171.
The physical data for
-cyano-
-hydroxy-
-methyl-N-(3-chlorophenyl)propenamide
(LFM-A10) is as follows: mp, 184-187 °C; IR (KBr), 3293, 2221, 1610, 1595, and 1557 cm
1; 1H NMR
(Me2SO-d6),
10.61 (s, 1H, NH),
7.76 (m, 1H, ArH), 7.42 (m, 1H, ArH), 7.33 (m, 1H, ArH), 7.16 (m, 1H,
ArH), 2.25 (s, 3H, CH3); MS (CI) m/z
[M]+, 236.
The physical data for
-cyano-
-hydroxy-
-methyl-N-(3-fluorophenyl)propenamide
(LFM-A11) is as follows: mp, 136-138 °C; IR (KBr), 3297, 2221, 1613, 1597, and 1567 cm
1; 1H NMR
(Me2SO-d6),
10.54 (s, 1H, NH),
7.54 (m, 1H, ArH), 7.33 (m, 2H, ArH), 6.93 (m, 1H, ArH), 2.27 (s, 3H,
CH3); GC/MS m/z 220 (M+), 111.
The physical data for
-cyano-
-hydroxy-
-methyl-N-[4-(trifluoromethoxy)phenyl]propenamide
(LFM-A12) is as follows: mp, 182-183 °C; IR (KBr), 3308, 2213, 1625, and 1580 cm
1; 1H NMR
(Me2SO-d6),
10.57 (s, 1H, NH),
7.90 (s br, 1H, OH), 7.64 (d, J = 8.7 Hz, 2H, ArH),
7.32 (d, J = 8.7 Hz, 2H, ArH), 2.25 (s, 3H,
CH3); GC/MS m/z 286 (M+), 177, 108.
The physical data for
-cyano-
-hydroxy-
-methyl-N-(2,5-dibromophenyl)propenamide
(LFM-A13) is as follows: mp, 148-150 °C; IR (KBr), 3353, 2211, 1648, and 1590 cm
1; 1H NMR
(Me2SO-d6),
11.41 (s, 1H, NH),
8.57 (d, J = 2.4 Hz, 1H, ArH), 7.55 (d,
J = 8.7 Hz, 1H, ArH), 7.14 (dd, J = 8.7, 2.4 Hz, 1H, ArH), 7.10 (s br, 1H, OH), 2.17 (s, 3H,
CH3); MS (EI) m/z 362 (M+ + 4), 360 (M+ + 2), 358 (M+), 253, 251, 249, 150.
The physical data for
-cyano-
-hydroxy-
-methyl-N-(phenyl)propenamide
(LFM-A14) is as follows: mp, 134-135 °C; IR (KBr), 3281, 2214, 1605, 1579, and 1554 cm
1; 1H NMR
(Me2SO-d6),
10.33 (s, 1H, NH),
7.51 (d, J = 7.5 Hz, 2H, ArH), 7.40 (s br, 1H, OH),
7.31 (dd, J = 7.5, 7.5 Hz, 2H, ArH), 7.11 (m, 1H, ArH),
2.26 (s, 3H, CH3); GC/MS m/z 202 (M+), 93.
 |
RESULTS AND DISCUSSION |
BTK Is an Anti-apoptotic Enzyme--
We first evaluated the
anti-apoptotic activity of BTK by comparing the effects of the
apoptosis-inducing agents C2-ceramide, vincristine, and
anti-Fas monoclonal antibody on wild-type DT40 chicken B lymphoma cells
to those on a BTK-deficient subclone of DT40 cells that was established
by homologous recombination knock-out (32). Ceramide, the product of
ceramide synthase and sphingomyelinase, has been shown to function as a
second messenger that transmits membrane-induced apoptotic signals,
including the Fas-mediated and tumor necrosis factor receptor-mediated
death signals, to downstream effectors (37). The use of vincristine, a
commonly used anti-leukemia/lymphoma drug, results in a
time-dependent accumulation of ceramide in treated cells
which leads to apoptosis. On agarose gels, DNA from Triton X-100
lysates of anti-Fas-treated BTK-deficient DT40 cells showed a
ladder-like fragmentation pattern consistent with apoptosis, whereas no
DNA fragmentation was observed in wild-type DT40 cells (Fig.
1A, lane 7 versus lane 14). Thus, the anti-Fas antibody
treatment caused apoptosis in BTK-deficient DT40 cells but not in
wild-type DT40 cells consistent with our recently reported observations
that BTK is an inhibitor of the Fas-associated death-inducing signaling
complex (20). Notably, treatment of BTK-deficient DT40 cells with
C2-ceramide (N-acetylsphingosine; a synthetic
cell-permeable ceramide analog) or vincristine was able to recapitulate
the effects of anti-Fas in inducing oligonucleosomal DNA fragmentation
on agarose gel electrophoresis, whereas wild-type DT40 cells were
resistant to both agents, confirming the function of BTK as a negative
regulator of apoptosis (Fig. 1A).

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Fig. 1.
The anti-apoptotic function of BTK.
Wild-type and BTK-deficient (BTK ) DT40 lymphoma B cells
(A) as well as BTK DT40 cells reconstituted
with wild-type or mutant human BTK (B) were treated with
C2-ceramide (C2-CER), vincristine
(VCR), or anti-Fas antibody, as described under
"Experimental Procedures." BTK-deficient DT40
(BTK ) cells expressing wild-type BTK,
BTK(Arg525 Gln), BTK(Arg28 Cys), and
BTK(Arg307 Ala) were designated as
BTK ,rBTK(WT), BTK ,rBTK(K ),
BTK ,rBTK(mPH) and BTK ,rBTK(mSH2),
respectively. Vehicle (0.1% Me2SO in phosphate-buffered
saline) treated as well as drug-treated cells were maintained in
culture medium for 24 h at 37 °C and 5% CO2 before
harvesting. DNA from Triton X-100 lysates was analyzed for
fragmentation, as described (32). bp, base pairs;
WT, wild type; M, size markers.
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In order to examine the participation of the various domains of BTK in
its anti-apoptotic function, we introduced wild-type human
BTK gene as well as human BTK genes harboring
mutations either in the catalytic domain (Arg525 to Gln),
SH2 domain (Arg307 to Ala), or PH domain (Arg28
to Cys) into the BTK-deficient DT40 cells (37). As evidenced in Fig.
1B, BTK-deficient DT40 cells reconstituted with wild-type human BTK gene (WT) did not undergo apoptosis
after treatment with C2-ceramide (lanes 2-4) or
vincristine (lanes 8-10), whereas DT40 subclones expressing
human BTK with mutations in the kinase (K
)
(lanes 5-7 and 11-13), SH2 (mSH2)
(lanes 15-17 and 21-23), or PH (mPH)
domains (lanes 18-20 and 24-26) did. Thus, the
kinase, SH2, and PH domains of BTK are all important and apparently
indispensable for its anti-apoptotic function.
Homology Model of BTK Kinase Domain--
The three-dimensional
coordinates of BTK used in the protein/inhibitor modeling studies were
constructed based on a structural alignment with the sequences of known
crystal structures for four protein kinase domains (kinase domains of
HCK (22), FGFR (23), IRK (24), and cAPK (25)), as detailed under
"Experimental Procedures." The modeled BTK kinase domain (Fig.
2A) has the expected protein
kinase fold with the catalytic site in the center dividing the kinase
domain into two lobes. It is composed of a smaller N-terminal lobe
connected by a flexible hinge to a larger C-terminal lobe. The
N-terminal lobe is rich in
-strands, whereas the C-terminal region
is mostly helical. The catalytic site is defined by two
-sheets that
form an interface at the cleft between the two lobes. It is in this
catalytic region where small molecule inhibitors can bind. Our modeling
studies revealed that the catalytic site of the BTK kinase domain is
composed of a distinct planar rectangular binding pocket near the hinge
region. The rectangular binding region is defined by residues
Leu460, Tyr476, Arg525, and
Asp539 which occupy the corners of the rectangle. The
dimensions of this rectangle are approximately 18 × 8 × 9 × 17 Å, and the thickness of the pocket is approximately 7Å
(Fig. 2B). The far left corner of the rectangle
can be visualized as beginning close to the hinge region at
Leu460 (shown in yellow, Fig. 2B) and
extending 8 Å toward the upper right to Asp539 (shown in
blue, Fig. 2B). This is the shortest side of the
binding pocket and is located closer to the inner core of the protein. The left side of the pocket, which is the longest, extends from Leu460 and traces 18 Å along the hinge region up to
Tyr476 (shown in green, Fig. 2B). The
right side of the rectangular pocket, opposite to the hinge
region, extends about 9 Å from Asp539 to
Arg525 (shown in pink, Fig. 2B),
which is immediately adjacent to the binding subsites for the sugar and
triphosphate groups of ATP. The hinge region of the binding site is
composed of residues 472-481. The solvent-exposed or fourth side of
the rectangle extends 17 Å along the slot-shaped opening to the
catalytic site from Tyr476 to Arg525. The
binding pocket is wider at the solvent-accessible region, it narrows
toward the innermost region of the binding site, and overall it is
relatively shallow with a thickness of about 7 Å.

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Fig. 2.
A, ribbon representation of the homology
model of the BTK kinase domain. The LFM-A13 molecule is
shown as a space filling model in the catalytic site of BTK. Prepared
using Molscript and Raster3D programs (38-40). B, space
filling representation of the backbone of the catalytic site residues
of the BTK kinase domain. The C- chain of BTK is represented as a
blue ribbon. Shown in yellow, green, pink, and
blue are the residues at the four corners of the
rectangular-shaped binding pocket (other residues in the
cavity are shown in gray). A ball and stick model
of the BTK inhibitor LFM-A13 is shown in
multicolor and represents the favorable orientation of this
molecule in the kinase active site of BTK. Prepared using InsightII
program (26).
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Although most of the catalytic site residues of the BTK kinase domain
were conserved relative to other tyrosine kinases, a few specific
variations were observed. Residues Asn526 and
Asp539 (opposite the hinge) are conserved in EGFR, IRK,
HCK, and BTK. Residue Thr474 in the hinge region changes to
Met in IRK, JAK1, and JAK3 and residue Tyr476 in the hinge
region changes to Leu in EGFR and IRK. Residue Ser538
changes to Gly in JAK1 and IRK, to Thr in EGFR, and to Ala in FGFR,
JAK3, and HCK. One region of binding site contains Cys481
in BTK which is more hydrophobic than the corresponding residue of PDGF
receptor (Asp), FGFR (Asn), and IRK (Asp). These residue identity
differences may provide the basis for designing selective inhibitors of
the BTK kinase domain.
Structure-based Design and Synthesis of LFM Analogs with Potent
BTK-inhibitory Activity--
In modeling studies aimed at identifying
LFM analogs with a high likelihood to bind favorably to the catalytic
site of the kinase domain of BTK, we chose to evaluate the estimated
Ki values that quantitate predicted binding
interactions between the inhibitor and residues in the catalytic site
of BTK. Each of the small molecule LFM analogs was individually modeled
into the catalytic site of the BTK kinase domain using an advanced docking procedure (see "Experimental Procedures"). The position of
quercetin in the HCK crystal structure (22) was used as a template to
obtain a reasonable starting point for the docking procedure. The
various docked positions of each LFM analog were qualitatively
evaluated using a scoring procedure and consequently compared with the
IC50 values of the compounds in cell-free BTK inhibition
assays. Table I shows the interaction
scores, calculated Ki values, and measured
IC50 values for LFM and its analogs with
BTK.
The inhibitors in our modeling studies were sandwiched by two regions
of mostly hydrophobic residues. The region above the docked inhibitor
consisted of residues Leu408, Val416, and
Lys430, and the residues below the docked inhibitor
included BTK residues Leu528, Ser538,
Gly480, and Cys481. Of all the reported
compounds evaluated in our modeling studies (Table I), we predicted
that compound LFM-A13 would provide the strongest binding to
BTK. The positions of the critical residues in the active site of the
BTK and the docked position of the lead compound LFM-A13 is
shown in Fig. 3. Of all the possible orientations of this molecule bound to the catalytic site, the one
shown in Fig. 3 showed the highest interaction score with BTK. This
high interaction score is indicative of an energetically favored
binding mode, with a correspondingly low calculated
Ki value of 1.4 µM. This binding
position of LFM-A13 is such that the aromatic ring of the
inhibitor faces the Tyr476 residue and the flexible side
chain extends toward the Asp539 and Arg525
residues. The aromatic ring is also sandwiched between the hydrophobic residues Leu408 and Val416 above and
Gly480 and Leu528 below. Residue
Ser538 lies below the flexible side chain of the inhibitor,
and the end of the side chain is located between residues
Asp539 and Arg525. This position closely
resembles that of the ATP analog position found in the IRK complex
crystal structure (24). According to our modeling studies, the O-3 atom
in the hydroxyl group of LFM-A13 would form a hydrogen bond
with Asp539:O and its O-4 atom would form a hydrogen bond
with Arg525:N.

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Fig. 3.
Docked position of the LFM-A13 molecule
(multicolor) at the catalytic site (blue
ribbon) of the kinase domain of BTK. Dashed lines
represent hydrogen bonds between LFM-A13 and the kinase
domain residues of BTK.
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Fig. 4 shows the superimposed docked
positions of our lead compound LFM-A13 in the catalytic site
of BTK, together with the control compounds LFM and
LFM-A12. The molecules LFM and LFM-A12
are docked such that they lie along the hinge region, corresponding to
the quercetin position in the HCK crystal structure. The aromatic ring
of these molecules is close to Tyr476, and the end of the
side chain is sandwiched between residues Asp539 and
Thr474. The CF3 group in these molecules points
toward the solvent-accessible region and is surrounded by
Leu408 above and Gly480 below. The OH group of
LFM is hydrogen-bonded to an oxygen atom of
Asp539, and for LFM-A12, the same group is
hydrogen-bonded to an oxygen atom of Thr474. All LFM
analogs listed in Table I, except LFM-A13, lie along the
hinge region like LFM or LFM-A12, and their side
chains are sandwiched between Asp539 and
Thr474. A comparison of the docked positions of
LFM, LFM-A12, and LFM-A13 in the BTK
active site shows that although the aromatic portion of the three
molecules are roughly in the same region (which is also true for the
other inactive LFM analogs), the side chain of LFM-A13 is
tilted away from those of the others and is sandwiched between residues
Asp539 and Arg525. This rotation is likely due
to a more favorable orientation of the 2,5-dibromo groups of
LFM-A13. This slightly tilted position and the larger
bromine groups afford two advantages for the interaction of
LFM-A13 with the active site residues of BTK. The first
advantage is that LFM-A13 is able to form two hydrogen bonds
with active site residues Asp539 and Arg525,
whereas the inactive LFM analogs form only one hydrogen bond
each with the Thr474 or Asp539 of BTK. The
second advantage for binding is the higher contact area of
LFM-A13 with active site residues of BTK, relative to the
other 12 inactive LFM analogs, which leads to a greater
hydrophobic interaction for LFM-A13. This feature is
reflected by the correspondingly higher lipophilic score for LFM-A13 in Table I.

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Fig. 4.
Superimposed docked positions of LFM
(purple), LFM-A12 (red), and LFM-A13
(multicolor) in the catalytic site of the kinase
domain of BTK.
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The results from the modeling studies prompted the hypothesis that
LFM-A13 would exhibit potent BTK inhibitory activity. In
order to test this hypothesis and validate the predictive value of the
described BTK homology model, we synthesized LFM-A13, LFM, and 13 other LFM analogs listed in Table I. The
structures of LFM, LFM-A12, and
LFM-A13 were determined by single crystal x-ray diffraction
(crystal data, experimental parameters, and refinement statistics for
these compounds are summarized in Table
II). All structures were found to have a
planar conformation, and all bond lengths and angles were in the
expected range. Fig. 5 shows an Oak Ridge
Thermal Ellipsoid Plot representation of the lead compound
LFM-A13. The crystal structure of LFM-A13 showed
that its molecular conformation was very similar to the
energy-minimized molecular coordinates that were generated and used for
docking studies with BTK. This conformational similarity with the
crystal structures indicated that the molecular models used for docking
were appropriate for modeling studies.

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Fig. 5.
Oak Ridge Thermal Ellipsoid Plot
picture of the crystal structure of the lead BTK
inhibitor, LFM-A13.
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Specific Inhibition of BTK by LFM-A13--
We first used
cell-free immune complex kinase assays to compare the effects of
LFM and 12 LFM analogs on the enzymatic activity of human
BTK immunoprecipitated from B18-2 cells (32) (i.e.
BTK-deficient DT40 chicken lymphoma B cells reconstituted with
wild-type human BTK gene). As shown in Table I, only
LFM-A13 exhibited significant BTK inhibitory activity with
an IC50 value of 6.2 ± 0.3 µg/ml (= 17.2 ± 0.8 µM). None of the other compounds inhibited BTK even
at concentrations as high as 100 µg/ml (i.e. at a range of
349 µM for LFM-A12 to 495 µM for
LFM-A14). LFM-A13 was also effective against
recombinant BTK expressed in a baculovirus vector expression system
with an IC50 value of 0.9 µg/ml (~2.5 µM,
Fig. 6A), as well as BTK
immunoprecipitated from NALM-6 human B-lineage ALL cell lysates (Fig.
6B). Furthermore, treatment of B18.2 cells (Fig.
6C) or NALM-6 cells (data not shown) with LFM-A13
resulted in a dose-dependent inhibition of cellular BTK
activity. The inhibitory activity of LFM-A13 against BTK was
specific since it did not affect the enzymatic activity of other
protein tyrosine kinases, including Janus kinases JAK1 and JAK2, Src
family kinase HCK, and receptor family tyrosine kinase IRK, at
concentrations as high as 100 µg/ml (~278 µM; Table III and Fig.
7).

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Fig. 6.
Effects of LFM-A13 on the tyrosine kinase
activity of BTK. A, a highly purified (>90%)
preparation of BTK produced in a baculovirus vector expression
system was treated for 1 h at room temperature with
LFM-A13 at the indicated concentrations. The enzymatic
activity of BTK was determined by measuring autophosphorylation in a
10-min kinase assay, as described under "Experimental
Procedures." B, BTK was immunoprecipitated from B18.2
cells (i.e. BTK DT40 cells reconstituted with
wild-type human BTK), treated with LFM-A13 or vehicle (0.1%
Me2SO in phosphate-buffered saline) for 1 h, and then
assayed for PTK activity, as measured by autophosphorylation as well as
phosphorylation of GST-Ig , which was used an exogenous kinase
substrate. C, B18.2 lymphoma B cells were treated with
LFM-A13, then lysed, and BTK immune complex kinase assays
and Western blots were performed as described under "Experimental
Procedures." CON, control.
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Table III
Interaction scores, calculated Ki values, and measured
IC50 values for LFM-13 with several different PTKs
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Fig. 7.
Effects of LFM-A13 on the tyrosine kinase
activity of JAK1, JAK3, HCK, and IRK. JAK1 and JAK3
immunoprecipitated from Sf21 insect ovary cells transfected with
the appropriate baculovirus expression vectors, HCK immunoprecipitated
from COS-7 cells transfected with the pSV7c-HCK plasmid, and IRK
immunoprecipitated from HepG2 hepatoma cells were treated with
LFM-A13, then subjected to in vitro kinase assays
as described under "Experimental Procedures."
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Structural Basis for the BTK Specificity of
LFM-A13--
Biological assays have shown LFM-A13 to
be a selective inhibitor of BTK, whereas it is a poor inhibitor of
EGFR, HCK, JAK1, JAK3, and IRK. To evaluate this selectivity, we
constructed a homology model of EGFR, JAK1, and JAK3 using homologous
crystal structure coordinates of protein kinases IRK (24), HCK (22), and cAMP-dependent protein kinase (25) as a template. The models were
then used to study the binding of small molecules such as LFM-A13 into the catalytic sites of these kinases and to understand better how LFM-A13 can inhibit BTK but not EGFR, IRK, JAK1, JAK3, or HCK. Our preliminary studies have identified three
factors that may contribute to the specificity of LFM-A13 for BTK. The small molecule LFM-A13 was docked into the kinase domains of IRK, HCK, JAK3, and EGFR. Table III shows the interaction scores, calculated Ki values, and
measured IC50 data for LFM-A13 with these
kinases. We postulate that the selectivity of LFM-A13 for
BTK results from favorable interactions with the BTK catalytic site
residues that are not present in the other kinases studied. There are
some residues in the BTK active site that differ from those of other
PTKs. These differences are illustrated in Fig.
8 which shows the backbone of the BTK
catalytic site, the residue differences between BTK and other kinases,
and the docked positions of LFM-A13 in the kinase domains of
BTK, HCK, JAK3, JAK1, EGFR, and IRK. We propose that the residue
differences shown at positions A, B, and C in
Fig. 8 may contribute to the specificity of LFM-A13 for BTK.
Kinases that are not inhibited by LFM-A13, such as IRK
(dark blue) and JAK1/JAK3 (pink), contain a
methionine residue at position A which protrudes into the
active site and prevents the close contact of small molecules like
LFM-A13 with the hinge region of the binding site. As a
result, LFM-A13 can lose favorable hydrophobic contact with
the hinge region of the kinase domains of these proteins and does not
bind to it tightly. Moreover, docking studies indicated that the
favorable position of LFM-A13 in the BTK kinase domain is
such that the side chain of the small molecule is located between
residues Asp539 and Arg525 and forms hydrogen
bonds with them. In addition, the aromatic residue, Tyr476,
at position B of BTK increases the hydrophobic interaction
between LFM-A13 and BTK, an interaction that is lost in EGFR
(red, Fig. 8) and IRK (dark blue, Fig. 8). This
is reflected by the lipophilic (hydrophobic interaction) scores shown
in Table III. While the Lipo scores ranged between 457 and 473 for
other kinases, the Lipo score for BTK was higher (more favorable) at
517. Finally, the Arg525 residue at position C
of BTK can hydrogen-bond to LFM-A13. This interaction is
lost in HCK (yellow), which contains an Ala at the
C position. The favorable position of LFM-A13 at the HCK kinase domain (shown in yellow) is such that the
small molecule is aligned along the hinge region. At this position
LFM-A13 does not form hydrogen bonds with HCK, which is not
the case for BTK. The longer side chain of Arg525 in BTK
(position C) is involved in hydrogen bonding with
LFM-A13, whereas HCK has an Ala at this position which is
not able to form the same hydrogen bond.

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Fig. 8.
Structural basis for the selectivity of
LFM-A13 for BTK. Shown in light blue is a trace of the
BTK homology model with selected residues at positions A, B,
and C, together with the docked position of the leflunomide
metabolite analog LFM-A13 (multicolor).
Shown in red is the docked position of
LFM-A13 with a model of EGFR and the residue difference
between EGFR and BTK at position B. Shown in
yellow is the docked position of LFM-A13 with the
crystal structure of HCK(22) and the residue difference between HCK and
BTK at position C. Shown in pink is the docked
position of LFM-A13 with models of JAK3/JAK1 and the residue
difference between JAK3/JAK1 and BTK at position A. Shown in
dark blue is the docked position of LFM-A13 with
the crystal structure of IRK(24) and the residue differences between
IRK and BTK at positions A and B.
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LFM-A13 Enhances the Sensitivity of B-lineage Acute
Lymphoblastic Leukemia (ALL) Cells to Ceramide- and Vincristine-induced
Apoptosis--
Patients with Philadelphia Chromosome (Ph+) ALL have a
dismal outcome after intensive multimodality treatment programs. The treatment failure of these patients could be overcome if the apoptotic threshold of their leukemic cells could be decreased. We set out to
determine if LFM-A13, by means of inhibiting the
anti-apoptotic tyrosine kinase BTK, could alter the sensitivity of the
Ph+ ALL cell line ALL-1 to C2-ceramide. As shown in Fig.
9, treatment with LFM-A13
significantly enhanced the chemosensitivity of ALL-1 cells to
ceramide-induced apoptosis, as evidenced by a greater percentage of
cells treated with LFM-A13 plus C2-ceramide, as
compared with cells treated with C2-ceramide alone or
LFM-A13 alone, showing dual PI/MC540 fluorescence (shown in
blue) consistent with advanced stage apoptosis. Furthermore, on agarose gels, DNA from Triton X-100 lysates of B18.2 chicken lymphoma B cells (i.e. BTK-deficient DT40 cells
reconstituted with wild-type human BTK gene; see also Fig. 1), NALM-6
human pre-B ALL cells, and ALL-1 cells showed a ladder-like
fragmentation pattern consistent with apoptosis after treatment with
LFM-A13 plus ceramide or LFM-A13 plus
vincristine, which was more pronounced than after treatment with
LFM-A13, ceramide, or vincristine alone (Fig.
10). These results demonstrated that LFM-A13 enhances the sensitivity of B-lineage
leukemia/lymphoma cells to both ceramide-induced and
vincristine-induced apoptosis. LFM-A13 could also have
clinical utility as a B-cell inhibitory agent in xenotransplantation
and treatment of autoimmune diseases.

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Fig. 9.
Effects of LFM-A13 on ceramide sensitivity of
human leukemia cells. Fluorescence-activated cell
sorter-correlated three-parameter (FSC, forward scatter
size; fluorescence from PI, propidium iodide, and fluorescence from
MC540 staining) displays of ALL-1 Ph/t(9;22)+ human ALL
cells stained with MC540 and PI 24 h after treatment with vehicle
(0.1% Me2SO in phosphate-buffered saline),
C2-ceramide (C2-CER) (10 µM),
LFM-A13 (200 µM), or LFM-A13 + C2-CER. The percentages indicate the fraction of cells at an early
stage of apoptosis, as measured by single MC540 fluorescence, and the
fraction of cells at an advanced stage apoptosis, as measured by dual
MC540/PI fluorescence.
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Fig. 10.
Chemosensitizing effects of LFM-A13.
BTK-deficient DT40 cells reconstituted with wild-type human
BTK gene (i.e. B18.2 clone) (A),
NALM-6 human pre-B ALL cells (B), and ALL-1 human
Ph+ ALL cells (C) were treated with
LFM-A13 (100 µM), vincristine (VCR)
(10 ng/ml), C2-ceramide (C2-CER) (10 µM), LFM-A13 (100 µM) + vincristine (10 ng/ml), LFM-A13 (100 µM) + C2-ceramide (10 µM) for 24 h at
37 °C. DNA from Triton X-100 lysates was analyzed for fragmentation,
as described (32). bp, base pairs.
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