Supplemental Materials & Methods
Compounds and reagents
NLE was prepared using the dried leaf powder from the capsules (Nature’s Way,
Springville, UT) and was made to a final concentration of 12.5% (w/v) in absolute alcohol
by incubating the leaf powder in ethanol overnight and collecting the supernatant after
centrifugation at 14,000 x g for 10 minutes, followed by filtration. Nimbolide, Bortezomib
and Ibrutinib (PCI-32765) were purchased from Biovision (Mountain View, CA, USA).
RPMI, penicillin, streptomycin, Tetramethylrhodamine, methyl ester (TMRM) and FBS
were purchased from Life technologies. Antibodies for Bcl, Mcl-1, Akt, p-Akt, Hsp-90,
Hsp-70, BTK, p-BTK, p53, p-PTEN, ERK, p-ERK, p73 and p65 were purchased from
Santa Cruz biotechnology (Dallas, TX). Antibodies for caspases 3, 9 and PARP-1 were
obtained from cell signaling technology (Danvers, MA).
Annexin V/propidium iodide
apoptosis staining kit was purchased from BD biosciences (San Jose, CA).
Viability assay
An MTS assay was performed wherein 20,000 cells/200L in quadruplicates were
incubated in 96 well plates with serially diluted bortezomib (0 – 1μM) and nimbolide
(0.05μM) at 37oC for 72h. MTS reagent (Molecular Probes) was added at 40 l/well, and
the plates were further incubated at 37oC for 3h. The color that developed was read at
490nm using BioTek synergy HT plate reader against blanks with no cells.
Annexin – V staining apoptosis assay
Apoptosis was measured by the annexin V binding assay kit from Pharmingen (San
Diego, CA) according to the manufacturer’s instructions and as previously described.1
Briefly, cells were washed with PBS and 1x106 cells were resuspended in 100μL of 1x
binding buffer. FITC-labeled Annexin V (5 µL) and propidium iodide [PI] (10μl) were
added to each sample and incubated in the dark for 15 minutes at room temperature.
Subsequently cells were analyzed by on BD accuri C6flow cytometry. Data from 10000
events per sample were collected and processed using FCS express software (DeNovo
Software).
Apoptosis ELISA
1
Induction of apoptosis was also determined by ELISA using a cell death ELISA detection
kit from Roche (cat# 11774425001). Briefly, 20,000 cells from control and drug treated
cell cultures were lysed in 200μl of lysis buffer and incubated at room temperature for
15min. The lysate was centrifuged at 200 xg for 5min, and the supernatant was added
to the streptavidin coated 96 well microplates (supplied with the kit) at 20 μl/well in
triplicates followed by 80 μl of the immunoreagent supplied. The plates were covered
and incubated at room temperature on a shaker for 2h with gentle shaking. The lysate
solution was removed from the wells, and the wells were washed with wash buffer three
times. Subsequently, 100 μl/well of ABTS enzyme substrate solution was added, and
plates were incubated for 10min at room temperature. The reaction was terminated by
adding 100 μl/well of the stop solution, and the color was measured using BioTek
synergy HT plate reader at 405nm. The data is the average OD405+SD of triplicate
wells.
Determination of Mitochondrial Outer Membrane Permeability [MOMP]
All tumor cell lines tested were treated with nimbolide and were tested for MOMP using
tetramethylrhodamine methyl ester [TMRM] (Invitrogen, Carlsbad, CA) as previously
described.2 TMRM was directly added to the cell cultures at 100nM concentrations and
incubated at 37°C in the dark for 15min.
At the end of the incubation, cells were
washed 2X with cold PBS containing 2% FBS and analyzed. The cells were washed for
fluorescence (FL2) and analyzed by BD accuriC6 flow cytometer and its software. Data
from at least 20,000 events per sample was collected and analyzed. TMRM-negative
(%) cells were calculated to determine % MOMP.
Immunoblot analysis
Protein content in the extracts was measured by the Bradford method using Bio-Rad
protein assay reagent. Aliquots of 30µg of total protein and 25μg of nuclear/cytoplasmic
protein were boiled in Laemmli sample buffer and loaded onto 10% SDS-PAGE gels and
transferred onto a PVDF membrane. Membranes were blocked for 1h in TBS/Tween 20
[TTBS] containing 1% nonfat dried milk and 1% BSA. Incubation with primary antibodies
was done overnight at C, followed by washing 3x with TTBS and incubation for 1h with
HRP-conjugated
secondary
antibody.
The
chemiluminescence (Thermo Scientific, IL).
2
blots
were
developed
using
Animal experiments
All animal experiments were performed with the approval of the Institutional Animal Care
and Use Committee of Mayo Clinic.
personnel.
Animals were monitored daily by animal care
Twenty female SCID mice (10 weeks of age) were subcutaneously
implanted with 1x106 RPCI-WM1 cells, which formed localized tumors that secreted
detectable human IgM in the serum by Day 14. On Day 10 post-implantation, the mice
were randomized into 3 groups and received either DMSO (n=6) or nimbolide formulated
in DMSO (100mg/kg, n=7; or 200mg/kg, n=7) at a final volume of 50ml every alternate
day for 26 days via intraperitoneal injection. Tumor size was measured every 7 days
using direct caliper measurements, and tumor volume was calculated using the formula
(width)2 x length/2. Human IgM levels’ using ELISA was recorded every 10 days. On
average, WM tumor tissues grew to a median of 3cm and IgM levels reached a median
of 823ng/mL by Day 30 post-implantation. On Day 37, mice were sacrificed, and final
tumor volume was measured in control and treatment arms. All images were obtained
using a Canon D40 digital camera.
Immunohistochemistry analysis
Immunohistochemistry was performed using IgM (Invitrogen 1:400), BCL-2 (DAKO predilute), Ki-67 (DAKO 1:100), P53 (Santa Cruz 1:3000), and Caspase 3 (Cell Signaling
1:100) antibodies. A complete description of the analysis is provided in the
supplementary methodology. Formalin fixed, paraffin embedded (FFPE) blocks were cut
at 5 microns on positively charged slides and placed into a 60oC degree oven for 1 hour
to dry. Slides were placed into xylene three times for 5 minutes each to remove paraffin.
Slides were placed into decreasing grades of alcohol for 15 dips each (100% and 95%
ETOH) to rehydrate tissues sections and were then placed into tap water for 5 minutes.
Slides were placed into PBS buffer with Tween 20 for 5 minutes, and then were placed
into 3% Hydrogen Peroxide for 5 minutes to block endogenous peroxidase. Slides were
placed into PBS buffer with Tween 20 until IHC staining was started. Slides were placed
onto a DAKO Immunostainer Plus and subsequently were scanned using the Leica
Biosystems Aperio Digital Image Scanner XT (Leica Microsystems Inc. Buffalo Grove, IL
USA). Positive Pixel Count from Aperio was used to determine intensity of antibody
staining. The Aperio algorithm was tweaked using a positive and negative IHC control,
which had all elements of positivity and negativity (0-3+). The algorithm was saved and
3
run on each slide to determine intensity of staining in negative cells (0), weak staining
cells (1+), moderately stained cells (2+) and strongly stained cells (3+).
Chemoinformatics Methods
Nimbolide was analyzed for chemoinformatic properties and physical descriptors.
Pharmocophore properties for nimbolide were then placed into comparison with a
pool of commercial drugs (>1 712 different drug molecules) and ranked based on
chemoinformatic statistics that fell within the range formed from greater than 95% of
commercial drugs. The initial structure was derived in ChemDraw and optimized in
chemBio 3D using GAMESS for optimization of the structure with both RHF/3-21G
level of theory and PM6, R-closed shell, PCM solvent for semi-empirical optimization
(Supplementary Figure 2).3,4 The root mean square deviation (r.m.s.d.) difference
between the two optimized structures was <0.05 Å.
The latter structure was
imported into Schrödinger’s Maestro module for chemical profiling with QikProp. 5
Additionally, we conducted a similarity comparison against the most common
pharmaceutical drugs and screened for the top five drugs with the most similar
physical descriptors. Such a comparison does not preclude a common activity, but
does indicate the likelihood of drug-like ability for nimbolide.
considerations
for
optimal
chemoinformatics
and
druglike
Additional
behaviour
were
investigated as previously described. 6-8
Docking Methods
Based on the chemoinformatic properties of nimbolide, its chemical space profile, and its
LASSO score; we generated a pool of potential protein partners for nimbolide-protein
docking examination. The top pre-screened proteins selected formed sets of protein
families and their multiple isoforms, which could be studied using all-atom molecular
dynamics and docking experiments. This pool comprised >50 proteins (top 5 proteins
shown in Supplementary Table 2). Docking of nimbolide across all selected proteins was
completed using methods previously described.9-15 The starting conformation of
nimbolide ligands was obtained by the method of Polak-Ribiere Conjugate Gradient
(PRCG) energy minimization with the OPLS 2005 force field for 5000 steps, or until the
energy difference between subsequent structures was less than 0.001 kJ/mol-Å using
PR-conjugate calculations.16
Based on nimbolides chemoinformatics properties,
chemical space profile and LASSO score; we generated a pool of potential protein
4
partners for nimbolide-protein docking examination.
The top pre-screened proteins
selected formed sets of families of proteins (along with multiple isoforms) to be studied
using all-atom molecular dynamics. Briefly, Schrödinger’s SiteMap module was used to
determine grid placement for the region of binding, which has overlap with known
binding sites. The binding site was generated via multiple overlapping grids with a
default rectangular box. Alternate binding sites were also observed and tested in silico.
Then a larger composite grid was generated such that several binding sites within a core
region were contained within the grid. Using this grid, nimbolide was docked using Glide
algorithms within Schrodinger suite as a virtual screening workflow (VSW). The docking
proceeded from lower precision (HTVS) through SP docking and Glide extra precision
(XP) (Glide, version 5.6, Schrödinger, LLC, New York, NY).17 The top 10 000 poses
were ranked for best scoring pose and unfavorable scoring poses were discarded. Each
conformer was allowed multiple orientations in the site. Site hydroxyls, such as serines
and threonines, were allowed to move with rotational freedom.
Induced fit docking
method was utilized within Schrodinger suite to allow larger side-chain re-orientation, as
needed. Hydrophobic patches were utilized within the VSW as an enhancement. Most
favorable docking results for nimbolide were retained and yielded >30 poses, which were
ranked by docking score. XP descriptors were used to obtain atomic energy terms like
hydrogen bond interaction, electrostatic interaction, hydrophobic enclosure, and pi-pi
stacking interaction that result during the docking run.17 The top docking poses were
chosen for analysis. Figures were generated using Maestro’s built in rendering software
(Maestro, v9.6, Schrödinger, LLC, New York, NY).
Statistical Analysis
Experiments were repeated a minimum of three times, with consistent results. Data
represented are given as the mean ± S.D. The statistical analysis was carried out using
a two-tailed unpaired Student's t test. A value of p < 0.05 was considered statistically
significant.
5
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