MOPS, Triton X-100 and TDP-Glucose were purchased from Sigma. MgCl2 and glycerol were from Fisher. NADPH was purchased from Roche. Black 384-well low volume non-binding polystyrene plates were from Corning (Corning 3676). The 384-well v-bottom polypropylene plates (Greiner 781280), 1536-well v-bottom polypropylene plates (Greiner 782270), and 384-well white sterile tissue culture treated plates (Greiner 781080) were all from Greiner.
4.2 Protein Expression and Purification
The clone expressing Salmonella enterica
RmlB used to make the TDP-KDX 15
was a gift from Jim Naismith, and RmlB was expressed and purified as described 15
. (The RmlB from M. tuberculosis
requires the addition of NAD for activity whereas NAD is tightly bound to the S. enterica
RmlB as purified and no additional NAD is required. As we preferred not to introduce NAD into the system and were not testing for inhibition of RmlB, the S. enterica
enzyme was used for substrate preparation). RmlC and RmlD from M. tuberculosis
were cloned and expressed in Escherichia coli
and purified as described 9
4.3 Synthesis of TDP-KDX
The substrate, TDP-6-deoxy-D
hexopyranosid-4-ulose (TDP-KDX), for the RmlC enzyme was synthesized enzymatically by converting d-TDP-glucose (2.5 mg) to TDP-KDX using 2.4 μg of purified RmlB from Salmonella typhimurium 15
in 50 mM MOPS buffer, pH 7.4, at 30 °C for 1 hr. Aliquots of TDP-KDX were stored at −80 °C.
4.4 Assay Optimization
The activity of the M. tuberculosis cell wall enzymes, RmlC and RmlD, towards TDP-KDX was measured by the decrease in fluorescence observed upon the oxidation of NADPH to NADP, at excitation and emission wavelengths of 340 and 460 nm, respectively. The Km for TDP-KDX (substrate) was determined by varying the TDP-KDX concentration from 0–1000 μM (10 point, 1.5-fold serial dilutions), while maintaining the NADPH concentration at 15 μM in a total volume of 10 μl. The NADPH (cofactor) Km was determined by varying NADPH concentration from 0–100 μM (12 point, 1.5-fold serial dilutions), while maintaining TDP-KDX concentration at 200 μM in a total volume of 10 μl. The data were fit using the computer program XLfit using the “General pharmacology model 250” (Michaelis-Menten model).
Based on the Km values of TDP-KDX and NADPH, assays were set up varying the TDP-KDX (100 and 200 μM) and NADPH (15 and 25 μM) concentrations as well as the concentrations of RmlC (8.75 × 10−5 to 5.25 × 10−4 μg/μl) and RmlD (2.175 × 10−4 to 1.31 × 10−3 μg/μl) in 50 mM MOPS, pH 7.4, containing 1 mM MgCl2, 10% glycerol and 0.01% Triton X-100, at 25 °C (data not shown). The assay was monitored by the decrease in fluorescence over 3 hr on a plate reader (Perkin Elmer Envision 2102 Multilabel reader). Based on concentrations that gave a linear assay time course, final assay conditions for the HTS in 384-well plates (Corning 3676) were selected: 200 μM TDP-KDX, 25 μM NADPH, 2.63 × 10−4 μg/μl RmlC and 6.55 × 10−4 μg/μl RmlD in a volume of 10 μl, and monitoring of the decrease in 340/460 fluorescence over 90 minutes at 25° C. Under these conditions the amounts of RmlC and RmlD were balanced as determined by a significant decrease in rate when the amount of either enzyme was diminished.
TDP, a known inhibitor of the RmlC/RmlD enzymes, was used in a quality control (QC) plate to monitor the sensitivity of the enzymes towards inhibition throughout the course of the HTS. The IC50 value of TDP was determined by dose response (16 point, 2-fold serial dilution, highest concentration of 30 mM) against 200 μM TDP-KDX, 15 μM NADPH, 1.8 × 10−4 μg/μl RmlC, and 4.4 × 10−4 μg/μl RmlD in a 384-well assay plate (Corning 3676). The change in fluorescence over 3 hr was monitored, and the IC50 was calculated by a non-linear fit of the rate of change in fluorescence against TDP concentration. Based on the observed IC50 of 340 μM, a solution of TDP in DMSO was added to a compound storage plate (Greiner 781280), set up to deliver a final assay concentration of 500 μM TDP, by pintool transfer of 0.12 μl (384-pin, V&P Scientific), expected to give slightly greater than 50% inhibition. The TDP storage plate contained 20 μl of 40.3 mM TDP in DMSO in columns 3–22, and 20 μl DMSO in columns 2 and 24 for controls (100% activity) and 20 μl DMSO in columns 1 and 23 for blanks (0% activity, no TDP-KDX). A TDP-containing QC plate was included in each HTS run and assayed using the same protocol as the test compound plates (see below).
4.5 High-Throughput Screening (HTS)
HTS of 201,368 compounds from the Molecular Libraries Screening Center Network (MLSCN) library was conducted in 384-well assay plates. The compounds were supplied as a smaller library (106,290 compounds) and as a larger library (214,178 compounds) that fully contained all of the 106,290 compounds of the first library but with no straightforward way to only screen the compounds not present in the smaller library. The entire smaller library was screened and 159,098 compounds from the larger library were screened of which 95,078 were not present in the smaller library leading to the screening of 201,368 unique compounds. The initial library was supplied by BioFocus DPI in 384-well storage plates and the expanded library supplied by BioFocus DPI in 1536-well compound storage plates. The 384-well library (supplied as 10 mM solutions in DMSO in v-bottom plates) was screened as mixtures of four compounds per well, by transfer of compounds from four 384-well compound storage plates into a single 384-well assay plate. Thus, 384-well dilution compound plates (Greiner 781280) were made at 0.5 mM in 20 μl DMSO (one compound per well) from the 10 mM plates supplied by BioFocus DPI. Mixtures were generated during HTS, using a pintool (384-pin, V & P Scientific) to transfer 0.12 μl of compound from the same well number (e.g. A3) from four different 384-well 0.5 mM dilution compound plates into a single well of a 384-well assay plate containing 4 μl of H2
O (again the same well number, e.g., A3). The final compound concentration was 5.5 μM per compound (22.0 μM total per well). A Multidrop reagent dispenser (Thermo) was used to dispense 5 μl of 83.5 mM MOPS buffer containing 2x RmlC, RmlD and NADPH, and a Multidrop Micro (Thermo) was used to dispense 1 μl of 83.5 mM MOPS buffer containing 10x TDP-KDX. Blank columns (1 and 23) contained 1 μl of 83.5 mM MOPS buffer instead of TDP-KDX. The final RmlC, RmlD, and NADPH concentrations in the 10.5 μl assay were 2.63 × 10−4
μg/μl, 6.53 × 10−4
μg/μl, and 25 μM, respectively, and the final concentration of TDP-KDX was 200 μM. The final buffer concentration was 50 mM MOPS buffer, pH 7.4 with 1 mM MgCl2
, 10% glycerol, and 0.01% Triton X-100. The assay was monitored by the change in NADPH fluorescence at 25 °C, measured at time 0 and after 90 minutes, at excitation and emission wavelengths of 340 and 460 nm, respectively. Data were analyzed in IDBS ActivityBase, and the percent inhibition of each compound was calculated from the change in fluorescence over 90 min (ΔSignal), calculated from t=0 and t=90 min reads, and the mean of the change in plate controls and blanks over 90 min, using the following equation:
The 159,098 compounds in the 1536-well library were supplied by BioFocus DPI as 2.5 mM solutions in DMSO, stored in 1536-well v-bottom compound plates. These compounds were also screened as mixtures of four compounds per well in 384-well assay plates. Mixtures were generated by pintool (384-pin) transfer of 0.091 μl of compound from each of the four quadrants of a 1536-well compound plate into a single well of a 384-well compound dilution plate (Greiner 781280) containing 25 μl of H2O. For example, compounds in wells A5, A6, B5, and B6 of a 1536-well compound plate were mixed in well A3 of a 384-well compound dilution plate. Four μl from each well of the compound dilution plate were transferred into a 384-well assay plate (Corning 3676) using a 384-tip pipetting head (PerkinElmer Evolution P3 Pipetting Platform). The well location of the compounds was unchanged during the transfer into the assay plate; for example, the mixture of four compounds in well A3 of a compound dilution plate was transferred to well A3 of the corresponding assay plate. The 384-well mixture dilution compound plate contained 9.1 μM of each compound and a total of 36.8 μM in 25 μl H2O, and the final assay plate contained 3.64 μM of each compound and a total mixture concentration of 14.6 μM in 10 μL in each well. The additions of RmlC, RmlD, and NADPH (5 μl) and TDP-KDX (1 μl), and the HTS assay were the same as in the HTS of the 384-well compound plates. The data were analyzed in IDBS ActivityBase, as described above.
The compounds that gave greater than 30 % inhibition in the HTS of the 384- and 1536-well compound plates were selected as hits and retested as single compounds. Compounds were ordered from BioFocus DPI, diluted into 384-well compound plates (0.5 mM), transferred by pintool into 4 μl of H2O in assay plates to give a final concentration of 5.5 μM in a final assay volume of 10 μl, and screened as described above. Each compound was tested in duplicate, the data were analyzed by IDBS ActivityBase, and compounds that gave >30% inhibition in both well locations were selected for further study in dose-response.
4.6 IC50 determination of HTS hits
Compounds for the dose-response testing were reordered from BioFocus DPI in 384-well plates, with 20 compounds at 10 mM in 10 μl DMSO in each plate in wells A3-A22. Dose-response compound plates were created by diluting each 10 mM compound solution to 2.5 mM with the addition of 30 μl of DMSO, followed by a 16-point 2-fold serial dilution using 30 μl disposable tips on the Evolution pipetting platform, to a final volume of 20 μl in each well with the highest concentration at 2.5 mM. Compounds from the dose-response compound plate were transferred twice by pintool (2 × 0.11 μl) into 4 μl of H2O in an assay plate, followed by addition of 5 μl of RmlC, RmlD, and NADPH and 1 μl of TDP-KDX as above for the HTS assay. Each dose-response plate was tested in triplicate using the protocol described above for HTS. The data were analyzed using ActivityBase. Each dose-response assay plate contained compounds in columns 3–22, controls (100% activity) in columns 2 and 24, and blanks (0% activity) in columns 1 and 23. Each compound column (3–22) contained 16 two-fold dilutions of a single compound, ranging in concentration from 55 μM to 1.7 nM. Percent activity was calculated for each concentration of each compound from the change in fluorescence over 90 min, as described above under HTS. IC50 values were calculated from a 4-parameter logistic fit of the change in percent activity with compound concentration using XLfit (IDBS).
Compounds that gave IC50 values of less than 20 μM were tested in buffer alone, in the dose-response assay format, to determine the fluorescence due to the compounds in the absence of enzymes, substrate, and NADPH. One μl of compound from each well of the dose-response compound plate was mixed with 18 μl of H2O in a V-bottom plate (Greiner 781280), and 4 μl of this dilution mixture was transferred into an assay plate (Corning 3676) containing 6 μl of 83.5 mM MOPS buffer, and the fluorescence at 340/460 nm was monitored over 90 min. The data were analyzed by Excel and dose-response curves were plotted
4.7 Acquisition and synthesis of compounds
The active compound SID 7975595 was obtained three ways: re-supply from the Molecular Libraries Screening Small Molecule Repository, from ChemBridge, and by chemical synthesis (see below). The highly active 77074 was obtained from ChemBridge and chemically synthesized (see below); the remaining compounds shown in were prepared by chemical synthesis.
4.8 Chemical synthesis
All solvents and chemicals were purchased from Sigma-Aldrich and Fisher Scientific except that 5-ethyl-5H-[1,2,4]triazino[5,6-b]indole-3-thiol was purchased from ChemBridge Corporation, CA, USA; 5-allyl-5H-[1,2,4]triazino[5,6-b]indole-3-thiol was purchased from Ryan Scientific, Inc., SC, USA; and 5-allyl-8-methyl-5H-[1,2,4]triazino[5,6-b]indole-3-thiol was purchased from Sigma-Aldrich (rare chemical library). 1H NMR spectra were recorded at 300 MHz on a Bruker ARX NMR instrument or 500 MHz on a Varian Inova NMR instrument. Accurate mass mass spectrometry in positive mode was performed on an Agilent 6220 TOF mass spectrometer equipped with a MultiMode source selected to be in the dual atmospheric pressure chemical ionization/electrospray ionization mode. Analytical RP-HPLC was conducted on a Shimadzu HPLC system with a Phenomenex C18 column (100Å, 3 μm, 4.6 × 50 mm), flow rate 1.0 mL/min and a gradient of solvent A (water with 0.1% TFA) and solvent B (acetonitrile): 0–2.00 min 100% A; 2.00–8.00 min 0–100% B (linear gradient). UV detection at 218 and 254 nm was used.
4.8.1. Synthesis of triazinoindol-benzimidazolones SID7975595, 77074, and 78531
Alkylation of the corresponding triazino-indol-3-thiols was performed as reported previously 16, 17
using optimized reaction conditions (). 5-Allyl-5H-[1,2,4]triazino[5,6-b]indol-3-thiol (0.242 g, 1 mmol), potassium hydroxide (65 mg, 1 mmol, 86.4%), and water (5 mL) were stirred at room temperature for 2h, followed by the addition of 1-(3-chloropropyl)-1,3-dihydro-2H-benzimidazol-2-one (0.234 g, 1 mmol, 90%). The resulting solution was stirred at 90°C for 24h. The reaction mixture was evaporated, and the crude product was purified by Biotage flash column chromatography to yield product 77074 as a light yellow powder.
1-(3-(5-ethyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)propyl)-1H-benzo[d]imidazol-2(3H)-one, SID7975595, 0.38 g (1 mmol scale), 93.9% yield. 1H NMR, 300 MHz (CDCl3): δ9.37 (s, 1H, NH), 8.42 (d, J = 7.6 Hz, 1H), 7.64–7.73 (m, 1H), 7.39–7.49 (m, 2H), 7.00–7.18 (m, 4H), 4.34 (q, J = 7.2 Hz, 2H, NCH2CH3), 4.14 (t, J = 6.9 Hz, 2H, NCH2), 3.46 (t, J = 7.0 Hz, 2H, SCH2), 2.40 (quintet, J = 7.0 Hz, CH2), 1.45 (t, J = 7.2 Hz, 3H, CH3). Mass spectrum m/z (M+Na)+ 427.1314 (427.1312 predicted). HPLC purity: >96% (monitored at 218 and 254 nm), tR = 5.92 min.
1-(3-(5-allyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)propyl)-1H-benzo[d]imidazol-2(3H)-one, 77074, 0.23 g (1 mmol scale), 55.2% yield. 1H NMR, 300 MHz (CDCl3): δ 8.41–8.47 (m, 1H), 8.29 (s, 1H, NH), 7.64–7.71 (m, 1H), 7.43–7.49 (m, 2H), 7.03–7.10 (m, 4H), 5.89–6.03 (m, 1H, CH=(Allyl)), 5.27 (d, J = 10.4 Hz, 1H, =CHa(Allyl)), 5.18 (d, J = 17.7 Hz, 1H, =CHb(Allyl)), 4.91 (d, J = 5.3 Hz, 2H, CH2(Allyl)), 4.12 (t, J = 6.9 Hz, 2H, NCH2), 3.44 (t, J = 7.0 Hz, 2H, SCH2), 2.38 (quintet, J = 7.0 Hz, 2H, CH2). Mass spectrum m/z (M+Na)+ 439.1315 (439.1312 predicted). HPLC purity: 100% (monitored at 218 and 254 nm), tR = 6.02 min.
1-(3-(5-allyl-8-methyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)propyl)-1H-benzo[d]imidazol-2(3H)-one, 78531, 40 mg (0.195 mmol scale), 47.6% yield. 1H NMR, 500 MHz (CDCl3): δ 8.52 (s, 1H, NH), 8.20 (s, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.02–7.08 (m, 4H), 5.89–5.97 (m, 1H, CH=(Allyl)), 5.24 (d, J = 10.5 Hz, 1H, =CHa(Allyl)), 5.13 (d, J = 17.1 Hz, 1H, =CHb(Allyl)), 4.85 (d, J = 5.4 Hz, 2H, CH2(Allyl)), 4.10 (t, J = 6.8 Hz, 2H, NCH2), 3.41 (t, J = 7.1 Hz, 2H, SCH2), 2.36 (quintet, J = 7.1 Hz, 2H, CH2). Mass spectrum m/z (M+Na)+ 453.1469 (453.1458 predicted). HPLC purity: 100% (monitored at 218 and 254 nm), tR = 6.23 min.
4.8.2. Synthesis of triazinoindol-benzimidazolone sulfonation derivatives 78532 and 78533
Sulfonation reaction was carried out according to a reported procedure 18
. Catalytic amounts (ca. 1 mg each) of sodium tungstate dihydrate (Na2
O), methyltrioctylammonium hydrogen sulfate ([CH3
), and phenylphosphonic acid (C6
) were added to a 25 mL flask, followed by the addition of 150 mg of aqueous 30% H2
. After the mixture was stirred vigorously at room temperature for 10 min, 1-(3-(5-allyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)propyl)-1H-benzo[d]imidazol-2(3H)-one (77074, 70 mg, 0.168 mmol) was added and stirred at room temperature for 24h. The resulting mixture was evaporated and purified by reverse phase Biotage flash C18 column chromatography to give product 78533 as a light yellow powder.
1-(3-(5-ethyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylsulfonyl)propyl)-1H-benzo[d]imidazol-2(3H)-one, 78532, 15 mg (0.0989 mmol scale), 34.7% yield. 1H NMR, 500 MHz (CDCl3): δ 9.34 (s, 1H, NH), 8.57 (d, J = 7.8 Hz, 1H), 7.86 (t, J = 7.8 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 6.99–7.10 (m, 4H), 4.53 (q, J = 7.3 Hz, 2H, NCH2CH3), 4.14 (t, J = 6.1 Hz, 2H, NCH2), 3.87 (t, J = 6.8 Hz, 2H, SO2CH2), 2.50 (quintet, J = 6.4 Hz, CH2), 1.52 (t, J = 7.3 Hz, 3H, CH3). Mass spectrum m/z (M+Na)+ 459.1213 (459.1210 predicted). HPLC purity: 100% (monitored at 218 and 254 nm), tR = 5.60 min.
1-(3-(5-allyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylsulfonyl)propyl)-1H-benzo[d]imidazol-2(3H)-one, 78533, 40 mg (0.168 mmol scale), 53.1% yield. 1H NMR, 500 MHz (CDCl3): δ 9.75 (s, 1H, NH), 8.55 (d, J = 7.6 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H), 7.54–7.62 (m, 2H), 7.00–7.15 (m, 4H), 5.93–6.02 (m, 1H, CH=(Allyl)), 5.31 (d, J = 10.3 Hz, 1H, =CHa(Allyl)), 5.23 (d, J = 17.1 Hz, 1H, =CHb(Allyl)), 5.09 (d, J = 4.2 Hz, 2H, CH2(Allyl)), 4.15 (br s, 2H, NCH2), 3.87 (br s, 2H, SO2CH2), 2.50 (br s, 2H, CH2). Mass spectrum m/z (M+Na)+ 471.1212 (471.1215 predicted). HPLC purity: 100% (monitored at 218 and 254 nm), tR = 5.72 min.
4.9 IC50 determination of triazinoindol-benzimidazolones
A slightly different method than that described above was used for determining the IC50 values of triazinoindol-benzimidazolones that were obtained after the screen was complete. Thus the IC50 values of compounds obtained from ChemBridge and by chemical synthesis were determined in 96-well plates by monitoring the conversion of NADPH to NADP via absorbance at 340 nm. The total assay volume was 200 μl of 50 mM 3-(N-morpholino)propanesulfonic acid buffer (MOPS) with 10% glycerol, 0.01% Triton X-100, 1 mM Mg2Cl2, at pH 7.4. Test compounds were added in 4 μl of DMSO at a range of concentrations. The remaining components were added to the following concentrations: RmlC (70 nM), RmlD (5 nM), TDP-KDX (200 μM), and NADPH (25 μM). The reaction was started by the addition of RmlC and continuously monitored for 80 min, after which the initial reactions velocities were determined and the IC50 values calculated using the program GraFit (Erithacus Software limited).
4.10 Determination of which Enzyme (RmlC or RmlD) is the Target of Inhibition
The 96-well plate method used to determine IC50 values of triazinoindol-benzimidazolones was performed. Thus, to determine which enzyme is inhibited by SID 7975595, the concentration of one enzyme was varied while maintaining the other constant, and vice versa, and the IC50 of compound SID 7975595 was monitored for changes in IC50 that correlated with changes in enzyme concentration. The RmlC enzyme concentration was held constant at 70 nM while varying RmlD concentration from 5 to 412 nM, and the RmlD concentration was held constant at 5 nM while varying RmlC concentration from 70 to 350 nM, in the presence of 200 μM TDP-KDX and 25 μM NADPH.
4.11 Direct Confirmation of inhibition of RmlC using GC/MS analysis to quantify the product TDP-6-deoxy-L-lyxo-hexopyranosid-4-ulose
An assay to confirm the inhibition of RmlC by SID 7975595 (at the concentrations of 0.25, 0.5, and 5 μM) in the absence of RmlD in which the RmlC product is measured by GC/MS after reduction of the 4-ketogroup and derivatization was performed as previously described 11
4.12 Determination of the mode of Inhibition by SID 7975595
To determine the mode of inhibition of SID 7975595, the Km of TDP-KDX was determined in the absence of inhibitor and in the presence of several concentrations of inhibitor. TDP-KDX concentration was varied from 0–100 μM in the presence of SID 7975595 concentrations of 0.1, 0.15, and 0.2 μM. The Km and Vmax were determined by non-linear regression using GraFit. The Km(obs) was plotted against the concentration of SID 7975595 and the Ki calculated from the slope, which equals Km/Ki, where Km is given by the y-axis intercept.
4.13 Determination of time dependence of the onset of inhibition of SID 7975595
Time-dependence of the onset of inhibition by compound SID 7975595 was studied by pre-incubating the compound with RmlC and RmlD enzymes (and NADPH) in 384 well plates for times ranging from 0 to 3 hr before adding TDP-KDX to initiate the enzymatic assay. One μl of compound SID 7975595 from each well of the dose-response compound plate (see above) was mixed with 18 μl per well of H2O in a 384-well plate (Greiner 781280) and 4 μl of the resulting diluted compound was transferred into a 384-well assay plate (Corning 3676) containing 5 μl of 2x RmlC, RmlD, and NADPH in 83.5 mM MOPS buffer and pre-incubated for 0–3 hr. After pre-incubation, TDP-KDX was added and the enzymes were assayed over 90 min, and IC50 data were analyzed as described above for IC50 determination of HTS hits.
4.14 Determination of the reversibility of SID 7975595 inhibition
The reversibility of inhibition by SID 7975595 was determined using the procedure of Copeland 19
. Thus SID 7975595 at 10 times its IC50
was incubated for 0 and 0.5 hr at 25°C with RmlC at 100 times its final assay concentration in 2 μl buffer, in Eppendorf microcentrifuge tubes. After incubation, the compound-RmlC mixture was diluted 100-fold to a final volume of 200 μl, RmlD, TDP-KDX, and NADPH were added, and the activity of the enzyme was measured by transferring 10 μl of the assay mixture into a 384-well assay plate and monitoring the decrease in NADPH fluorescence over 200 min as described above. Control assays were set-up in the absence of compound.
4.15 Modeling the binding of triazinoindol-benzimidazolones in the active site of RmlC
Docking of SID 7975595 and its analogs was performed using both Glide 20
and Autodock Vina 13
. As a control experiment, TDP-Rha was re-docked into RmlC using both programs. The RmlC dimer structure was taken from pdb entry 2ixc (5). Protonation states of protein residues were initially assigned based on pKa calculations using PROPKA 21, 22
. Key residues at the active site were then inspected visually to make sure that their protonation states were consistent with experimental findings. In particular, Lys72 was given a +1 charge despite of a predicted pKa of ~7, and His62, His119 were kept neutral with protons on their ε-nitrogen. The protein was then prepared using the Schrodinger Protein Preparation Wizard 23–25
and AutodockTools 26
for Glide and Autodock Vina docking, respectively. Initial structures of SID 7975595 and its analogs listed in were prepared using Ligprep 27
or DiscoveryStudio 28
. Docking with Glide was performed using both the standard precision (SP) and the extra precision (XP) modes, with the ligand van der Waals radii scaled by 0.8. Docking with Autodock Vina was performed with an exhaustiveness factor of 8.
4.16 MIC Determination
The MIC of SID 7975595 and structural analogs against Mycobacterium tuberculosis
(H37Rv) was determined by the microbroth dilution method as described by Sun et al 29
4.17 Cytotoxicity Studies with Human Aortic Endothelial (HAE) Cells
Cytotoxicity of compound SID 7975595 in mammalian cells was tested in HEA cells by seeding 1000 cells/25 μl/well in white 384-well tissue culture treated plates and incubating at 37 °C for 24 hr before treating with compound and incubating for an additional 24 hr at 37 °C and measuring luminescence. To prepare SID 7975595, 3μl of 10 mM SID 7975595 in DMSO was mixed with 47 μl of EGM-2 endothelial cell media and 25 μl of this mixture was transferred into 25 μl of media for a 16-point, 2-fold serial dilution ranging in concentration from 600 μM to 18 nM. As controls, 6 μl of doxorubicin (positive control) and DMSO (solvent control) were mixed with 94 μl of media and 25 μl of each was transferred into 25 μl media for a 16 point, two-fold serial dilution, where the doxorubicin concentrations were the same as the SID 7975595 concentrations (600 μM to 18 nM) and the DMSO only concentration was the same as the DMSO concentration in both the SID 7975595 and doxorubicin tests. Five μl of each of the serial dilutions were added to cells in 25 μl media for final compound concentrations of 100 μM to 3 nM. Luminescence was measured on the Envision microplate reader after the addition of 30 μl of CellTiter-Glo and incubation for 10 min. Each plate was tested in triplicate.