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Supplemental Material for:Blinded Potency Comparison of Transthyretin Kinetic Stabilizers by Subunit Exchange in Human PlasmaLuke T. Nelson1, Ryan J. Paxman1, Jin Xu1, Bill Webb2, Evan T. Powers1 and Jeffery W. Kelly1, 31Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA2Center for Metabolomics, The Scripps Research Institute, La Jolla, CA, USA3The Skaggs Institute for Chemical Biology, The Scripps Research Institute, CA, USA Detailed Materials and Methods SectionDual-FLAG-tagged WT TTR Expression and PurificationThe plasmid encoding the dual-FLAG-tagged WT TTR (FT2-WT TTR) insert was transformed into BL21(DE3) E. coli by heat shock, and the resulting colonies were used to grow an overnight culture in LB (30 mL) containing 100 ?g/mL Ampicillin. The cultures (2 x 1 liter) were treated at 1:100 with starter culture and cells were grown at 37 °C with shaking (180 rpm) until an O.D. 600 of 0.5 was reached, at which point the culture was induced with 1 mL of 1 mM isopropyl β-D-1-thio-galactopyranoside (IPTG) and incubated overnight at 25 °C with shaking. Cells were pelleted via centrifugation, resuspended in 20 mL of ion exchange (IEX) buffer A (25 mM Tris, pH 7.8), and the cells were lysed by sonication on ice. Following clearing of the lysate by centrifugation, the supernatant was saturated with 50% ammonium sulfate at 4°C with shaking for 1 h. After additional centrifugation (19,000×g for 20 min), the precipitate was removed, and the supernatant was saturated with ammonium sulfate at 90% for 1 h. After a final round of centrifugation, the precipitated TTR was resuspended in 20 mL of IEX buffer A and dialyzed against 4 L of IEX buffer A at 4°C overnight. The dialyzed protein was then loaded onto a 30 mL anion exchange column (GE Life Sciences) using an ?kta pure protein purification system (GE Life Sciences) and eluted using a linear gradient of 0% - 100% IEX buffer B (25 mM Tris, 1 M NaCl, pH 7.8). The TTR from the anion exchange column was then loaded onto a size exclusion column (GE Life Sciences) and eluted with size exclusion chromatography (SEC) buffer (50 mM NaPO42-, 100 mM NaCl, pH 7.8). The collected TTR was then dialyzed for a final time against IEX buffer A. Plasma Preparation for Experiments 1 and 2Blood from healthy volunteers was obtained from The Scripps Research Institute’s Normal Blood Donor Services Center. Blood was collected in tubes with EDTA, followed by centrifugation at 1500×g for 20 min. The resulting supernatant (plasma) was carefully removed and centrifuged for an additional 20 min to remove any remaining cells. Plasma was transferred to 1.5 mL cryovials with a cap and stored at ?80°C. For experiment 1, plasma samples from nine healthy individuals were thawed at ambient temperature, separated into groups of three, and the plasma in each group was pooled. The plasma was then centrifuged for 30 min at 19,000×g and filtered through a 0.22 m filter. The molar absorptivity (ε) of FT2-WT TTR (85720 M-1cm-1) tetramers in IEX buffer A was used to calculate the FT2-WT TTR concentration. The recombinant FT2-WT TTR protein (552 L) at a concentration of 54 M was added to the plasma (29.45 mL) for a final concentration of 1 M FT2-WT TTR to initiate the subunit exchange reaction. Once the subunit exchange reaction had been initiated, 499 L aliquots of plasma were added in triplicate to dilute the 1 L kinetic stabilizer stocks originally at 10 mM, 5 mM, 2.5 mM, and 0.5 mM to afford the 20 M, 10 M, 5 M, and 1 M final concentrations. To yield the 30 M kinetic stabilizer final concentration, 332.33 L of plasma was added to 1 L of 10 mM kinetic stabilizer stock solution. For each measurement, one aliquot from each of the three plasma pools was removed for chromatography at each time point and kinetic stabilizer concentration and subunit exchange was arrested by the addition of A2 as described below. Thus experiment 1 was performed in biological triplicate. One aliquot from each of the three plasma pools was removed for chromatography for the DMSO or vehicle control at each timepoint and was added to the “chromatography preparation solution” defined below as well.For experiment 2, plasma samples from six healthy donors were thawed and combined into a single plasma pool. The plasma was then centrifuged for 30 min at 19,000×g and filtered through a 0.22m filter. The molar absorptivity (ε) of FT2-WT TTR tetramers (85720 M-1cm-1) in IEX buffer A was used to calculate the FT2-WT TTR concentration. The FT2-WT TTR protein (9.2 L) was added at a concentration of 54 M to the 490.8 L of plasma to yield a final concentration of 1 M FT2-WT TTR. The plasma / recombinant FT2-WT TTR exchange solution was then used to dilute drug stocks to their final concentrations of 30 M, 20 M, 10 M, 5 M, and 1 M, prepared separately for experiment 2, but in the same fashion as for experiment 1. For each measurement, two aliquots of the stock were removed and were added to the “chromatography preparation solution” defined below to provide data in technical duplicate. Aliquots of the plasma / recombinant FT2-WT TTR exchange solution (9 μL) were added to aliquots of the fluorogenic molecule A2 (5 mM in 1 ?L DMSO) [Choi S, Ong DS, Kelly JW. “A stilbene that binds selectively to transthyretin in cells and remains dark until it undergoes a chemoselective reaction to create a bright blue fluorescent conjugate” J. Am. Chem. Soc. 2010; 132: 16043-16051]. A2 immediately arrests subunit exchange upon addition while allowing the conjugation reaction to proceed to completion at 25 °C for 3 h, resulting in complete covalent modification of the two thyroxine binding sites within the TTR tetramer by A2. After fixation with A2, samples were flash frozen in liquid nitrogen and stored at -80 °C until later injection on the UPLC. The A2 conjugation reactions were thawed on ice and the samples were diluted by adding 40 μL of IEX buffer A. This conjugate sample in IEX buffer A (40 μL) was injected onto a Waters Acquity H-Class Bio-UPLC instrument employing a Waters Protein-Pak Hi Res Q, ion exchange column (5 ?m, 4.6 mm x 100 mm). Samples were separated using a linear 24% to 39% IEX buffer B gradient over 29 min (flow 0.5 mL/min). The area of each peak was determined utilizing the Waters integration tool employing fluorescence detection, wherein an excitation wavelength of 328 nm and an emission wavelength of 430 nm were used. All subunit exchange experiments in plasma were carried out at 25 °C because subunit exchange is fast enough to be practically measured on a laboratory timescale at this temperature. A previous study showed that the rate of WT TTR subunit exchange in plasma in the presence of 10 M tafamidis was 2.6-fold faster at 25 °C than at 37 °C [Rappley I, Monteiro C, Novais M, et al. “Quantification of Transthyretin Kinetic Stability in Human Plasma Using Subunit Exchange” Biochemistry 2014; 53: 1993-2006].Processing the Peak Area DataPeak areas in all ion exchange chromatograms from subunit exchange chromatograms were measured using the integration tool on our Waters Acquity H-Class Bio-UPLC. The proportion, p, of non-tagged TTR was calculated for each subunit exchange experiment using the chromatogram for the earliest time point as follows:Where Ai is the area of the ith peak in the chromatogram (note that under ideal conditions at time = 0, before any exchange has occurred, this simplifies to p = A1/(A1+A5)). The expected peak areas of all five peaks at full exchange were then calculated by using Equation 1 below:(1)Where Pr (X = k) is the proportion of TTR tetramers having k flag-tagged subunits at equilibrium, and n = 4. In particular, the expected final proportion of heterotetramers with 2 flag-tagged subunits (n = 2, k = 2), which corresponds to the expected area of peak 3 in the ion exchange chromatograms relative to the total area for all peaks, was termed the “exchange coefficient” for the time course. After determining the peak areas for all blinded samples at all time points of a subunit exchange time course, the area of peak 3 was divided by total area at each time point and further divided by the exchange coefficient to yield the fraction exchange values that are plotted in Figure 2 and Supplemental figures S1 and S2, with 0 representing no exchange and 1 representing 100% exchange. In other words:Where fex,t is the fraction of exchange at time t, Ai,t is the area of peak i at time t, and Pr (X = 2) is the exchange coefficient (the expected final proportion of heterotetramers with 2 flag-tagged subunits). Once the full subunit exchange time courses had been measured for all replicates of each kinetic stabilizer and data processed to determine rates of exchange, the drugs were unblinded. Global fits of subunit exchange data.The rate of subunit exchange of TTR tetramers is: Where fex is the fraction of exchange, funbound is the fraction of TTR tetramers that are not ligand bound, kdiss is the dissociation rate for TTR tetramers, t is the time in hours and a is the final plateau value for the fex curve (a is expected to be close to 1 in all cases). The apparent subunit exchange rate constant is kex = funboundkdiss. The value of funbound at a given concentration of tetrameric TTR, albumin, and ligand can be determined by solving the mass balance equations for each of these species, which are: Where quantities in brackets refer to molar concentrations (“Alb” refers to albumin), Kd1 is the dissociation constant for the first kinetic stabilizer binding to TTR, Kd2 is the dissociation constant for the second kinetic stabilizer binding to TTR, and Kd,Alb is the dissociation constant for kinetic stabilizer binding to albumin. These equations can be solved for [TTR]free and then the value of funbound = [TTR]free/[TTR]total. The values of Kd1 and Kd2 for all ligands were taken from literature sources as discussed in the Results section. The value of [Alb]total was assumed to be 650 ?M (which corresponds to 43 g/L; the reference range for albumin is 35-50 g/L), while [TTR]total was determined by measuring the concentration of TTR tetramer determined from the t = 0 UPLC trace + 1 ?M to account for the added FLAG-tagged TTR. Best-fit values for kdiss, Kd,Alb and a were then determined by globally fitting the fraction exchange data at all concentrations of each ligand using the function NonlinearModelFit as implemented in Mathematica 12 as we have described previously [Wiseman RL, Green NS, Kelly JW. “Kinetic stabilization of an oligomeric protein under physiological conditions demonstrated by a lack of subunit exchange: implications for transthyretin amyloidosis” Biochemistry 2005; 44: 9265-9274].Kinetic stabilizer purity assessment A purity assessment of the four kinetic stabilizers employed was performed by reverse phase HPLC using an Agilent 1260 HPLC system. Compound purity was assessed by UV absorption at wavelengths: 254, 280, and 360 using an XTERRA MS C8 column, (3.5 ?m particle size) 4.6 x100 mm; Mobile phase: water/MeCN with 0.1% TFA; Gradient: 20% MeCN (0 min)-20% increasing to 100% (20 min)-maintaining100% (30 min) using a flow rate oj 1 mL/min. Compound purities were also assessed by 1H NMR. All four compounds were determined to have a purity >98%. HPLC traces for all stabilizers are presented in Supplemental Figure S3. For the one kinetic stabilizer that we synthesized de novo (AG10), we present the 1H- and 13C-NMR spectra in Supplemental Figures S4 and S5 and high-resolution mass spectrometry data in Supplemental Figure S6.Kinetic stabilizer concentration determinations The concentration of the TTR kinetic stabilizers being compared were quantified by three independent methods to be sure that accurate concentrations were employed in plasma in experiments 1 and 2 summarized in Supplemental Figures S1 and S2, respectively. In concentration determination method 1, each kinetic stabilizer was dried under vacuum overnight, weighed out on an analytical balance, and concentration was assumed to be consistent with the measured mass of compound added to the stock solution using analytical best practices. In concentration determination method 2, the kinetic stabilizer concentrations prepared by mass were determined using a Waters TQ-XS triple quad mass spectrometer coupled to a Waters Acquity UPLC stack by injecting 5 ?L of a sample (concentration was determined by method 1). Data was acquired in negative MRM mode using the following transitions: Diflunisal 248.8 → 156.7 (quant), 184.7 (qual); Tolcapone 271.8 → 241.8 (quant), 254.8 (qual); AG10 290.96 → 110.95 (quant), 110.95 (qual); Tafamidis 305.7 → 261.1 (quant), 433.98 (qual). Separations were achieved with a BEH C18 1.7 ?m column, 2.1 × 100 mm and mobile phase A of water/0.1% formic acid, mobile phase B = CH3CN/0.1% formic acid using gradient: 95:5 for one minute, then changing to 2:98 over the next five minutes, holding at 2:98 for three minutes, then back to 95:5. The flowrate was 0.6 ml/min. Observed peak areas were compared to a calibration curve generated independently by the Scripps Mass Spectrometry Center to calculate the concentrations of the stock solutions. Compound concentrations were also confirmed by NMR to establish purity and dryness of the compounds and to use a method for determining concentration that is independent of mass measurements performed on a balance. Compounds were assessed using a Bruker AVIII HD-600 with a cryoprobe using a relaxation time of 50 seconds to allow for quantitative comparison of proton integrations. Compounds were compared to an internal standard, maleic acid, with a known concentration and also compared with a solution of caffeine of known concentration using the same maleic acid internal standard. Relative peak integrations determined that the kinetic stabilizer concentrations employed in experiments 1 and 2 were within 5% of the anticipated concentration according to method 1. Sample blindingSamples were blinded to everyone by one of us (R.P.), who made the stock solution for each of the four kinetic stabilizers, and who assigned a random letter to each stock solution. The samples were only unblinded after peak area processing was completed by a different member of our team (L.T.N.).Study ApprovalBlood from healthy volunteers was obtained from The Scripps Research Institute’s Normal Blood Donor Services Center. This ex vivo study using deidentified plasma samples was approved by the institutional review board at The Scripps Research Institute in the context of NIH grant DK 046335.Expanded Results Section Regarding % of WT TTR Subunit Exchange after 48hThere is relatively little difference in the % of subunit exchange after 48 h (the half life of TTR in human blood) when comparing AG10, tafamidis and tolcapone at the 20 and 30 ?M plasma concentrations (Supplemental Figure S7). At the 10 ?M kinetic stabilizer concentration, AG10 displays less (3.83 (mean) ± 1.49 %) TTR subunit exchange in plasma after 48 h of subunit exchange, relative to tafamidis (9.01 (mean) ± 0.68 %) and tolcapone (8.25 (mean) ± 0.74 %) (Supplemental Figure S7).Supplemental Methods.Synthesis of AG10. AG10 was synthesized following the literature route, summarized in Supplemental Scheme 1 below. Detailed procedures follow Supplemental Scheme 1.Supplemental Scheme 1. Synthesis of AG10 a) 1,3-dibromopropane, K2CO3, DMF, rt, 16 h; b) acetylacetone, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), toluene, 50 oC, 6 h; c) hydrazine hydrate, ethanol, 90oC, 16 h; d) LiOH.H2O, THF, water, rt, 18 h. Methyl 3-(3-bromopropoxy)-4-fluorobenzoate (1) To a solution of methyl 4-fluoro-3-hydroxybenzoate (1.7 g, 10.0 mmol) and 1,3-dibromopropane (4.06 ml, 40 mmol, 4 equiv) in DMF (25 ml) was added K2CO3 (2.76 g, 20.0 mmol, 2 equiv). The reaction mixture was stirred at 25 °C for 16 h then filtered to remove most of K2CO3. The solution was removed under high vacuum rotary evaporation and the resulting residue was purified by silica gel column chromatography using a Combiflash system (0-15% EtOAc in hexanes) to afford compound 1 (2.47 g, 85% yield) as viscous colorless oil; 1H NMR (500 MHz, CDCl3) δ 7.67 (dd, J = 8.1, 2.0 Hz, 1H), 7.64 (ddd, J = 8.4, 4.5, 2.1 Hz, 1H), 7.11 (dd, J = 10.7, 8.4 Hz, 1H), 4.22 (t, J = 5.8 Hz, 2H), 3.91 (s, 3H), 3.63 (t, J = 6.3 Hz, 2H), 2.41 – 2.33 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 166.26, 156.82, 154.80, 146.86, 146.78, 126.75, 126.72, 123.61, 123.54, 116.31, 116.15, 116.02, 115.99, 66.88, 52.43, 32.29, 29.78. (ESI-MS) m/z: 290/292 [M+H]+.Methyl 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoate (3) To a solution of 1 (2.0 g, 6.87 mmol, 1 equiv) in toluene (15 ml) was added the mixed solution of acetyl acetone (1.37 ml, 13.74 mmol, 2 equiv) and DBU (2.05 ml, 13.74 mmol, 2 equiv) in toluene (5 ml) via syringe slowly over 10 min. The reaction mixture was stirred at 50 °C for 6 h then cooled to 25 °C. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (silica gel, 0-20% EtOAc in hexanes) to afford compound 2 which was used in the next step directly. Hydrazine hydrate (0.47 ml, 7.55 mmol, 2.5 equiv) was added to a solution of 2 (938 mg, 3.02 mmol) in ethanol (8 ml) and the reaction was heated at 90 °C for 16 h and then cooled to 25 °C. The solvent was removed under reduced pressure and the residue was purified by flash column chromatography (silica gel, 0-10% MeOH in CH2Cl2) to afford compound 3 (614 mg, 66% in one step, or 33% in two steps); 1H NMR (400 MHz, CDCl3) δ 7.66 – 7.57 (m, 2H), 7.11 (dd, J = 10.9, 8.0 Hz, 1H), 4.01 (t, J = 6.0 Hz, 2H), 3.89 (s, 3H), 2.59 (t, J = 7.2 Hz, 2H), 2.20 (s, 6H), 2.04 – 1.92 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 166.34, 156.61, 154.93, 147.01, 144.37, 142.22, 126.63, 126.60, 123.15, 123.10, 116.14, 116.01, 115.69, 115.67, 114.06, 67.91, 52.35, 29.62, 18.96, 10.76. (ESI-MS) m/z: 307 [M+H]+.3-(3-(3,5-Dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoic acid (AG10) To a suspension of 3 (564 mg, 1.84 mmol, 1 equiv) in a mixture of THF (10 ml) and water (10 ml) was added LiOH.H2O (155 mg, 3.68 mmol, 2 equiv). The reaction mixture was stirred at room temperature (25 °C) for 18 h, and carefully acidified to pH 3-4 using 1N aqueous HCl. The mixture was extracted with EtOAc (3 x 50 ml) and the combined organic extract was washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 0-25% MeOH in CH2Cl2) to give AG10 (375 mg, 70% yield) as a white powder. The final product was further purified by preparative HPLC (Phenomenex Gemini-NX C18, 30x250 mm, 20% to 95% gradient MeCN in water with 0.1% TFA) before subjecting it to biological tests. 1H NMR (500 MHz, MeOD) δ 7.68 – 7.61 (m, 2H), 7.19 (dd, J = 10.9, 8.4 Hz, 1H), 4.03 (t, J = 5.9 Hz, 2H), 2.61 (t, J = 7.3 Hz, 2H), 2.15 (s, 6H), 2.01 – 1.94 (m, 2H). 13C NMR (151 MHz, MeOD) δ 168.87, 157.79, 156.12, 148.30, 148.23, 143.34, 128.71, 128.68, 124.33, 124.28, 116.97, 116.84, 116.80, 116.78, 115.18, 68.89, 30.67, 19.68, 10.43. HRMS (ESI-TOF) m/z: calcd for C15H18FN2O3: 293.1296; found 293.1304 (M+H)+. The 1H- and 13C-NMR spectra for AG10 are shown in Supplemental Figures S4 and S5 respectively. The high-resolution mass spectrum is depicted in Supplemental Figure S6.Supplemental Figures.Supplemental Figure S1. TTR subunit exchange kinetics in the presence of kinetic stabilizers at varying concentrations, experiment 1. Time courses of subunit exchange between endogenous plasma TTR (~ 3 ?M) and added dual-flag tagged WT-TTR (1 ?M) in the presence of kinetic stabilizers at the indicated concentrations. Measurements were performed in biological triplicate using three pooled human plasma samples, each derived from three distinct healthy donors. The x-axis shows time in hours; the y-axis shows the fraction of exchange as calculated from the area under peak 3 in ion exchange chromatograms (see Figure 1C for a sample chromatogram and the Materials and Methods for the procedure to calculate fraction exchange). (A) Subunit exchange time courses in the presence of kinetic stabilizers at a concentration of 30 ?M. (B) As in panel A, but at a stabilizer concentration = 20 ?M. (C) As in panel A, but at a stabilizer concentration = 10 ?M. (D) As in panel A, but at a stabilizer concentration = 5 ?M. (E) As in panel A, but at a stabilizer concentration = 1 ?M. The same DMSO control time course is shown in each plot. Red diamonds = tafamidis; blue squares = AG10; inverted green triangle = tolcapone; purple circle = diflunisal; black triangle = DMSO control. Data points represent the mean and error bars the standard deviations of the triplicate data. The curves in each plot represent the best fits to the data based on the model described in the text.Supplemental Figure S2. TTR subunit exchange kinetics in the presence of kinetic stabilizers at varying concentrations, experiment 2. As described in the legend of Supplemental Figure S1, except that measurements were performed in technical duplicate using a single pooled human plasma sample derived from six distinct donors. Red diamonds = tafamidis; blue squares = AG10; inverted green triangle = tolcapone; purple circle = diflunisal; black triangle = DMSO control. Data points represent the mean and error bars the range of the duplicate data. The curves in each plot represent the best fits to the data based on the model described in the text.Supplemental Figure S3. Purity of kinetic stabilizers as assessed by HPLC. Reverse phase HPLC traces of (A) diflunisal, (B) tafamidis, (C) tolcapone and (D) AG10 demonstrating their purity. Peaks were detected by absorbance at 254 nm (similar chromatograms were obtained with detection at 280 nm and 360 nm). 756046133163700Supplemental Figure S4. 1H NMR (500 MHz, MeOD) of synthesized AG10: δ 7.68 – 7.61 (m, 2H), 7.19 (dd, J = 10.9, 8.4 Hz, 1H), 4.03 (t, J = 5.9 Hz, 2H), 2.61 (t, J = 7.3 Hz, 2H), 2.15 (s, 6H), 2.01 – 1.94 (m, 2H).80946990678000Supplemental Figure S5. 13C NMR (151 MHz, MeOD) of synthesized AG10: δ 168.87, 157.79, 156.12, 148.30, 148.23, 143.34, 128.71, 128.68, 124.33, 124.28, 116.97, 116.84, 116.80, 116.78, 115.18, 68.89, 30.67, 19.68, 10.43.Supplemental Figure S6. HR-MS (ESI-TOF) of synthesized AG10: AG10 was analyzed on an Agilent 6230 TOF LC/MS with a Dual AJS ESI ion source mass spectrometer coupled to an Agilent 1200 series LC stack. A DMSO stock solution of AG10 (3 ?L) was directly infused into the instrument in a mobile phase consisting of 80% acetonitrile / 0.1% formic acid and 20% Water / 0.1%formic acid. M/Z: calcd for C15H18FN2O3: 293.1296; found 293.1304 (M+H)+.Supplemental Figure S7. The extent of TTR subunit exchange as a function of various kinetic stabilizer concentrations after 48 h. Fraction exchange after 48 h for endogenous TTR (~ 3 ?M) in plasma with added dual-flag tagged WT-TTR (1 ?M) in the presence of kinetic stabilizers at varying concentrations using the triplicate data from “experiment 1” and the duplicate data from “experiment 2”. (A) Stabilizer concentration = 30 ?M. (B) Stabilizer concentration = 20 ?M. (C) Stabilizer concentration = 10 ?M. (D) Stabilizer concentration = 5 ?M. (E) Stabilizer concentration = 1 ?M. Error bars represent the standard deviation from the combined data from experiments 1 and 2. Red bars = tafamidis; blue bars = AG10; green bars = tolcapone; purple bars = diflunisal; gray bars = DMSO control. The same DMSO control is shown in each chart. ................
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