Quantification of transthyretin kinetic stability in human plasma using subunit exchange

Abstract

The transthyretin (TTR) amyloidoses are a group of degenerative diseases caused by TTR aggregation, requiring rate-limiting tetramer dissociation. Kinetic stabilization of TTR, by preferential binding of a drug to the native tetramer over the dissociative transition state, dramatically slows the progression of familial amyloid polyneuropathy. An established method for quantifying the kinetic stability of recombinant TTR tetramers in buffer is subunit exchange, in which tagged TTR homotetramers are added to untagged homotetramers at equal concentrations to measure the rate at which the subunits exchange. Herein, we report a subunit exchange method for quantifying the kinetic stability of endogenous TTR in human plasma. The subunit exchange reaction is initiated by the addition of a substoichiometric quantity of FLAG-tagged TTR homotetramers to endogenous TTR in plasma. Aliquots of the subunit exchange reaction, taken as a function of time, are then added to an excess of a fluorogenic small molecule, which immediately arrests further subunit exchange. After binding, the small molecule reacts with the TTR tetramers, rendering them fluorescent and detectable in human plasma after subsequent ion exchange chromatography. The ability to report on the extent of TTR kinetic stabilization resulting from treatment with oral tafamidis is important, especially for selection of the appropriate dose for patients carrying rare mutations. This method could also serve as a surrogate biomarker for the prediction of the clinical outcome. Subunit exchange was used to quantify the stabilization of WT TTR from senile systemic amyloidosis patients currently being treated with tafamidis (20 mg orally, once daily). TTR kinetic stability correlated with the tafamidis plasma concentration.

Figure 1

Subunit exchange measures the stability of recombinant TTR tetramers. (A) The top left panel shows a ribbon diagram of the TTR tetramer with individual subunits shown in different colors. Arrowheads indicate the thyroxine-binding pockets. The top right panel shows a cartoon of the same tetramer. The weak dimer–dimer interface is indicated with a dotted line. The bottom panel shows a schematic of the steps involved in dissociation, reassociation, and subunit exchange of untagged WT TTR subunits (green squares) and dual-FLAG-tagged (FT₂) WT TTR subunits (orange squares). (B) Recombinant WT and FT₂·WT TTR were mixed in equal amounts and separated by ion exchange chromatography after exchange for 7 days. The initial populations of homotetramers mix to create heterotetramers incorporating zero to four FT₂·WT TTR subunits. Green squares represent untagged WT TTR subunits and orange squares FT₂·WT TTR subunits. (C) To quantify subunit exchange, the relative area of peak 1 at time zero is calculated and used in the binomial equation to calculate the expected area of peak 3 at equilibrium. At each time point, the fraction exchange is calculated by comparing the actual relative area of peak 3 at that time point to the expected relative area of peak 3 at equilibrium (see the text for details).

Figure 2

Subunit exchange is a robust measure of TTR tetramer kinetic stability at various ratios of tagged to untagged tetramer concentrations. (A) After incubation in standard phosphate buffer for 8 days, different ratios of the starting concentrations of WT and FT₂·WT TTR yield different ratios of the five tetramer peaks. (B) The kinetics of subunit exchange, as measured by fraction exchange of peak 3, were followed over a time course of 8 days for the indicated ratios of WT to FT₂·WT TTR. Symbols represent means; error bars represent the SEM, and lines represent fitted curves. (C) The time course of appearance or disappearance of the five tetramer peaks in absolute terms varies according to the molar ratio of the initial populations of WT and FT₂·WT TTR (top panels). However, the time course of the increase in fraction exchange calculated for each peak is very similar (bottom panels): (left) 5 μM WT TTR and 5 μM FT₂·WT TTR, (middle) 5 μM WT TTR and 2 μM FT₂·WT TTR, and (right) 5 μM WT TTR and 1 μM FT₂·WT TTR. Symbols represent means; error bars represent the SEM, and lines represent fitted curves.

Figure 3

A2 can be used to quantify TTR subunit exchange in blood plasma. (A) Samples were incubated with A2 or vehicle control for 3 h at 25 °C. The recombinant WT TTR homotetramer peak in standard phosphate buffer (···) is detected at an elution time of 4.5 min. In healthy donor plasma (green line), a similar peak is detected at 4.25 min. This TTR·A2 conjugate peak is absent in plasma from a TTR knockout (KO) mouse (orange line) and in human plasma that was incubated with vehicle without A2 (purple line). In all plasma samples, A2 also detects a peak eluting around 1–3 min (peak 0). The inset shows the structure of A2. (B) Healthy donor plasma was incubated at 25 °C without (red) or with 10 μM tafamidis (black). At time zero, A2 (30 μM) was added, and the sample was diluted with 5 volumes of sodium phosphate buffer (50 μM, pH 7.6) and immediately placed in the UPLC autosampler to acquire the first time point. Subsequent time points were acquired automatically from the same vial at the indicated times. The area of peak 1 was calculated for each time point. Symbols represent means; error bars represent the SEM. (C) At time zero, 1 μM FT₂·WT TTR was added to healthy donor plasma to initiate the subunit exchange reaction. An aliquot was immediately removed, and A2 was added at a final concentration of 30 μM (A2 at 0 h, solid green line). Both samples were incubated at 25 °C for 24 h. At 24 h, a second aliquot was removed and A2 was added at a final concentration of 30 μM and the mixture incubated at 25 °C for 3 h to allow for complete covalent modification of TTR by A2 (A2 at 24 h, dotted black line). Each sample was then analyzed by ion exchange chromatography. (D) Recombinant WT TTR (5 μM) was incubated with FT₂·WT TTR (5 μM) for 96 h. At each time point, subunit exchange was assessed by measuring Trp fluorescence (solid black line) or A2·TTR conjugate fluorescence (dotted red line). Symbols represent means; error bars represent the SEM, and lines represent fitted curves. (E) Healthy donor plasma was incubated with FT₂·WT TTR for 21 days. The sample was incubated with A2 (30 μM) and then separated by ion exchange chromatography (top panel). Peaks were collected from four identical injections, pooled, concentrated, and separated by SDS–PAGE followed by Western blotting for TTR or FLAG-tagged TTR (bottom panels). The expected pattern of WT and FT₂·WT TTR monomers is detected in peaks 1–5. Total human plasma (Plsm) was included as a control.

Figure 4

The small molecule tafamidis stabilizes TTR tetramers when added ex vivo in human plasma. (A) Subunit exchange was assessed in the presence of 1 μM FT₂·WT TTR in plasma from healthy normal donors (orange) and in standard phosphate buffer containing equal concentrations of recombinant WT and FT₂·WT TTR (green). Symbols represent individual data points; lines represent fitted curves. (B) Recombinant WT TTR (3 μM) was added to endogenous WT TTR (3.8 μM) in a sample of healthy donor plasma (red). FT₂·WT TTR (1 μM) was then added, and subunit exchange was assessed at the indicated time points. Recombinant WT TTR (3 μM) was also incubated with 1 μM FT₂·WT TTR in standard phosphate buffer (green) as a control. The averages of the data for recombinant TTR in standard phosphate buffer (dark gray) and for endogenous TTR in plasma (light gray) from panel A are shown for comparison. Symbols represent individual data points; lines represent fitted curves. (C) Recombinant WT TTR (4 or 3 μM, as indicated) was mixed with FT₂·WT TTR (1 μM) and the indicated concentrations of tafamidis in buffer, and subunit exchange was assessed at the indicated time points. Symbols represent individual data points; lines represent fitted curves. (D) Tafamidis was added ex vivo to healthy donor plasma at the indicated concentrations and incubated for 30 min at room temperature. FT₂·WT TTR (1 μM) was then added, and subunit exchange was assessed at the indicated time points. Symbols represent individual data points; lines represent fitted curves. Rate constants and relative fraction exchange were calculated for each condition and are listed in Table 2. (E) Data from panels C and D and Figure S4 of the Supporting Information were graphed on the same set of axes to highlight the difference in the sensitivity of TTR, in buffer and in plasma, to kinetic stabilization by tafamidis.

Figure 5

Sensitivity of TTR subunit exchange to incubation temperature is attenuated by tafamidis stabilization. Tafamidis was added at the indicated concentrations to plasma from three healthy donors and incubated for 30 min at room temperature. Each sample was then split into two aliquots for incubation at 25 or 37 °C. FT₂·WT TTR (1 μM) was added to each aliquot. (A) Subunit exchange was assessed at the indicated time points over a time course of 8 days. Filled symbols and solid lines represent data from 25 °C; empty symbols and dotted lines represent data from 37 °C. Symbols represent means; error bars represent the SEM, and lines represent fitted curves. (B) The rate of tetramer dissociation (k_ex) was calculated for each sample using a least-squares fit to a one-phase association model in GraphPad Prism. Filled symbols represent data from 25 °C; empty symbols represent data from 37 °C, and error bars represent the SEM.

Figure 6

TTR kinetic stabilization by orally administered tafamidis can be measured directly in patient plasma. (A and B) Plasma samples were obtained from SSA patients treated with 20 mg of tafamidis once daily (SSA + tafamidis, green triangles), SSA patients not treated with tafamidis (SSA no tafamidis, orange squares), and age-matched controls (WT ctrls, black circles). For each sample, subunit exchange was quantified at 48 (A) and 96 h (B), and fraction exchange was calculated as described. Differences between groups were assessed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. (C) The rate of tetramer dissociation (k_ex) was calculated for each sample from each time point using the equation k_ex,t=t = −ln(1 – FE_t=t)/t. The k_ex values calculated from the 48 h subunit exchange incubation (filled symbols) and from the 96 h subunit exchange incubation (empty symbols) were plotted on the same set of axes, with each individual patient represented by a different color. Differences between groups were assessed by one-way ANOVA followed by Tukey’s post hoc test. (D) The k_ex values calculated from the 48 h subunit exchange data were plotted as a function of the concentration of tafamidis measured in each sample by HPLC. The data (excluding the outlier with a calculated k_ex of 0) were fit by a linear regression model, yielding a calibration line with the equation y = −0.00014x + 0.00318 (R² = 0.929).