Article Figures & Data
Figures
- Fig. 1.
PF-06649298 and PF-06761281 behave as state-dependent or mixed uncompetitive/noncompetitive SLC13A5 inhibitors. (A) Structure of PF-06649298. (B) 14C-citrate uptake inhibition using 20 μM 14C-citrate in the presence or absence of 230 μM cold citrate by PF-06649298 or PF-06761281. (C) Inhibition of 20 μM 14C-citrate uptake by increasing concentrations of cold citrate, as indicated on the graph, in the presence or absence of PF-06649298 or PF-06761281. Data are normalized to maximal uptake for each condition. (D) Structure of PF-06761281. (E) 14C-citrate uptake using 20 μM 14C-citrate in the presence of increasing concentrations of cold citrate, as indicated on the graph, and in the presence or absence of PF-06649298. (F) C14-citrate uptake using 20 μM 14C-citrate in the presence of increasing concentrations of cold citrate, as indicated on the graph, in the presence or absence of PF-06761281. Corresponding IC50 values are represented in Tables 1 and 2 (averages ± S.E.M. from three independent experiments).
- Fig. 2.
PF-06649298 and PF-06761281 are low-affinity partial substrates but potent SLC13A5 inhibitors in the membrane depolarization assay. (A) Concentration-dependent membrane depolarization induced by citrate (216 ± 13 μM; n = 5) and succinate (IC50 = 1018 ± 275 μM; n = 3) in HEK293TRex-TO/hSLC13A5 cells. (B) Representative traces showing the citrate-, PF-06649298-, and PF-0676128-induced depolarization (increase in fluorescence) in the FLIPR Tetra. Note that membrane depolarization induced by citrate is sustained, but responses to PF-06649298 and PF-06761281 are considerably smaller and transient in nature. (C) Concentration dependence of the PF-06649298- and PF-06761281-induced depolarization. Both compounds are weak substrates inducing <25% of the citrate-induced membrane depolarization. Symbols represent the mean ± S.E.M. for n = 4 independent experiments. (D) Concentration-dependent inhibition of citrate (400 μM)-induced depolarization after preincubation with PF-06649298 (IC50 = 0.74 ± 0.23 μM) and PF-6761281 (IC50 = 0.36 ± 0.05 μM). Symbols represent the mean ± S.E.M. for n = 3 independent experiments. Corresponding EC50 and IC50 values are represented in Table 3 (averages ± S.E.M. from three independent experiments).
- Fig. 3.
PF-06649298 and PF-06761281 inhibition is highly dependent on citrate concentration in the membrane potential assay. Representative traces showing the 1.25 mM citrate-induced depolarization in HEK293TRex-TO/hSLC13A5 cells in the presence of vehicle, 0.8 μM PF-06649298 (A) or 0.3 μM PF-0676128 (B). The effect of various concentrations of PF-06649298 (C) or PF-0676128 (D) on the citrate concentration–response curve. Note that inhibition of citrate-induced depolarization is highly dependent on the citrate concentration for both compounds. Symbols represent the mean ± S.E.M. for n = 3 independent experiments.
- Fig. 4.
PF-06649298 and PF-06761281 induce transient transporter currents in HEK293TRex-TO/hSLC13A5. Representative recordings are shown in (A)–(E). Citrate or test compound was applied for 1 s to cells held at −60 mV. Currents induced by PF-06649298 and PF-06761281 were normalized to the peak current elicited by citrate in the same cell, and the average ± SEM normalized current is plotted against the compound concentration in (F). EC50 values for peak responses were approximately >300 and 124 μM, respectively, for PF-06649298 and PF-06761281. Control = 0.3% DMSO. Symbols represent the mean ± S.E.M. for n = 2-3 cells/concentration.
- Fig. 5.
PF-06649298 and PF-06761281 inhibit hSLC13A5 in a concentration- and time-dependent manner when applied extracellularly. Representative recordings are shown in (A) and (B). The 10 mM citrate-induced currents were elicited in the absence of PF-06649298 and PF-06761281, and again in the presence of PF-06649298 and PF-06761281 after ∼5 minutes of preincubation with the inhibitors. Citrate or citrate plus compound was applied for 10 seconds to cells held at −60 mV. For both compounds, block was modest at the start of the 10-second citrate application but increased during the 10-second citrate application. The percentage of inhibition by each compound was calculated at the start (peak) and the end of the citrate response [steady-state (ss)] and plotted against the compound concentration in (C). The percentage of inhibition in time-matched control experiments (5-minute incubation with buffer or 0.1% DMSO) is also shown (control). Symbols represent the mean ± S.E.M. for n = 2-4 cells/concentration.
- Fig. 6.
PF-06649298 and PF-06761281 inhibit hSLC13A5 in a concentration- and time-dependent manner when applied intracellularly. Representative recordings are shown in (A) and (B). PF-06649298 (50 μM) and PF-06761281 (50 μM) were included in the pipette solution, and 10 mM citrate-induced currents were elicited every 60 seconds for 15 minutes, starting immediately after breakin (∼0 minutes). Citrate was applied for 10 seconds to cells held at −60 mV. Block developed over time (with dialysis). At ∼15 minutes, the block for both was modest at the start of the 10-second citrate application but increased during the 10-second citrate application. The percentage of inhibition by each compound was calculated at the start (peak) and the end of the citrate response [steady state (ss)] and was plotted against the compound concentration in (E). For time-matched controls (control), pipettes contained either KF solution or KF plus 0.1% DMSO. Symbols represent the mean ± S.E.M. for n = 4–9 cells/concentration (except n = 1 for 5 μM). A two-pulse protocol [see inset below (C) and (D)] was used to measure recovery of peak responses after the removal of citrate. The first pulse was 10 mM citrate long enough (5 seconds) to allow block to develop to inhibitors. A second 1-second pulse of 10 mM citrate was applied after a variable (1–10 seconds) washout period to evaluate recovery from block. The entire protocol was run in the continuous presence of 50 μM PF-06649298 and PF-06761281 in the pipette solution [depicted by open bars above (C) and (D)]. Symbols represent the mean ± S.E.M. for n = 3 cells/compound. Representative recordings are shown in (C) and (D). The percentage of recovery was plotted against the recovery interval in (F). Recovery from block occurs rapidly after the removal of citrate for both compounds. Tau values were 2.7 and 2.0 seconds, respectively, for PF-06649298 and PF-06761281.
- Fig. 7.
Cartoon summarizing the proposed mechanism of action of PF-06649298 and PF-06761281 on hSLC13A5. At rest, SLC13A5 resides in an outward-facing conformation (A). Citrate (green circles) and PF compounds (red circles) bind to the outward-facing conformation with relatively low affinity (B) and are transported into the cell. Inside the cell, citrate and PF compounds dissociate from the inward facing transporter (C), and the resulting unbound, inward-facing transporter (D) returns to the resting conformation. Due to their polar nature and low membrane permeability, PF compounds accumulate inside the cell, where they can preferentially interact with an allosteric site on the substrate-bound, inward-facing transporter to inhibit transporter function (C*).
Tables
IC50 (μM) in presence of 20 μM C14-citrate 20 μM C14-citrate + 230 μM cold citrate PF-06649298 10.5 ± 3.0 0.58 ± 0.07 PF-06761281 3.4 ± 1.8 0.67 ± 0.09 Data are averages ± S.E.M. from three independent experiments combined.
- TABLE 2
Citrate potency in the 20 μM C14-citrate uptake assay in the absence or presence of various concentrations of PF-06649298 and PF-06761281
Vehicle PF-06649298 5 μM PF-06649298 2 μM PF-06761281 2 μM PF-06761281 1 μM IC50 (μM) 745 ± 111 196 ± 3**** 422 ± 36** 112 ± 11**** 226 ± 12*** Hill slope 2.25 ± 0.12 1.02 ± 0.02 0.86 ± 0.04 0.97 ± 0.02 1.3 ± 0.06 Data are averages ± S.E.M. from three independent experiments with P values.
↵** P < 0.01; *** P < 0.001; **** P < 0.0001, one-way ANOVA with Dunnett post hoc test.
EC50 agonist mode membrane potential (μM) Citrate Succinate PF-06649298 PF-06761281 216 ± 13 1018 ± 275 N.D. 4 ± 2 IC50 antagonist mode membrane potential (μM) Citrate Succinate PF-06649298 PF-06761281 N.D. N.D. 0.74 ± 0.23 0.36 ± 0.05 N.D., not determined. Data are averages ± S.E.M. from three independent experiments.
Data Supplement
Files in this Data Supplement:
- Supplemental Figures -
Supplemental Figure 1 - Functional validation of HEK293TRex-TO/hSLC13A5 cells
Supplemental Figure 2 - Succinate is a competitive inhibitor of citrate uptake at hSLC13A5
- Supplemental Figures -
In this issue
State-Dependent Inhibition of SLC13A5
