Article Figures & Data
Figures
- Fig. 1.
Inhibition of CRT-1-mediated [3H]creatine uptake. (A) Structure of creatine and of commercially available nitrogenous compounds, which were screened for inhibition of CRT-1. GAA = guanidinoacetate; GPA = guanidinopropionate; GBA = guanidinobutyrate; ATPCA = 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid; 1 = 2-(N-benzylcarbamimidamido)acetic acid; 2 = 2-(2H-1,2,3-triazol-2-yl)acetic acid; 3 = 2-(5-amino-2H-1,2,3,4-tetrazol-2-yl)acetic acid; 4 = 2-(5-amino-3-methyl-1H-pyrazol-1-yl)acetic acid; 5 = 3-amino-1-(carboxymethyl)-1H-1,2,4-triazole-5-carboxylic acid; 6 = 4-(carbamimidamidomethyl)benzoic acid; 7 = (2S)-2-carbamimidamidopentanedioic acid; 8 = 3-amino-1,2,4-triazole-5-carboxylic acid hemihydrate; 9 = 2-aminobenzimidazol; 10 = 4-guanidinobenzoic acid. (B) Inhibition of [3H]creatine-uptake by HEK293 cells transiently expressing CRT-1 by 1 mM of various nitrogenous compounds (Cyclocr = cyclocreatine; Gua-Succ = guanidio-succinate). Specific [3H]creatine uptake in the presence of these compounds was normalized to the uptake in their absence. Control uptake (Ctrl) amounted to 9.16 ± 1.54 pmol min−1.10−6 cells and set to 100% to account for for interassay variations. Bars represent arithmetic means ± S.D. from three independent experiments. (C) Uptake of [3H]creatine by HEK293 cells transiently expressing CRT-1 in presence of increasing concentrations of various guanidinocarboxylic acids. Specific [3H]creatine uptake in the presence of inhibitors was normalized to the uptake in their absence. This control uptake was 10.4 ± 4.4 pmol min−1.10−6 cells and set to 100% to account for interassay variations. The lines were drawn by fitting the data points to the equation for a monophasic inhibition curve. The extracted concentrations required for IC50 are given in Table 1. Data are arithmetic means from three independent experiments; error bars indicate S.D.
- Fig. 2.
Patch clamp-recordings of currents elicited by creatine and creatine analogs in HEK293 cells expressing CRT-1. (A) The schematic representation illustrates the approach to measuring transporter-associated currents: for each translocated substrate CRT-1 cotransports 1 Cl− and 2 Na+ ions. If the substrate is electroneutral (such as creatine), this results in a net movement of one positive charge. Continuous transporter cycling thus leads to steady-state currents, which can be recorded by the patch-clamp technique. The scheme also indicates the extra- and the intracellular ion concentrations and the membrane potential used in panels (B) and (C). (B) Representative current traces measured by patch-clamping of CRT-1 expressing (left hand side) and untransfected (right hand side) HEK293 cells: cells were first superfused with a saturating concentration (1 mM) of creatine (Cr). After subsequent washout with external solution cells were then subjected to saturating concentrations of 3 mM GAA, 1 mM GPA, 1 mM ATPCA, 3 mM GBA, 1 mM cyclocreatine or 1 mM compound 1. White boxes indicate the superfusion steps. (C) Amplitudes of steady state currents through CRT-1 elicited by GAA (n = 9; mean ± S.D.: −11.3 ± 4.1 pA), GPA (n = 5; −19.4 ± 11.5 pA), GBA (n = 9; −9.1 ± 3.3 pA), ATPCA (n = 5; −8.3 ± 3.4 pA), cyclocreatine (n = 5; −17.9 ± 12.7 pA) and compound 1 (n = 18; −0.25 ± 1.06 pA) in HEK293 cells transfected with CRT-1 at the concentrations given in (B). Amplitudes were paired to current amplitudes triggered by 1 mM creatine for each individual cell: statistically significant differences were found only for compound 1 (Wilcoxon signed rank test, P < 0.0001, ****).
- Fig. 3.
Interaction of CRT-1 with creatine and the active compounds identified as substrates in present study. (A) The homology model structure of human CRT-1 used for the docking procedure is based on the structure of human SERT (Clarke et al., 2024); the best docked poses of the nine novel compounds are overlaid in the S1 binding site. The ligands are colored in tan (with nitrogens and oxygens in blue and red, respectively). CRT-1 is shown in ribbon representation. The helices are colored in tan, with the exception of the helices lining the S1, which are colored according to the following scheme: TM1 (green), TM3 (orange), TM6 (blue), TM8 (violet), and TM10 (pink). (B) Legend for the ligand interaction diagrams shown in C–H. (C) Docking of creatine into the S1 binding site of CRT-1, as determined by Clarke et al. (2024), shown in a three-dimensional representation (left); the two-dimensional map (right) visualizes the nature of the interactions. Helix coloring is according to the scheme in (A). Clarke et al. (2024) identified two major binding poses for creatine, which are shown here and designated pose 1 (top) and pose 2 (bottom). (D–H) Top docked pose for each of the novel compounds identified as substrates, according to the HADDOCK docking score and distance calculations (see Methods). The pose is shown in three dimensions on the left, with the helices colored according to (A) and the ligand colored in tan. Two-dimensional interaction maps are shown on the right. Grey spheres indicate atoms and functional groups on the ligands, which are solvent exposed.
- Fig. 4.
Interaction of CRT-1 with creatine and the active compounds identified as blockers in the present study. Top docked poses of the three true inhibitors, i.e., compound 1 (A) and the two enantiomers of MIPA572 (B, C) and of MIPA573(D, E). Docking to the SERT-based homology model of CRT-1 was done as outlined in the legend to Fig. 3A and under Materials and Methods. Helices and the ligand interaction diagrams are colored as in Fig. 3.
- Fig. 5.
Schematic representation of the synthesis and structures of the designed and synthesized novel guanidinocarboxylic acids (A) and substrate uptake by (B, C) and transport-associated currents through CRT-1 (D) in the presence of candidate CRT-1 ligands. (A) The synthetic procedures are described in detail under Materials and Methods. (B) Inhibition of [3H]creatine uptake into HEK293 cells transiently expressing CRT-1 by the newly synthesized candidate compounds shown in Fig. 3. Specific uptake of [3H]creatine uptake was measured in the absence [buffer control = Ctrl (KRH) and DMSO control = Ctrl (DMSO)] and in the presence of these compounds (1 mM) was divided by the uptake in their absence. Control uptake (Ctrl) amounted to 8.99 ± 1.96 pmol min−1.10−6 cells (standard) and was set to 100% to account for interassay variation. MIPA573 was dissolved in DMSO; as a reference, uptake was also determined in the presence of 2% DMSO to account for carryover (DMSO); this reference amounted to (and 7.29 ± 1.03 pmol min−1 10−6 cells. Bars show arithmetic means ± S.D. from four independent experiments. (C) Specific [3H]creatine uptake by HEK293 cells heterologously expressing CRT-1 in the presence of increasing concentrations of MIPA572, MIPA573, MIPA574, GiDi1254 and GiDi1257. Specific [3H]creatine uptake in the presence of (putative) inhibitors was normalized to the uptake in their absence. This control uptake was 7.9 ± 1.8 pmol min−1.10−6 (buffer control) and 6.8 ± 1.5 pmol min−1 10−6 (DMSO 2%) and set to 100% to account for interassay variations. The data points were fitted to the equation for a monophasic inhibition curve. The IC50 values were estimated as 1.19 ± 0.01 mM, 500 ± 57 μM, 1.15 ± 0.15 mM, 390 ± 93 μM and 14.8 ± 2.4 μM for MIPA572, MIPA573, MIPA574, GiDi1254, and GiDi1257, respectively. Because the concentration of [3H]creatine used in this experiment (0.01 μM) was far below its KM, the IC50 values are virtually identical to Ki values. Data are arithmetic means from four independent experiments; error bars indicate S.D. (D) Patch-clamp recordings of transport-associated currents through CRT-1. Currents were recorded in untransfected HEK293 cells and in HEK293 cells transiently expressing CRT-1 in the cell-attached configuration as outlined in Fig. 2A. Cells were first superfused with a saturating concentration (500 μM) of creatine (Cr). Following washout of creatine with external solution, cells were then superfused with saturating concentrations (given above the white boxes, which indicate the superfusion steps) of MIPA572 (n = 9), MIPA573 (n = 19), MIPA574 (n = 10), GiDi1253 (n = 11), GiDi1254 (n = 12), and GiDi1257 (n = 21). Representative traces are shown on the left-hand side for each compound; the right-hand spaghetti plots compare the amplitudes of the steady-state currents recorded in the presence of individual compounds to those elicited by creatine in the same cell. Statistically significant differences were found by the Wilcoxon signed-rank test between creatine and GiDi1253 (P = 0.001), GiDi1254 (P = 0.002), GiDi1255 (0.0005), MIPA572 (P = 0.0039), and MIPA573 (P < 0.0001); n.s., not significant, *P ≤ 0.01; ***P ≤ 0.001, ****P ≤ 0.0001.
- Fig. 6.
Inhibition of creatine uptake in hippocampal neurons of neonatal rats by compound 1. Rat hippocampal neurons were transfected with a plasmid encoding the fluorescent protein PercevalHR (referred to as “Perceval”), which allows for monitoring the ratio of cytosolic ATP/ADP by recording fluorescence (F488/F403) emitted at 525 nm after excitation at 488 nm and 403 nm. (A) Change in ATP/ADP ratio induced by superfusion of hippocampal neurons expressing PercevalHR neurons with 10 μM of the creatine kinase inhibitor FDNB: the actual ATP/ADP ratio F was normalized to F0, i.e., the F488/F403 recorded at time point 0 prior to addition of FDNB (application indicated by black bar). Shown are the means ± S.D. recorded in independent experiments from three neurons. (B and C) Change in ATP/ADP ratio upon superfusion of hippocampal neurons with compound 1. Neuronal cultures were allowed to equilibrate after removal of the medium by superfusion with (creatine-free) buffer. The ATP/ADP-ratio (F488/F403) of neurons expressing PerecvalHR was recorded for 2 minutes to verify a stable baseline; thereafter, superfusion with compound 1 (5 mM) was initiated and the ATP/ADP-ratio (F488/F403) was continuously recorded. Data are means ± S.D. recorded in independent experiments from 11 neurons. The actual ATP/ADP ratio F was normalized to F0 as in panel A. The spaghetti plot in panel C summarizes the F488/F403 ratio for each individual neuron recorded prior to addition of compound 1 (-compound 1) and after 400 seconds of superfusion with compound 1 (+compound 1). The difference was statistically significant (paired t test, P = 0.0088; ***P ≤ 0.01). (D and E) Change in ATP/ADP-ratio of hippocampal neurons superfused with 250 μM creatine in the absence and presence of 5 mM compound 1. Neurons were first superfused for 8 minutes in the absence and presence of compound 1 (5 mM) as shown in panel C to reach a stable baseline fluorescence ratio (the last 200 seconds of this equilibration period are shown). Thereafter, the superfusion with 250 μM creatine (indicated by black bar) was initiated in the absence (black trace) and continuous presence of 5 mM compound 1 (blue trace, Com1). The F488/F403 ratio was normalized as in panel A to the F488/F403 ratio recorded at time point 0 (“F0”, i.e., prior to the start of the superfusion with creatine). Sown are the means ± S.D. recorded in independent experiments from 11 and 12 neurons in the absence and presence of compound 1, respectively. In pane E, the initial ATP/ADP-ratio, i.e., prior to addition of creatine, and the ATP/ADP-ratio after 600 seconds was plotted for each individual neuron imaged in panel D (- creatine = time point 0; + creatine = time point 400 seconds later). A statistically significant difference was only seen in the absence of compound 1 (paired t test, P = 0.001; ****; n.s., not significant).
Tables
- TABLE 1
Inhibitor constants for inhibition of CRT-1 mediated or tissue creatine uptake determined experimentally and compiled from the literature
Experiment Literature Ki (μM) Ki (μM) KM (creatine) (μM) Reference GAA 106.7 ± 14.8 494 46 Dodd et al., 2010 3000 500 Fitch et al., 1968 GPA 15.7 ± 1.4 200 500 Fitch et al., 1968 450 21 Ku and Passow, 1980 23 ± 4 46 Schloss et al., 1994 22 20 Dai et al., 1999 9 — Al-Khawaja et al., 2018 19 ± 1 48 ± 7 Farr et al., 2022 GBA 108.4 ± 18.8 349 20 Dai et al., 1999 Compound 1 407.8 ± 52.1 — — Cyclocreatine — 185 20 Dai et al., 1999 167 ± 10 48 ± 7 Farr et al., 2022 ATPCA — 65 — Al-Khawaja et al., 2018 105 ± 7 48 ± 7 Farr et al., 2022 N-ethyl-GAA — 1000 500 Fitch et al., 1968 ATPCA II — 670 — Al-Khawaja et al., 2018 Taurocyamine/GES — 1377 20 Dai et al., 1999 Ki values (±S.D.) of CRT-1 mediated [3H]creatine uptake given in the second column (from left) were obtained from the experiments described in Fig. 1C. The third column from the left lists Ki values obtained from the literature (IC50 values obtained from the literature were converted to Ki values using the Cheng-Prusoff equation). The affinity of creatine determined in the respective studies (fourth column) serve as an interassay comparator. Italic references (Fitch et al., 1968; Ku and Passow, 1980 and Dodd et al., 2010) report endogenous creatine uptake by rat skeletal muscle, erythrocytes and hippocampal neurons, respectively. In the other references (Schloss et al., 1994; Dai et al., 1999; Al-Khawaja et al., 2018; Farr et al., 2022), creatine uptake was determined in cells heterologously expressing CRT-1. ATPCA II = 6-amino-2,3,4,5-tetrahydropyridine-3-carboxylic acid hydrochloride; GES, guanidinoethane sulfate.
Data Supplement
- Supplemental Data -
Supplementary Figure 1. Structures of compounds previously reported to inhibit CRT 1 or creatine uptake.
Supplementary Table 1. Compilation of compounds, which were tested for inhibition of creatine uptake and inhibited uptake by ≥25%.
Supplementary Table 2. Compilation of compounds, which were tested for inhibition of creatine uptake and failed to suppress uptake by ≥20%.
Supplementary Figure 2. Mapping of ligand interaction sites.
- Supplemental PDB -
ATPCA.
- Supplemental PDB -
Compound1.
- Supplemental PDB -
GiDi1254.
- Supplemental PDB -
GiDi1257.
- Supplemental PDB -
hCRT1_Homology_Model.
- Supplemental PDB -
MIPA574.
- Supplemental PDB -
R-enantiomer_MIPA573.
- Supplemental PDB -
R-enantiomer_MIPA574.
- Supplemental PDB -
S-enantiomer_MIPA572.
- Supplemental PDB -
S-enantiomer_MIPA573.
- Supplemental PDB -
R-enantiomer_MIPA572.
- Supplemental Data -
