Pyrazolyl-pyrimidones inhibit the function of human solute carrier protein SLC11A2 (hDMT1) by metal chelation

Abstract

Solute carrier proteins (SLCs) control fluxes of ions and molecules across biological membranes and represent an emerging class of drug targets. SLC11A2 (hDMT1) mediates intestinal iron uptake and its inhibition might be used to treat iron overload diseases such as hereditary hemochromatosis. Here we report a micromolar (IC₅₀ = 1.1 μM) pyrazolyl-pyrimidone inhibitor of radiolabeled iron uptake in hDMT1 overexpressing HEK293 cells acting by a non-competitive mechanism, which however does not affect the electrophysiological properties of the transporter. Isothermal titration calorimetry, competition with calcein, induced precipitation of radioactive iron and cross inhibition of the unrelated iron transporter SLC39A8 (hZIP8) indicate that inhibition is mediated by metal chelation. Mapping the chemical space of thousands of pyrazolo-pyrimidones and similar 2,2'-diazabiaryls in ChEMBL suggests that their reported activities might partly reflect metal chelation. Such metal chelating groups are not listed in pan-assay interference compounds (PAINS) but should be checked when addressing SLCs.

Fig. 1. Structure and reported activity of DMT1 inhibitors. Data for inhibition of Fe²⁺ uptake into transfected HEK cells measured by calcein fluorescence assay (1–3) from ref. 14 and 15 or radioactive ⁵⁵Fe²⁺ uptake assay (4 and 5) from ref. 17.

Scheme 1. Representative synthesis of pyrazolyl-pyrimidones at the example of 13 and 19.

Fig. 2. Chemical structures and X-ray crystallography of analogs of pyrazole 5. Carbon is in gray, nitrogen in blue, oxygen in red and chlorine in green.

Fig. 3. Characterization of Fe²⁺ transport inhibition by 1 and 13 in X. laevis oocytes (collected from frogs under license number BE60/18 conceded by the Veterinärdienst des Kantons BE, Sekretariat Tierversuche). (a) Upper panel: Average ⁵⁵Fe²⁺-uptake (20 μM) by hDMT1-expressing and non-injected oocytes in the absence and the presence of the indicated compounds (10 μM). Data was normalized to ⁵⁵Fe²⁺-uptake (pmol min^–1) in the absence of compounds represented as mean ± SD (16–29 oocytes). Lower panel: Net uptake of ⁵⁵Fe²⁺ (20 μM) by hDMT1 in the absence and the presence of the indicated compounds (10 μM). For each batch of oocytes, uptake values were corrected for unspecific iron uptake in non-injected oocytes. Data were normalized to the mean iron uptake by hDMT1 WT (pmol min^–1) and represented as mean ± SD (14–34 oocytes). (b and c) Electrophysiology. Oocytes were held at –50 mV (V_h) and a voltage-step protocol was applied (V_m = –150 mV to +50 mV) before and after the addition of Fe²⁺ (20 μM) in the absence or the presence of the indicated compounds (10 μM). For each trace, recorded currents were corrected for the background currents observed without substrate. (b) Representative traces recorded with hDMT1 injected oocytes (upper panel) and non-injected oocytes (lower panel). (c) Upper panel: Average current traces of the response of hDMT1 injected oocytes to the indicated compounds. Data was normalized to Fe²⁺ (20 μM) evoked currents at –150 mV and represented as mean ± SD (8 to 13 oocytes). Lower panel: Average dose–response (0.01–10 μM) of the inhibition of the indicated compounds over the Fe²⁺ (20 μM) evoked currents at –70 mV. Data was normalized to Fe²⁺ (20 μM) + compound (0.01 μM) evoked currents and represented as mean ± SD (4 oocytes). Kinetic parameters were obtained by fitting experimental results to a 4-parameter sigmoidal curve (black line). Compound 13 did not fit to the equation while for compound 1 the obtained IC₅₀ was 0.14 μM. All the experiments were performed with oocytes from at least 3 different oocyte batches. Statistical differences were assessed using T-test or Mann–Whitney U test (Fe²⁺-uptake by hDMT1 WT vs. non-injected oocytes or vs. compound); p > 0.05 = ns; p < 0.001 = ***.

Fig. 4. Chemical evidence of divalent metal complexation by hDMT1 inhibitors. (a and b) Isothermal titration calorimetry of pyrazole 13 (0.4 mM) with Cd²⁺ (5 mM) in aqueous buffer pH 7.4 and pH 5.5, 25 °C. (c) Calcein quenching assay in aqueous buffer pH 5.5 (2 μM Fe²⁺, 10 μM ligand, 1 μM calcein). Fe²⁺ (2 μM), ascorbic acid (200 μM) and compounds (20 μM) were incubated for 5 min before the addition of calcein (1 μM) in Krebs buffer at pH 5.5. Fluorescence was measured after 5 min incubation. (d and e) Removal of radioactive ⁵⁵Fe²⁺ from buffers upon incubation with ligands and centrifugation. ⁵⁵Fe²⁺ (1 μM) precipitation upon interaction with the indicated compounds (10 μM). ⁵⁵Fe²⁺ and the indicated compounds were incubated for 15 min, then, an aliquot of the solution supernatant was taken for determination of the remaining ⁵⁵Fe²⁺. Data was normalized to the values obtained for the Fe²⁺ + DMSO (0.1%) solution and represented as mean ± SD (N = 15; 2 independent experiments). Statistical differences were assessed using T-test or Mann–Whitney U test (Fe²⁺+ DMSO vs. each compound); p > 0.05 = ns; p < 0.05 = *; p < 0.001 = ***.

Fig. 5. Cross reactivity of hDMT1 inhibitors over the functional activity of the divalent metal transporter hZIP8. (a) ⁵⁵Fe²⁺ uptake (1 μM) by HEK293T cells (source: American Type Culture Collection, catalog no. CRL-3216) transiently transfected with the empty vector (Ev), hDMT1 or hZIP8. Uptake assay was performed at the optimal pH for the functional activity of each transporter (pH 5.5 and pH 7.4 respectively). The indicated compounds (10 μM) were pre-incubated for 5 min, and then, for 15 min with both compound and ⁵⁵Fe²⁺. Measured ⁵⁵Fe²⁺ was corrected by subtracting the background iron uptake measured in non-transfected cells. The data was normalized to the Fe²⁺ uptake measured in the presence of DMSO (0.1%) and represented as mean ± SD (N = 5–16; obtained from 2 independent experiments). IC₅₀ determination for pyrazolyl-pyrimidone 13 (b) and bipyridine (c) in HEK293T cells transiently transfected with hDMT1 (left panels) or hZIP8 (right panels). ⁵⁵Fe²⁺ uptake (1 μM) was measured in the presence of the indicated compound concentration ranges, and the measurements were corrected by subtracting the background iron uptake measured in non-transfected cells. IC₅₀ values were calculated by fitting the data to a sigmoidal 4-parameter equation. Representative experiments are depicted, while IC₅₀ values were calculated as mean ± SD of 3 different experiments.

Fig. 6. (a) Acid–base titration of 3, 5, 13 and neutral and basic forms of 3, 5, 13. (20 μmol of compound in 10 mL (10% DMSO, ∼2 mM), 3 and 5 were acidified with HCl (50 μmol)). (b) Acid-base equilibria for inhibitors 3, 5 and 13 highlighting potentially metal chelating nitrogen atoms in red. (c) TMAP of our inhibitors combined with 169 pyrazolyl-pyrimidones and 1717 pyrazolyl-pyridines found in ChEMBL, color coded by source: cyan = pyrazolyl-pyridines from ChEMBL, blue = pyrazolyl-pyrimidones from ChEMBL, red = this work. Insert: Close-up view around pyrazolyl-pyrimidones color-coded by target class: red = transporter, cyan = enzyme, magenta = unclassified protein, grey = unclassified target. The interactive TMAP including further color-codes (target classes, QED, etc.) is accessible at: ; http://tm.gdb.tools/dmt1_inhibitors/pyrimidones_pyridines/