Inhibitors of Human Divalent Metal Transporters DMT1 (SLC11A2) and ZIP8 (SLC39A8) from a GDB-17 Fragment Library

Abstract

Solute carrier proteins (SLCs) are membrane proteins controlling fluxes across biological membranes and represent an emerging class of drug targets. Here we searched for inhibitors of divalent metal transporters in a library of 1,676 commercially available 3D-shaped fragment-like molecules from the generated database GDB-17, which lists all possible organic molecules up to 17 atoms of C, N, O, S and halogen following simple criteria for chemical stability and synthetic feasibility. While screening against DMT1 (SLC11A2), an iron transporter associated with hemochromatosis and for which only very few inhibitors are known, only yielded two weak inhibitors, our approach led to the discovery of the first inhibitor of ZIP8 (SLC39A8), a zinc transporter associated with manganese homeostasis and osteoarthritis but with no previously reported pharmacology, demonstrating that this target is druggable.

Keywords: chemical space; fragments; inhibitors; iron transport; membrane transporters.

Figure 1

Assembly of GDB‐17 fragment screening set and discovery of divalent metal transporter inhibitors.

Figure 2

Comparison of purchased fragments with fragments from FDB‐17 and with commercial fragments. (a) TMAP (tree‐map) layout of the 1,676 purchased fragments (orange+red) including its diversity subset of 511 compounds (red) combined with 1,700 randomly selected fragments from FDB‐17 (blue) and from commercial fragments (cyan). Each point corresponds to a molecule. The active compounds are shown in magenta. An interactive version of this TMAP is accessible at https://tm.gdb.tools/fragment_project/tmap/1.7k_pcc_fdb_mcf.html (b) Analysis of molecular shingles from each subset. The shingle counts are given as the average±standard error across 10 different selections of FDB‐17 subsets and commercial fragments. (c) Histogram of fsp³C (fraction of sp³ carbon atoms). (d) Nearest neighbor similarity analysis in terms of Jaccard distance (d_j) according to the molecular fingerprint MAP4. (e–h) Histograms of HAC (heavy atom count), HBD (hydrogen bond donor atom count), HBA (hydrogen bond acceptor atom count), and CLogP (calculated octanol:water partition coefficient). The standard error in histogram values across the ten FDB‐17 subsets (blue lines) and the ten commercial fragment set (cyan lines) were below 1 % for all histograms and are not shown except for the nearest neighbor Jaccard distance histogram.

Figure 3

Discovery of DMT1 and ZIP8 inhibitors by GDB fragment screening. IC₅₀ curves determined for compounds 1 a (a) and 2 (c) using HEK293 cells stably overexpressing DMT1. Cells were pre‐incubated for 5 min in the presence of the indicated concentrations of 1 a and 2. Next, radioactive ⁵⁵Fe²⁺ was added (1 μM), and cells were incubated for 15 minutes. Each data point represents the Mean±SD (N=6–8) of the ⁵⁵Fe²⁺‐uptake determined in the presence of the indicated compound concentrations. (b) Structure of analogs 1 b–1 n found to be inactive against DMT1. (d) Representative trace of the Cd²⁺‐flux recorded in HEK293T cells transiently transfected with ZIP8 or the empty vector. Cd²⁺‐flux was monitored using the FLIPR Calcium 5 Assay Kit. A baseline was recorded for 30 sec, then, cells were incubated for 5 minutes in the presence of rac‐3 (50 μM), and finally, Cd²⁺ (5 μM) was added, and the signal (AU) was recorded for 15 minutes. (e) IC₅₀ curve determined for rac‐3 measuring the Cd²⁺‐uptake in the presence of the indicated compound concentrations. Cd²⁺‐uptake was determined as the Area Under the Curve (AUC) of the change in florescence intensity observed upon substrate addition (459–750 s). Each data point represents the Mean±SD (N=6–8) of the AUC determined in the presence of each compound concentration. (f) Calcein quenching assay at pH 7.4. Ligand (20 μM), Fe²⁺ (4 μM) and ascorbic acid (400 μM) were preincubated for 5 min. Then, an equal volume of calcein (2 μM) in uptake solution was added to give the final concentrations of 1 μM calcein, 10 μM rac‐3 or bipyridine, 2 μM Fe²⁺ and 200 μM ascorbic acid, which were incubated for 5 min before the fluorescence measurement. Data was obtained from an experiment performed in quintuplicate, and results are presented as the Mean±SD. See Supporting Information for details. (g) Structure of inhibitor rac‐ 3 and analogs 4, 9 and 10. IC₅₀ values in (a), (c) and (e) are the Mean±SD (N=4–6) of the IC₅₀ values calculated from two independent experiments performed in triplicate.

Figure 4

Characterization of ZIP inhibition by rac‐3. (a) Average Cd²⁺‐flux recorded in the absence or presence of rac‐3 in HEK293T cells transiently transfected with DMT1, ZIP2, or ZIP14. Cd²⁺‐uptake was determined as the Area Under the Curve (AUC) of the change in florescence intensity observed upon substrate addition. Results were expressed as % of the Cd²⁺‐flux recorded in the absence of rac‐3. Data from three independent experiments performed in triplicate are represented as Mean±SD (N=12–33). (b) Enantioselective synthesis and activity of (R)‐3 and (S)‐3. Conditions: a) i) (S)‐α‐methyl‐4‐methoxybenzylamine, conc. aq. HCl, toluene, reflux overnight, ii) NaBH₄, EtOH, −30 °C – RT, overnight, iii) HCl, MeOH, toluene, RT, 60 min. (59 %); b) same as a) using (R)‐α‐methyl‐4‐methoxybenzylamine (66 %); c) BCl₃, CH₂Cl₂, 10 °C, overnight (R: 18 %, S: 47 %). (c–d) detoxification gene expression assay with ZIP8 and ZIP14. MT2A gene expression in HEK293T cells transiently transfected with ZIP8 (blue, left panel), ZIP14 (green, right panel) or the empty vector (EV, red) treated for 2 hours with the indicated solutions. Expression of MT2A mRNA was determined by Real‐Time PCR. Obtained Ct values for MT2A gene were normalized to the housekeeping gene GAPDH following the ΔCt method. Data was obtained from 5 independent experiments performed in triplicate, and results are presented as the Mean±SD (N=10–13). Statistical differences were determined by t‐test or Mann‐Whitney U test ((a) Cd²⁺ vs Cd²⁺+rac‐ 3; (c–d) Zn²⁺ (10 μM) vs. each other condition), p values are indicated on top of the corresponding graphs.