High-affinity inhibitors of human NAD-dependent 15-hydroxyprostaglandin dehydrogenase: mechanisms of inhibition and structure-activity relationships

Abstract

Background: 15-Hydroxyprostaglandin dehydrogenase (15-PGDH, EC 1.1.1.141) is the key enzyme for the inactivation of prostaglandins, regulating processes such as inflammation or proliferation. The anabolic pathways of prostaglandins, especially with respect to regulation of the cyclooxygenase (COX) enzymes have been studied in detail; however, little is known about downstream events including functional interaction of prostaglandin-processing and -metabolizing enzymes. High-affinity probes for 15-PGDH will, therefore, represent important tools for further studies.

Principal findings: To identify novel high-affinity inhibitors of 15-PGDH we performed a quantitative high-throughput screen (qHTS) by testing >160 thousand compounds in a concentration-response format and identified compounds that act as noncompetitive inhibitors as well as a competitive inhibitor, with nanomolar affinity. Both types of inhibitors caused strong thermal stabilization of the enzyme, with cofactor dependencies correlating with their mechanism of action. We solved the structure of human 15-PGDH and explored the binding modes of the inhibitors to the enzyme in silico. We found binding modes that are consistent with the observed mechanisms of action.

Conclusions: Low cross-reactivity in screens of over 320 targets, including three other human dehydrogenases/reductases, suggest selectivity of the present inhibitors for 15-PGDH. The high potencies and different mechanisms of action of these chemotypes make them a useful set of complementary chemical probes for functional studies of prostaglandin-signaling pathways.

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Figure 1. Quantitative high-throughput screen of 15-PGDH.

A. Plot of the Z' factor associated with each plate, showing high stability over the entire duration of the screen (completed in five days). The average Z' was 0.86. B. Hit frequency for the library of tested compounds, measured as the distribution of compounds according to binned potencies. C. Typical effect of a non-fluorescent screening hit (inhibitor 13, titrated between 3.5 nM and 57.5 µM) on the time course of NAD⁺-reduction upon addition of PGE₂. D. Dose-dependent reduction in enzyme activity caused by compound 13, as detected during the screen.

Figure 2. IC₅₀ from re-testing for selected inhibitors of human 15-PGDH identified in the quantitative high-throughput screen.

Shown are the IC₅₀ values for select compounds arranged by cluster number, along with their PubChem Chemical Identifiers (CID).

Figure 3. Correlation between thermal stabilization of 15-PGDH in presence of NAD⁺ (A) or NADH (B) and inhibitory pIC₅₀ for screening hits from different structural clusters.

Thermal stabilization is expressed as a shift in the midpoint of the unfolding transition of the protein (ΔT_m). ΔT_m and pIC₅₀ (-log(IC₅₀) values are means of at least three independent measurements. The inhibitors are represented by color-coded symbols according to their chemical clusters, respectively (supplementary information Table S1): blue, cluster 1; dark green, cluster 2; orange, cluster 3; purple, cluster 4; light blue, cluster 5; light green, cluster 8; grey, cluster 9; dark yellow, cluster 11; red, singletons (a T_m could not be determined for the tested compounds in clusters 6, 7 & 10). The dotted line in each graph A & B denotes correlations for the compounds in cluster 1, respectively. Numbers within the graphs assign the positions for the inhibitors of special interest (see text).

Figure 4. Effects of the re-synthesized compounds on stability and activity of human 15-PGDH.

A. Thermal stability of the protein in dependence of substrate and inhibitors, without cofactor (grey bars), and in presence of NAD⁺ (light blue bars) as well as in presence of NADH (purple bars). Shown are sets of these data, respectively, for each of the tested compounds as denoted on the Y axis. Bars represent averages of three independent experiments, with standard deviations displayed as error bars. B. Inhibitor dose-dependent reduction of the enzyme activity caused by compounds 13 (black triangles), 72 (blue squares) and 61 (red diamonds), respectively. Plotted values are means of three independent experiments, with standard deviations displayed as vertical error bars. Dashed or dotted lines, in colors corresponding to the data points, result from least-squares non-linear fitting of the data to the Hill equation (see Materials & Methods), respectively. C & D. Michaelis-Menten plots of the enzyme activity at varying concentrations of substrate, PGE₂, in absence of inhibitor (black symbols) or in presence of compounds 61 (C) or 13 (D) at concentrations close to (blue symbols) or above (red symbols) their IC₅₀ values, specified in table 3. The inset graph in D shows the data from the main graph as Lineweaver-Burk plot, demonstrating competitive inhibition from independence of the Y-axis intercept (1/V _max) of the inhibitor concentration. All values are means of three independent measurements; standard deviations are displayed as vertical error bars.

Figure 5. Structure of the homodimer of human 15-PGDH in complex with NAD⁺ (PDB: 2GDZ).

Displayed are the two protomers with their backbones in grey and blue, respectively. The cofactor is depicted in a ball-and-stick representation and color-coded by atom type (green, carbon; red, oxygen; blue, nitrogen; orange, phosphate). The C-terminus of each protomer whose residues originate from the cloning procedure (see text) is highlighted in orange.

Figure 6. Binding of the inhibitors 61 (A) and 13 (B) in the active site of 15-PGDH as predicted by docking studies.

The view shows a cut-through into the substrate pocket of 15-PGDH, with the volume of the pocket indicated by a green mesh. Key amino acid residues (see text) are labeled. Compounds and amino acid sidechains are in ball and stick representation, with the atoms color-coded: blue, nitrogen; red, oxygen; silver, bromide; white, carbon atoms in side chains; yellow, carbon atoms in compounds; green, carbon atoms in the cofactor. The figures were created using ICM (Molsoft, LLC).

Figure 7. Proposed mechanism of prostaglandin dehydrogenation (after [31]) and inhibitory mechanisms of action for compounds 61 and 13.

Compound 61 mimics the substrate (left panels), consistent with experimental data showing favored binding to the NAD⁺-bound form of 15-PGDH, and its competitive mechanism of inhibition with respect to PGE₂. Compound 13 mimics the product of oxidation (right panels), consistent with its favored binding to the NADH-bound form of 15-PGDH, and its uncompetitive mechanism of inhibition with respect to PGE₂.