Selective, high-affinity inhibitors for human 15-hydroxyprostaglandin dehydrogenase (15-PGDH) are desirable as tools to facilitate the mapping of prostaglandin signaling pathways in vitro and in vivo. Using a quantitative high-throughput screen approach we have found several new chemotypes that inhibit 15-PGDH with high affinity. Detailed biophysical analyses have demonstrated the strong stabilizing effect of these molecules on the enzyme. Based on the results from our detailed kinetic analyses a number of inhibition modes appeared possible, but the data are consistent with compounds 13 and 72 not inhibiting competitively, while a competitive mechanism of inhibition appeared likely for compound 61.
In order to gain better understanding of the distinct mechanisms of action of the inhibitors, it is helpful to examine the catalytic mechanism of 15-PGDH. The SDR enzymes have been shown to follow a common sequential ordered bi-bi-reaction mechanism, involving sequential cofactor binding, substrate binding, catalysis, product release, and finally release of co-product 
, as shown in the reaction coordinate (). In analogy with other SDRs, binding of the NAD+
cofactor is expected to alter the local electrostatic environment of the catalytic Tyr151 residue, favoring the deprotonated state. After substrate binding, the reaction proceeds by deprotonation of the substrate 15-OH group by Tyr151, facilitating hydride transfer from the substrate to NAD+
, and forming the product complex consisting of protonated Tyr151, NADH, and ketone.
Figure 7 Proposed mechanism of prostaglandin dehydrogenation (after ) and inhibitory mechanisms of action for compounds 61 and 13.
One of the inhibitor chemotypes identified from the high throughput screen is a series of imidazopyridines, represented by the most potent analogue 61, with an IC50 of 25 nM and an estimated Ki of 5 nM. The biophysical data show a strong preference for 61 in stabilizing 15-PGDH complexed with NAD+ as compared with NADH.
The results of the docking experiments provide support for a competitive mechanism of inhibition by compound 61. The docking directs the imidazopyridine group into hydrogen bonding interactions with the catalytic residues Tyr151 and Ser138. In the co-complex with NAD+, as discussed above, the deprotonated form of Tyr151 is favored: the resultant oxyanion is able to interact with the protonated form of the pyridine ring, while the hydroxy group of Ser138 is able to donate a hydrogen bond to the adjacent imidazole nitrogen. The much weaker stabilization observed with NADH implies that the protonated form of Tyr151 interacts considerably more weakly with the pyridyl nitrogen.
In addition to providing a convincing basis for interaction of the heterocyclic motif with the enzyme's catalytic machinery, this binding orientation also favorably directs the bromo-substituent into the nearby hydrophobic pocket, and places the S-linked cyanobenzyl group into the substrate tunnel leading towards the exterior of the protein, which contains side chains available for stacking interactions (Phe185, Tyr217). Taken together, the data argue that the position for binding of compound 61 along the reaction coordinate of 15-PGDH is at step 2, i.e., occurs to the complex of 15-PGDH•NAD+ (left panel in ). This interpretation is consistent with the observed uncompetitive mechanism of action with respect to NAD+, since the higher affinity of the inhibitor compared to the substrate is expected to increase the affinity for NAD+.
The majority of the remaining inhibitor hits fall into three structural clusters (clusters 1, 2 and 4; supplementary information Table S1
), all containing an amide moiety as the common feature (). Structure-activity relationships indicate that activity is favored when the nitrogen of the amide forms part of a saturated heterocycle, most commonly piperidine. In all of the active amides identified, the carbonyl is linked to a ring, which is aromatic in the most potent analogues. The highest potency is achieved when this ring is linked to a further aryl ring, with a variety of linkers being tolerated. In compound 13
, this linker is incorporated into the central fused benzimidazole ring system.
The results of the docking experiments are consistent with the observed structure-activity relationships and provide help with the decision about which of the mechanisms of inhibition that are possible according to the kinetic analyses would be most likely for the inhibitors. In the model of compound 13
docked into 15-PGDH, the oxygen of the amide carbonyl presents two lone pairs that can accept hydrogen bonds from both the catalytic Ser138 and Tyr151 residues, mimicking the ketone product of oxidation of the PGE2
substrate. Tyr151 is only able to donate this hydrogen bond when protonated, which is favored by binding of NADH, while binding of NAD+
favors the deprotonated state. Hence, compound 13
acts as a product analogue in the sequential ordered bi-bi catalysis mechanism, and a noncompetitive mode of inhibition with respect to PGE2
and an uncompetitive mode with respect to NAD+
would be expected 
. Our kinetic analysis is consistent with this mode, identifying uncompetitive inhibition with respect to the cofactor, while equal probability was found for noncompetitive, uncompetitive and mixed-type inhibition with respect to the substrate (). Uncompetitive inhibition by the compounds of the series with respect to NAD+
is, furthermore, consistent with the observed enhanced stabilization of 15-PGDH when the latter is bound to NADH as compared with NAD+
; this stabilization of product-bound 15-PGDH ultimately results in a suppression of enzymatic turnover.
The preference for small alicyclic amides is also explained by the docking model: the piperidine moiety of compound 13 is relatively tightly accommodated in the hydrophobic pocket adjacent to the catalytic residues (). Consistent with this binding mode, inhibitory potency is weaker for secondary or tertiary straight-chain amides, and further diminished or abolished with bicyclic amides that can clearly not be accommodated in this pocket (see ). As described above, the structure-activity relationships indicate that activity is retained with a variety of groups attached to the amide carbonyl carbon atom, although there is a preference for aromatic groups, and in general these groups are substituted in such a way as to be able to adopt an extended conformation. Such a conformation is consistent with the docking mode, placing the extended group into the substrate tunnel leading towards the exterior of the protein. The availability of side chain groups capable of stacking interactions (Phe185, Tyr217) adds further support to this proposed binding mode ().
shares aspects of its mechanism with compound 13
, notably a strong preference for NADH in co-stabilization of 15-PGDH ( and ) and a cofactor-uncompetitive inhibition mechanism. With respect to the substrate PGE2
the kinetic analysis favored the noncompetitive mode, consistent with the expectation for a product-analogous binding mode (see above). Although compound 72
does not contain an amide group, the fused 1,2,4-triazole is capable of acting as a bioisostere, with a lone pair of electrons on each of the adjacent nitrogen atoms mimicking the two lone pairs of the carbonyl oxygen in 13
(Supplementary Information Figure S3
). Thus, in the docking experiments, the triazole moiety accepts two hydrogen bonds from Ser138 and the protonated form of Tyr151; the remaining hydrophobic and stacking interactions are remarkably similar to those modeled for compound 13
. Therefore, both compounds 13
bind at step 4 along the 15-PGDH reaction coordinate (), mimicking the product, favoring co-complex formation with NADH and behaving in an uncompetitive manner with respect to the PGE2
The compound library used for screening of 15-PGDH in this study has been, so far, tested in screens against over 320 targets (http://pubchem.ncbi.nlm.nih.gov/
). This list includes two other proteins that are involved in prostaglandin signaling, the EP2 prostaglandin-E2
receptor and the M1-muscarinic receptor. The list also includes two SDR enzymes, hydroxyacyl dehydrogenase (HADH2, also known as type-10 hydroxysteroid dehydrogenase, HSD17β10; PubChem Assay ID 886) and type-4 hydroxysteroid dehydrogenase (HSD17β4; PubChem Assay ID 893), and a medium-chain dehydrogenase/reductase (MDR), aldehyde dehydrogenase 1 (ALDH1A1; PubChem Assay ID 1030), each of which was tested using a similar experimental protocol to that used for 15-PGDH. As the results in show, all lead inhibitors identified in this study were largely inactive against ALDH1A1: compounds 61
showed a flat response, while compound 13
displayed only a shallow curve with an approximate IC50
of 36 µM. Against the two hydroxysteroid dehydrogenases (HSD17β10 and HSD17β4), both compounds 13
were inactive up to the top concentration of 57.5 µM.
Selectivity profiling of key inhibitors against related dehydrogenase/reductase enzymes.
In summary, the new inhibitor chemotypes identified in this study provide a set of tools, with complementary mechanisms of inhibition, for functional studies on the role of 15-PGDH in prostaglandin signaling pathways. The lead compounds that have been characterized in detail exhibit nanomolar affinities. The existence of disease scenarios involving over-activation of 15-PGDH and in which selective inhibition of the enzyme would be beneficial, is currently unclear. Work published by Tai and co-workers suggested that certain prostate cancers might involve higher-than-normal activity of 15-PGDH 
, and for such cases application of inhibitors based on the chemotypes identified in this study may prove useful. The potential utility of the probes is reinforced by the low cross-reactivity of these compounds in screens against multiple targets, among them receptors with a role in prostaglandin signaling and dehydrogenase enzymes utilizing the same cofactors, suggesting selectivity of the inhibitors for 15-PGDH. The availability of multiple inhibitors with diverse chemical structures for cellular studies enhances confidence in any response observed being due to on-target effects; the likelihood for such diverse compounds having an identical off-target activity profile is very small. While it is difficult to anticipate differences in the cellular response between inhibitors showing contrasting modes of action, one might expect the noncompetitive compounds to demonstrate inhibition independently of the substrate concentration. This may help to avoid problems similar to those encountered, for example, with ATP-competitive protein kinase inhibitors, where high inhibitor concentrations are required in cells to compete against the endogenous co-factor. Finally, in this work we present the novel crystal structure of human 15-PGDH, which will serve as a basis for further studies on the function, mechanism and inhibitor design for this enzyme.