Urmi Dhagat

Structure-Guided Design, Synthesis, and Evaluation of Salicylic Acid-Based Inhibitors Targeting a Selectivity Pocket in the Active Site of Human 20α-Hydroxysteroid Dehydrogenase (AKR1C1)

The first design, synthesis, and evaluation of human 20alpha-hydroxysteroid dehydrogenase (AKR1C1) inhibitors based on the recently published crystal structure of its ternary complex with inhibitor are reported. While the enzyme-inhibitor interactions observed in the crystal structure remain conserved with the newly designed inhibitors, the additional phenyl group of the most potent compound, 3-bromo-5-phenylsalicylic acid, targets a nonconserved hydrophobic pocket in the active site of AKR1C1 resulting in 21-fold improved potency (K(i) = 4 nM) over the structurally similar 3alpha-hydroxysteroid dehydrogenase isoform (AKR1C2). The compound is hydrogen bonded to Tyr55, His117, and His222, and the phenyl ring forms additional van der Waals interactions with residues Leu308, Phe311, and the nonconserved Leu54 (Val in AKR1C2). Additionally, the metabolism of progesterone in AKR1C1-overexpressed cells was potently inhibited by 3-bromo-5-phenylsalicylic acid, which was effective from 10 nM with an IC(50) value equal to 460 nM.

A Salicylic Acid-Based Analogue Discovered from Virtual Screening as a Potent Inhibitor of Human 20α-Hydroxysteroid Dehydrogenase

20alpha-hydroxysteroid dehydrogenase (AKR1C1) plays a key role in the metabolism of progesterone and other steroid hormones, thereby regulating their action at the pre-receptor level. AKR1C1 is implicated in neurological and psychiatric conditions such as catamenial epilepsy and depressive disorders. Increased activity of AKR1C1 is associated with termination of pregnancy and the development of breast cancer, endometriosis and endometrial cancer. Inhibition of the undesired activity of AKR1C1 will help reduce risks of premature birth, neurological disorders and the development of cancer. In order to identify potential leads for new inhibitors of AKR1C1 we adopted a virtual screening-based approach using the automated DOCK program. Approximately 250,000 compounds from the NCI database were screened for potential ligands based on their chemical complementarity and steric fit within the active site of AKR1C1. Kinetic analysis revealed 3,5-diiodosalicylic acid, an analogue of salicylic acid, as a potent competitive inhibitor with respect to the substrate 5beta-pregnane-3alpha,20alpha-diol with a K(i) of 9 nM. Aspirin, which is a well known salicylic acid-based drug, was also found to inhibit AKR1C1 activity. This is the first report to show aspirin (IC(50)=21 microM) and its metabolite salicylic acid (IC(50)=7.8 microM) as inhibitors of AKR1C1.

Inhibitors of human 20α-hydroxysteroid dehydrogenase (AKR1C1).

Human 20α-hydroxysteroid dehydrogenase (AKR1C1), a member of the aldo-keto reductase (AKR) superfamily, is one of four isoforms (with >84% amino acid sequence identity) existing in human tissues. AKR1C1 most efficiently reduces biologically active progesterone and 5α-pregnan-3α-ol-20-one into their corresponding 20α-hydroxysteroids among the isoforms. The enzyme also accepts endogenous and xenobiotic non-steroidal carbonyl compounds as the substrates. In addition to the up-regulation of the AKR1C1 gene in cancer cells, the enzymes over-expression in the cells of lung, ovary, uterine cervix, skin and colon carcinomas was reported to be associated with resistance against several anticancer agents. Thus, AKR1C1 may be a marker of the above cancers and a target of poor prognosis in cancer therapy. The recently determined X-ray crystal structures of AKR1C1/NADP(+)/20α-hydroxyprogesterone and AKR1C1/NADP(+)/3,5-dichlorosalicylic acid ternary complexes have provided a strong foundation for structure-based design methods to improve inhibitor selectivity and potency. In this review we provide an overview of the different types of AKR1C1 inhibitors and an update on the design of potent and selective inhibitors based on the crystal structure of the enzyme-inhibitor complex. Article from the Special issue on Targeted Inhibitors.

Selectivity determinants of inhibitor binding to human 20alpha-hydroxysteroid dehydrogenase: crystal structure of the enzyme in ternary complex with coenzyme and the potent inhibitor 3,5-dichlorosalicylic acid

The crystal structure of human 20alpha-hydroxysteroid dehydrogenase (AKR1C1) in ternary complex with the coenzyme NADP (+) and the potent inhibitor 3,5-dichlorosalicylic acid was determined at a resolution of 1.8 A. The inhibitor is held in place by a network of hydrogen bonding interactions with the active site residues Tyr55, His117, and His222. The important role of the nonconserved residues Leu54, His222, Leu306, and Leu308 in inhibitor binding and selectivity was determined by site-directed mutagenesis.

Molecular determinants for the stereospecific reduction of 3-ketosteroids and reactivity towards all-trans-retinal of a short-chain dehydrogenase/reductase (DHRS4)

DHRS4, a member of the short-chain dehydrogenase/reductase superfamily, reduces all-trans-retinal and xenobiotic carbonyl compounds. Human DHRS4 differs from other animal enzymes in kinetic constants for the substrates, particularly in its low reactivity to retinoids. We have found that pig, rabbit and dog DHRS4s reduce benzil and 3-ketosteroids into S-benzoin and 3alpha-hydroxysteroids, respectively, in contrast to the stereoselectivity of human DHRS4 which produces R-benzoin and 3beta-hydroxysteroids. Among substrate-binding residues predicted from the crystal structure of pig DHRS4, F158 and L161 in the animal DHRS4 are serine and phenylalanine, respectively, in the human enzyme. Double mutation (F158S/L161F) of pig DHRS4 led to an effective switch of its substrate affinity and stereochemistry into those similar to human DHRS4. The roles of the two residues in determining the stereospecificity in 3-ketosteroid reduction were confirmed by reverse mutation (S158F/F161L) in the human enzyme. The stereochemical control was evaluated by comparison of the 3D models of pig wild-type and mutant DHRS4s with the modeled substrates. Additional mutation of T177N into the human S158F/F161L mutant resulted in almost complete kinetic conversion into a pig DHRS4-type form, suggesting a role of N177 in forming the substrate-binding cavity through an intersubunit interaction in pig and other animal DHRS4s, and explaining why the human enzyme shows low reactivity towards retinoids.

Inhibition of 3(17)α-hydroxysteroid dehydrogenase (AKR1C21) by aldose reductase inhibitors

Mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) is a member of the aldo-keto reductase superfamily that catalyses the oxido-reduction of steroid hormones such as estrogens, androgens and neurosteroids. Inhibitors of aldose reductase (AR), a member of the same superfamily, were evaluated against AKR1C21. Models of the enzyme-inhibitor complexes suggest that Tyr118 and Phe311 are important residues for inhibitor recognition and orientation in the active site of AKR1C21.

Structure-based optimization and biological evaluation of human 20α-hydroxysteroid dehydrogenase (AKR1C1) salicylic acid-based inhibitors.

The tertiary structure of the Leu308Val mutant of human 20α-hydroxysteroid dehydrogenase (AKR1C1) in complex with the inhibitor 3,5-dichlorosalicylic acid (DCL) has been determined. Structures and kinetic properties of the wild-type and mutant enzymes indicate that Leu308 is a selectivity determinant for inhibitor binding. The Leu308Val mutation resulted in 13-fold and 3-fold reductions in the inhibitory potencies of DCL and 3-bromo-5-phenylsalicylic acid (BPSA), respectively. The replacement of Leu308 with an alanine resulted in 473-fold and 27-fold reductions in the potencies for DCL and BPSA, respectively. In our attempts to optimize inhibitor potency and selectivity we synthesized 5-substituted 3-chlorosalicylic acid derivatives, of which the most potent compound, 3-chloro-5-phenylsalicylic acid (K(i) = 0.86 nM), was 24-fold more selective for AKR1C1 relative to the structurally similar 3α-hydroxysteroid dehydrogenase (AKR1C2). Furthermore, the compound inhibited the metabolism of progesterone in AKR1C1-overexpressed cells with an IC(50) value equal to 100 nM.

Structure of rat aldose reductase-like protein AKR1B14 holoenzyme: Probing the role of His269 in coenzyme binding by site-directed mutagenesis

Rat aldose reductase-like protein (AKR1B14) is the ortholog of mouse vas deferens protein (AKR1B7) playing roles in detoxification of reactive aldehydes and synthesis of prostaglandin F(2α). The crystal structure of the binary complex (AKR1B14-NADPH) was determined at 1.86Å resolution, and showed that the adenine ring and the 2-phosphate group of the coenzyme formed π-stacking and electrostatic interactions with the imidazole ring and ND1 atom, respectively, of His269, which is not conserved in other aldose reductase-like proteins. The interactions were supported by site-directed mutagenesis of His269 to Arg, Phe and Met, which increased the K(m) for NADPH by 4, 7 and 127-fold, respectively. This is the first report of the tertiary structure of a rodent AKR1B7 ortholog, which describes the role of a novel dual interaction for the non-conserved His269 in coenzyme binding.

Activation of aldo-keto reductase family member 1B14 (AKR1B14) by bile acids: Activation mechanism and bile acid-binding site

Aldo-keto reductase (AKR) 1B14, a rat ortholog of mouse androgen-dependent vas deferens protein (AKR1B7), is involved in the synthesis of prostaglandin F(2α) and detoxification of 4-oxononenal formed by lipid peroxidation. The NADPH-linked reductase activity of AKR1B14 was activated by various bile acids. Although the activation was increased by decreasing pH from 9.0 to 6.0, the concentrations giving maximum stimulation (2- to 18-fold) were 0.2-6.0 μM for bile acids at pH 7.4. Kinetic analyses of the activation by glycochenodeoxycholic acid in the forward and reverse reactions, together with fluorescence changes and protection against 4-oxononenal-induced inactivation by bile acid, indicate that the bile acid binds to the enzyme and its coenzyme binary complex as a non-essential activator. The bile acid binding to AKR1B14 mainly accelerates the NADP(+) dissociation, the rate-limited step of the enzyme reaction. AKR1B7 was also activated by bile acids, but the activation was low and independent of pH. The mutagenesis of His269 and Leu267 of AKR1B14 into the corresponding residues (Arg and Pro, respectively) of AKR1B7 resulted in low and pH-independent activation by bile acids. The results, together with the docking of the bile acid in the recently determined crystal structure of AKR1B14, identify the bile acid-binding site of which His269 plays a key role in significant activation through its electrostatic interaction with the carboxyl group of bile acid, facilitating the release of NADP(+).

Structure of 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) holoenzyme from an orthorhombic crystal form: an insight into the bifunctionality of the enzyme.

Mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) is a bifunctional enzyme that catalyses the oxidoreduction of the 3- and 17-hydroxy/keto groups of steroid substrates such as oestrogens, androgens and neurosteroids. The structure of the AKR1C21-NADPH binary complex was determined from an orthorhombic crystal belonging to space group P2(1)2(1)2(1) at a resolution of 1.8 A. In order to identify the factors responsible for the bifunctionality of AKR1C21, three steroid substrates including a 17-keto steroid, a 3-keto steroid and a 3alpha-hydroxysteroid were docked into the substrate-binding cavity. Models of the enzyme-coenzyme-substrate complexes suggest that Lys31, Gly225 and Gly226 are important for ligand recognition and orientation in the active site.