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Structure-function relationships for the selective inhibition of human 3β-hydroxysteroid dehydrogenase type 1 by a
Jenny H Pham1, Catherine M Will2, Vance L Mack1
1Department of Biomedical Sciences, Macon, GA, 31207, USA.
Abstract:
3β-Hydroxysteroid dehydrogenase type 1 (3β-HSD1) is selectively expressed in human placenta, mammary glands and breast tumors in women. Human 3β-HSD2 is selectively expressed in adrenal glands and ovaries. Based on AutoDock 3 and 4 results, we have exploited key differences in the amino acid sequences of 3β-HSD1 (Ser194, Arg195) and 3β-HSD2 (Gly194, Pro195) by designing a selective inhibitor of 3β-HSD1. 2,16-Dicyano-4,5-epoxy-androstane-3,17-dione (16-cyano-17-keto-trilostane or DiCN-AND) was synthesized in a 4-step procedure from androstenedione. In purified 3β-HSD inhibition studies, DiCN-AND competitively inhibited 3β- HSD1 with Ki=4.7μM and noncompetitively inhibited 3β-HSD2 with a 6.5-fold higher Ki=30.7μM. We previously reported similar isoenzyme-specific inhibition profiles for trilostane. Based on our docking results, we created, expressed and purified the chimeric S194G-1 mutant of 3β-HSD1. Trilostane inhibited S194G-1 (Ki=0.67μM) with a noncompetitive mode compared to its 6.7-fold higher affinity, competitive inhibition of 3β-HSD1 (Ki=0.10μM). DiCN-AND inhibited S194G-1 with a 6.3-fold higher Ki (29.5μM) than measured for 3β-HSD1 (Ki=4.7μM) but with the same competitive mode for both enzyme species. Since DiCN-AND noncompetitively inhibits 3β-HSD2, which has the Gly194 and Pro195 of 3β-HSD2 in place of the Ser194 and Arg195 in 3β-HSD1, this suggests that Arg195 alone in 3β-HSD1 or S194G-1 is required to bind DiCN-AND in the substrate binding site (competitive inhibition). However, both Ser194 and Arg195 are required to bind trilostane in the 3β-HSD1 substrate site based on its noncompetitive inhibition of S194G-1 and 3β-HSD2. In support of this hypothesis, DiCN-AND inhibited our chimeric R195P-1 mutant noncompetitively with a Ki=41.3μM (similar to the 3β-HSD2 inhibition profile). Since DiCN-AND competitively inhibited S194G-1 that still contains R195 but noncompetitively inhibited R195P-1 that still contains S194, our data provides strong evidence that the Arg195 being mutated to Pro195 (as present in 3β-HSD2) shifts the inhibition mode from competitive to noncompetitive in 3β-HSD1. This supports the key role of Arg195 in 3β-HSD1 for the high affinity, competitive binding of the trilostane analogs. Our new structure/function information for the design of targeted 3β-HSD1 inhibitors may lead to important new treatments for the prevention of spontaneous premature birth.
Insights
Researchers designed a selective inhibitor, DiCN-AND, targeting 3β-Hydroxysteroid dehydrogenase type 1 (3β-HSD1) based on amino acid differences. This work may lead to new treatments for preventing premature birth.
Area of Science:
- Biochemistry and enzymology
- Steroid hormone metabolism
- Drug discovery and medicinal chemistry
Background:
- 3β-Hydroxysteroid dehydrogenase type 1 (3β-HSD1) is crucial in placenta, mammary glands, and breast tumors.
- 3β-HSD1 and 3β-HSD2 exhibit distinct tissue expression patterns and amino acid sequences, particularly at positions 194 and 195.
- Targeting 3β-HSD1 offers potential therapeutic strategies for conditions like breast cancer and premature birth.
Purpose of the Study:
- To design and synthesize a selective inhibitor of 3β-HSD1 by exploiting sequence differences with 3β-HSD2.
- To investigate the structure-activity relationship of novel inhibitors, specifically DiCN-AND and trilostane analogs.
- To elucidate the role of specific amino acid residues (Ser194, Arg195) in enzyme inhibition and selectivity.
Main Methods:
- Computational docking studies (AutoDock 3 and 4) to identify key amino acid differences.
- Chemical synthesis of 2,16-dicyano-4,5-epoxy-androstane-3,17-dione (DiCN-AND).
- Enzyme inhibition assays using purified 3β-HSD1, 3β-HSD2, and engineered mutants (S194G-1, R195P-1).
Main Results:
- DiCN-AND selectively inhibited 3β-HSD1 competitively (Ki=4.7μM) over 3β-HSD2 (noncompetitive, Ki=30.7μM).
- Mutagenesis studies revealed that Arg195 in 3β-HSD1 is critical for competitive inhibition by DiCN-AND.
- Trilostane's inhibition mode shifted from competitive to noncompetitive upon mutation of Ser194 to Glycine in 3β-HSD1, indicating a different binding mechanism.
Conclusions:
- The study successfully designed a selective 3β-HSD1 inhibitor, DiCN-AND, leveraging key amino acid differences.
- Arg195 plays a pivotal role in the competitive binding of trilostane analogs to 3β-HSD1.
- These findings provide valuable structure-function insights for developing targeted 3β-HSD1 inhibitors for potential therapeutic applications, including preventing premature birth.
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