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Discovery of desketoraloxifene analogues as inhibitors of mammalian, Pseudomonas aeruginosa, and NAPE phospholipase D enzymes.

Scott SA, Spencer CT, O'Reilly MC, Brown KA, Lavieri RR, Cho CH, Jung DI, Larock RC, Brown HA, Lindsley CW - ACS Chem. Biol. (2014)

Bottom Line: In order to better understand the biology and therapeutic potential of these less explored PLD enzymes, small molecule tools are required.Selective estrogen receptor modulators (SERMs) have been previously shown to inhibit mammalian PLD (PLD1 and PLD2).This finding represents an important first step toward the development of small molecules possessing universal inhibition of divergent PLD enzymes to advance the field.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, ‡Department of Chemistry, §Department of Biochemistry, ∥Vanderbilt Institute of Chemical Biology, Vanderbilt University , Nashville, Tennessee 37232, United States.

ABSTRACT
Phospholipase D (PLD) hydrolyses cellular lipids to produce the important lipid second messenger phosphatidic acid. A PLD enzyme expressed by Pseudomonas aeruginosa (PldA) has been shown to be important in bacterial infection, and NAPE-PLD has emerged as being key in the synthesis of endocannabinoids. In order to better understand the biology and therapeutic potential of these less explored PLD enzymes, small molecule tools are required. Selective estrogen receptor modulators (SERMs) have been previously shown to inhibit mammalian PLD (PLD1 and PLD2). By targeted screening of a library of SERM analogues, additional parallel synthesis, and evaluation in multiple PLD assays, we discovered a novel desketoraloxifene-based scaffold that inhibited not only the two mammalian PLDs but also structurally divergent PldA and NAPE-PLD. This finding represents an important first step toward the development of small molecules possessing universal inhibition of divergent PLD enzymes to advance the field.

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Structuresand PLD activities of known SERMS 7–10. These scaffolds represent a second chemotype, other thanthat of 1–6, that has been shownto inhibit mammalian PLD.
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fig3: Structuresand PLD activities of known SERMS 7–10. These scaffolds represent a second chemotype, other thanthat of 1–6, that has been shownto inhibit mammalian PLD.

Mentions: Selective estrogen receptor modulators (SERMs) have longbeen usedfor the successful treatment of breast cancer (Figure 3). Two of the most commonly used SERMs are raloxifene (7)21 and tamoxifen (8).22 Both compounds were developed asantiestrogens acting at the estrogen receptor but have since beenshown to have differential effects. Raloxifene is often used in thetreatment of osteoporosis in postmenopausal women and is also usedas a preventative measure for women at a high risk for breast cancerdevelopment, whereas tamoxifen has been used for more than 30 yearsfor the treatment of ER+ breast cancer. Both 7 and 8 have been shown to have antiproliferative effects in ERnegative breast cancer, suggesting that there is an alternative mechanismof action for these compounds.23 A previousreport from our lab suggests that a potential ER-independent mechanismfor the efficacy of these SERMs may be due to off-target inhibitionof mammalian PLD.24 In that study, both 7 and 4-hydroxytamoxifen (9), the active cellularmetabolite of 8, were shown to modestly inhibit mammalianPLD activity, both in cells and in vitro, whereas 8 stimulated PLD2 and showed no effect on PLD1. Desketoraloxifene(10) was developed by Eli Lilly in the late 90s as araloxifene analogue that lacked the important carbonyl hinge in theparent molecule,25 thus leading to a SERMwith unique, planar topology.26 This modificationmade the analogue a much stronger activator in the AP-1 site of ERαcompared to that of ERβ, which is the opposite profile of 8. This new analogue had differential SERM efficacy with stimulatoryactivity in the uterus, similar to 9, but retained raloxifene-likecharacter by remaining estrogenic in the bone.25 Analogue 10 was synthesized following theliterature route, and its ability to inhibit PLDs was assessed; gratifyingly,in our exogenous PLD assays, 10 was a modest inhibitorof mammalian PLD1 (IC50 = 6.1 μM) and PLD2 (IC50 = 2.6 μM). However, as with the structurally distinctmammalian PLD inhibitors 1–6, theSERM-based PLD inhibitors 7–10 hadalso never been evaluated as inhibitors of either PldA or NAPE-PLD.


Discovery of desketoraloxifene analogues as inhibitors of mammalian, Pseudomonas aeruginosa, and NAPE phospholipase D enzymes.

Scott SA, Spencer CT, O'Reilly MC, Brown KA, Lavieri RR, Cho CH, Jung DI, Larock RC, Brown HA, Lindsley CW - ACS Chem. Biol. (2014)

Structuresand PLD activities of known SERMS 7–10. These scaffolds represent a second chemotype, other thanthat of 1–6, that has been shownto inhibit mammalian PLD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4336625&req=5

fig3: Structuresand PLD activities of known SERMS 7–10. These scaffolds represent a second chemotype, other thanthat of 1–6, that has been shownto inhibit mammalian PLD.
Mentions: Selective estrogen receptor modulators (SERMs) have longbeen usedfor the successful treatment of breast cancer (Figure 3). Two of the most commonly used SERMs are raloxifene (7)21 and tamoxifen (8).22 Both compounds were developed asantiestrogens acting at the estrogen receptor but have since beenshown to have differential effects. Raloxifene is often used in thetreatment of osteoporosis in postmenopausal women and is also usedas a preventative measure for women at a high risk for breast cancerdevelopment, whereas tamoxifen has been used for more than 30 yearsfor the treatment of ER+ breast cancer. Both 7 and 8 have been shown to have antiproliferative effects in ERnegative breast cancer, suggesting that there is an alternative mechanismof action for these compounds.23 A previousreport from our lab suggests that a potential ER-independent mechanismfor the efficacy of these SERMs may be due to off-target inhibitionof mammalian PLD.24 In that study, both 7 and 4-hydroxytamoxifen (9), the active cellularmetabolite of 8, were shown to modestly inhibit mammalianPLD activity, both in cells and in vitro, whereas 8 stimulated PLD2 and showed no effect on PLD1. Desketoraloxifene(10) was developed by Eli Lilly in the late 90s as araloxifene analogue that lacked the important carbonyl hinge in theparent molecule,25 thus leading to a SERMwith unique, planar topology.26 This modificationmade the analogue a much stronger activator in the AP-1 site of ERαcompared to that of ERβ, which is the opposite profile of 8. This new analogue had differential SERM efficacy with stimulatoryactivity in the uterus, similar to 9, but retained raloxifene-likecharacter by remaining estrogenic in the bone.25 Analogue 10 was synthesized following theliterature route, and its ability to inhibit PLDs was assessed; gratifyingly,in our exogenous PLD assays, 10 was a modest inhibitorof mammalian PLD1 (IC50 = 6.1 μM) and PLD2 (IC50 = 2.6 μM). However, as with the structurally distinctmammalian PLD inhibitors 1–6, theSERM-based PLD inhibitors 7–10 hadalso never been evaluated as inhibitors of either PldA or NAPE-PLD.

Bottom Line: In order to better understand the biology and therapeutic potential of these less explored PLD enzymes, small molecule tools are required.Selective estrogen receptor modulators (SERMs) have been previously shown to inhibit mammalian PLD (PLD1 and PLD2).This finding represents an important first step toward the development of small molecules possessing universal inhibition of divergent PLD enzymes to advance the field.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, ‡Department of Chemistry, §Department of Biochemistry, ∥Vanderbilt Institute of Chemical Biology, Vanderbilt University , Nashville, Tennessee 37232, United States.

ABSTRACT
Phospholipase D (PLD) hydrolyses cellular lipids to produce the important lipid second messenger phosphatidic acid. A PLD enzyme expressed by Pseudomonas aeruginosa (PldA) has been shown to be important in bacterial infection, and NAPE-PLD has emerged as being key in the synthesis of endocannabinoids. In order to better understand the biology and therapeutic potential of these less explored PLD enzymes, small molecule tools are required. Selective estrogen receptor modulators (SERMs) have been previously shown to inhibit mammalian PLD (PLD1 and PLD2). By targeted screening of a library of SERM analogues, additional parallel synthesis, and evaluation in multiple PLD assays, we discovered a novel desketoraloxifene-based scaffold that inhibited not only the two mammalian PLDs but also structurally divergent PldA and NAPE-PLD. This finding represents an important first step toward the development of small molecules possessing universal inhibition of divergent PLD enzymes to advance the field.

Show MeSH
Related in: MedlinePlus