Surprising unreactivity of cholesterol-5,6-epoxides towards nucleophiles

Abstract

This article is available online at http://www.jlr.org thesis ( 2 ). We showed that ChEH was fully inhibited by therapeutic doses of AEBS ligands, including drugs belonging to different pharmacological classes and widely used nutritional compounds. These compounds include tamoxifen (tam), one of the main drugs used for the fi rstline and long-term hormone therapy of breast cancers; raloxifene, which has been approved for the prevention of osteoporosis ( 3 ); amiodarone, which is widely used for the prevention and the treatment of anti-arrhythmia ( 4 ); trifluoroperazine, an antipsychotic drug used for the treatment of schizophrenia ( 5 ); and the omega-3 fatty acid docosahexaenoic acid, which is widely proposed as dietary supplement with health benefi ts ( 2 ). Extensive knowledge of the mechanism of action of all these molecules is required to prevent toxicity to patients submitted to longterm treatment with such inhibitors of ChEH.AEBS/ChEH ligands, such as tam and raloxifene, were recently shown to induce cell differentiation and death of breast cancer cells through a mechanism involving sterol accumulation and autoxidation ( 6-8 ) leading to the production of 5,6-EC, suggesting that 5,6-EC accumulation is involved in their anticancer and chemopreventitive activity. This is contrary to the initial hypothesis that the biological function of ChEH was to detoxify 5,6-EC. This hypothesis emerged because it was initially postulated that 5,6-EC could, like other chemicals bearing epoxide groups, be potent alkylating substances and, hence, carcinogenic ( 9 ).Abstract We recently established that drugs used for the treatment and the prophylaxis of breast cancers, such as tamoxifen, were potent inhibitors of cholesterol-5,6-epoxide hydrolase (ChEH), which led to the accumulation of 5,6 ␣ -epoxy-cholesterol (5,6 ␣ -EC) and 5,6 ␤ -epoxy-cholesterol (5,6 ␤ -EC). This could be considered a paradox because epoxides are known as alkylating agents with putative carcinogenic properties. We report here that, as opposed to the carcinogen styrene-oxide, neither of the ECs reacted spontaneously with nucleophiles. Under catalytic conditions, 5,6 ␤ -EC remains unreactive whereas 5,6 ␣ -EC gives cholestan-3 ␤ ,5 ␣ -diol-6 ␤ -substituted compounds. These data showed that 5,6-ECs are stable epoxides and unreactive toward nucleophiles in the absence of a catalyst, which contrasts with the well-known reactivity of aromatic and aliphatic epoxides. These data rule out 5,6-EC acting as spontaneous alkylating agents. In addition, these data support the existence of a stereoselective metabolism of 5,6 ␣ -EC. Cholesterol-5,6-epoxide hydrolase (ChEH; EC 3.3.2.11) catalyzes the stereoselective hydration of cholesterol-5,6-epoxide diastereoisomers (5,6-EC: 5,6 ␣ -EC and 5,6 ␤ -EC) into cholestane-3 ␤ ,5 ␣ ,6 ␤ -triol (CT) ( 1 ). We recently reported the molecular identifi cation of this enzyme and established that ChEH activity was carried out by the microsomal antiestrogen binding site (AEBS), which consists of two subunits: the 3 ␤ -hydroxyste...

The 2014 Bernard B. Brodie Award Lecture—Epoxide Hydrolases: Drug Metabolism to Therapeutics for Chronic Pain

“…Surprisingly, the molecular entity responsible for the ChEH activity was in fact the antiestrogen binding site, a multidomain protein that has high affinity for the pharmaceutical tamoxifen, which is composed of two enzymes, For years, it had been believed that 5,6-cholesterol epoxides had a role in carcinogenesis (Petrakis, 1986;Poirot and Silvente-Poirot, 2013). Although early studies indicated that 5,6-cholesterol epoxide binds covalently to nucleophilic DNA (Blackburn et al, 1979;Sevanian and Peterson, 1984), 5,6-cholesterol epoxide is relatively unreactive with nucleophiles, only forming adducts with mercaptoethanol and aminoethanol under catalytic conditions (Paillasse et al, 2012). Despite this lack of chemical reactivity, an undescribed enzyme was found to catalyze the addition of histamine to 5,6a-cholesterol epoxide, resulting in Dendrogenin A (DDA).…”

Dendrogenin A arises from cholesterol and histamine metabolism and shows cell differentiation and anti-tumour properties

“…One peculiarity of epoxide-bearing substances is their instability due to the high reactivity of the epoxide ring towards nucleophiles including amines, thiols and hydroxyl groups that are found in bio-macromolecules and biological media1314. Surprisingly, 5,6-ECs were found to be different from other epoxide-bearing substances in that they do not react spontaneously with nucleophiles, including amines, thus making 5,6-ECs extremely stable in biological media1115.…”

From tamoxifen to dendrogenin A: The discovery of a mammalian tumor suppressor and cholesterol metabolite

“…This hypothesis was chemically tested but no reactions occurred between 5,6a-EC or 5,6b-EC and nucleophiles containing amine or thiol groups, establishing a surprising non-spontaneous reactivity of 5,6-EC towards nucleophiles [37,38]. In the presence of a catalyst (heat or a Lewis acid), a conjugation reaction did take place but only between 5,6a-EC and nucleophiles, with 5,6b-EC remaining totally unreactive under the conditions tested [37,38]. Interestingly, while three different substitution products at the C6 were possible [10], only one was obtained, with a 6b substitute product resulting from the trans diaxial ring opening of the epoxide by the primary (a) amine of HA.…”