Novel Biosynthetic Pathway for Secosteroids and the Skin

描述（由申请人提供）：细胞色素P450scc系统可以有效地将7-脱氢胆固醇（7- dhc）转化为7-脱氢孕烯醇酮（7- dhp）和羟化维生素D2和D3以及麦角甾醇。因此，我们发现了产生新的生物活性产品的新的甾体/副甾体生成途径。皮肤途径将主要引起体内、自动或旁分泌方式的作用。在SLOS中，UVB光诱导的转化（仅限于皮肤）将与来自体循环的底物（5,7 -甾体二烯）一起产生维生素dl化合物。这些新途径可以产生许多分子，其结构取决于类固醇生成系统的局部类型和活性，以及UVB光子的获取。为了验证这一假设，设计了以下具体目标。1. 测试P450scc衍生的20-羟基维生素D3和20,22二羟基维生素D3, 17a，24-二羟基麦角甾醇及其光解衍生物，以及20-羟基维生素D2和17a， 20-二羟基维生素D2的生物活性。上述效果将与母体化合物，即维生素D3和2以及麦角甾醇进行比较。2. 检测7-DHP及其羟基衍生物及其光解（uvb诱导）转化的副甾体产物的生物活性；3. 7-DHP酶转化为羟基衍生物的表征，特别是在位置17,20,21和11。研究将包括标准的合成，以充分表征反应产物的化学结构和性质。4. 测试UVB诱导7-DHP及其羟基衍生物或17a24 -二羟基麦角甾b环分子内重排的预测作用，从而产生预期的vitDL -衍生物。还将对所得分子的结构稳定性进行测试；5. 确定P450scc在体内5,7 -二烯醇、维生素D和麦角甾醇代谢中的作用：a)与肾上腺细胞系相比，P450scc在体外培养皮肤细胞中转化7- dhc的特性。还将研究细胞暴露于紫外线辐射后产生的类固醇。b) P450scc在培养皮肤细胞中对维生素D和麦角甾醇转化的表征。6. 确定7-DHP、其羟基和次级衍生物以及维生素D（修饰或未修饰）对P450scc介导的胆固醇孕烯醇酮生成的影响。

DESCRIPTION (provided by applicant): Cytochrome P450scc system can efficiently transform 7-dehydrocholesterol (7-DHC) to 7- dehydropregnenolone (7-DHP) and hydroxylate vitamin D2 and D3, and ergosterol. Thus, we uncovered novel steroido/secosteroidogenic pathways generating new biologically active products. The cutaneous pathway would elicit mostly intra-, auto- or paracrine modes of action. In SLOS, UVB light-induced conversions (exclusive for the skin) would proceed with substrates derived from the systemic circulation (5, 7-steroidal dienes) to generate vitDL compounds. These novel pathways could generate a number of molecules with structures determined by the local type and activity of steroidogenic system, as well as the access to UVB photons. To test this hypothesis the following specific aims have been designed. 1. Testing the biological activity of P450scc derived 20-hydroxyvitamin D3 and 20,22 dihydroxyvitamin D3, 17a,24- dihydroxyergosterol, its photolytic derivative, and of 20-hydroxyvitamin D2 and 17a, 20-dihydroxyvitamin D2. The above effects will be compared to the parent compounds, i.e. vitamins D3 and 2, and ergosterol.; 2. Testing the biological activity of 7-DHP, its hydroxyderivatives and the secosteroidal products of their photolytical (UVB-induced) transformation; 3. Characterization of the enzymatic conversion of 7-DHP into hydroxy-derivatives, particularly at positions 17, 20, 21 and 11. The studies will include synthesis of the standards to fully characterize the chemical structures and properties of the reaction products. 4. Testing the predicted action of UVB in inducing B-ring intramolecular rearrangements of 7-DHP and its hydroxy- derivatives or 17a,24-dihydroxyergostero resulting in the anticipated vitDL - derivatives. Tests of the structural stability of the resulting molecules will also be performed; 5. Defining a role for the P450scc in the in vivo metabolism of 5, 7- dienesterols, vitamin D and ergosterol: a) characterization of 7-DHC transformation by P450scc in vivo in cultured skin cells in comparison to an adrenal cell line. Secosteroids production after exposure of the cells to ultraviolet radiation will also be investigated.; b) characterization of vitamin D and ergosterol transformation by P450scc in cultured skin cells. 6. Defining the effect of 7-DHP, its hydroxy and seco-derivatives and of vitamin D (modified or unmodified) on P450scc mediated pregnenolone production from cholesterol.