Schuster Inge, Egger Helmut, Reddy G Satyanarayana, Vorisek Georg
Institute of Pharmaceutical Chemistry, University Vienna, Althanstrasse 15, 1090 Vienna, Austria.
Recent Results Cancer Res. 2003;164:169-88. doi: 10.1007/978-3-642-55580-0_13.
1alpha,25(OH)2D3 exerts antiproliferative, differentiating effects on many cell types, including cancer tissues. In most of its target cells, levels of 1alpha,25(OH)2D3 are regulated by local synthesis via CYP27B and metabolism via CYP24. Rapidly induced by vitamin D, CYP24 repeatedly hydroxylates the vitamin D side chain and ultimately terminates hormonal activity. Aiming at increased hormone levels, lifetime and function, numerous vitamin D analogs have been synthesized with structural modifications, which impede oxidation of the vitamin D side chain. Our group followed a different strategy, namely, blocking 1,25(OH)2D3 metabolism with inhibitors of CYP24. As appropriate inhibitors, we exploited compounds termed azoles, which directly bind to the heme iron of the CYPs via an azole nitrogen and to other parts of the substrate site. We synthesized some 400 azoles and tested their potential to selectively inhibit CYP24, but not hormone synthesis by the related CYP27B. Using primary human keratinocyte cultures as the source of CYP24 and CYP27, we discovered some 50 inhibitors of CYP24 with IC50 values in the nanomole range and selectivities up to 60-fold. As the first representative of selective CYP24 inhibitors, VID400 underwent preclinical development. In human keratinocytes, VID400 stabilized levels of endogenously produced 1alpha,25(OH)2D3, and thereby strongly amplified and prolonged expression of CYP24, a surrogate marker of hormonal function. In parallel, antiproliferative activity showed up at 100-fold or more lower concentrations of 1alpha,25(OH)2D3. This data suggests that CYP24 inhibitors could become attractive drugs in antiproliferative therapy, used as single entities to increase or extend endogenous hormone function or in combination with low doses of potent analogs. Moreover, we used selective inhibitors as valuable tools to (a) elucidate regulatory mechanisms of vitamin D synthesis and metabolism, (b) determine intrinsic activities of the otherwise highly transient vitamin D metabolites and (c) model the active sites of CYP24 and CYP27B.
1α,25(OH)₂D₃ 对包括癌组织在内的多种细胞类型具有抗增殖和分化作用。在其大多数靶细胞中，1α,25(OH)₂D₃ 的水平通过 CYP27B 的局部合成和 CYP24 的代谢来调节。CYP24 受维生素 D 快速诱导，反复使维生素 D 侧链羟基化并最终终止激素活性。为了提高激素水平、延长激素寿命并增强其功能，人们合成了许多结构经过修饰的维生素 D 类似物，这些修饰阻碍了维生素 D 侧链的氧化。我们团队采用了不同的策略，即使用 CYP24 抑制剂来阻断 1,25(OH)₂D₃ 的代谢。作为合适的抑制剂，我们选用了一类称为唑类的化合物，它们通过唑氮直接与细胞色素 P450（CYPs）的血红素铁结合，并与底物位点的其他部分结合。我们合成了约 400 种唑类化合物，并测试了它们选择性抑制 CYP24 而不抑制相关 CYP27B 合成激素的潜力。以原代人角质形成细胞培养物作为 CYP24 和 CYP27 的来源，我们发现了约 50 种 CYP24 抑制剂，其半数抑制浓度（IC50）值在纳摩尔范围内，选择性高达 60 倍。作为选择性 CYP24 抑制剂的首个代表，VID400 进入了临床前开发阶段。在人角质形成细胞中，VID400 稳定了内源性产生的 1α,25(OH)₂D₃ 的水平，从而强烈放大并延长了 CYP24 的表达，CYP24 是激素功能的替代标志物。与此同时，在 1α,25(OH)₂D₃ 浓度低至其 100 倍或更低时就出现了抗增殖活性。这些数据表明，CYP24 抑制剂可能成为抗增殖治疗中有吸引力的药物，可单独使用以增强或延长内源性激素功能，或与低剂量强效类似物联合使用。此外，我们将选择性抑制剂用作有价值的工具来：(a) 阐明维生素 D 合成和代谢的调节机制；(b) 确定原本高度短暂的维生素 D 代谢产物的内在活性；(c) 模拟 CYP24 和 CYP27B 的活性位点。
