Bergstrom J D, Bostedor R G, Masarachia P J, Reszka A A, Rodan G
Infectious Disease, Merck Research Laboratories (R80-A14), Rahway, New Jersey, 07065, USA.
Arch Biochem Biophys. 2000 Jan 1;373(1):231-41. doi: 10.1006/abbi.1999.1502.
Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.
阿仑膦酸钠是一种含氮双膦酸盐，是一种用于治疗和预防骨质疏松症的强效骨吸收抑制剂。最近的研究结果表明，阿仑膦酸钠和其他含氮双膦酸盐会抑制类异戊二烯生物合成途径，并干扰蛋白质异戊二烯化，这是由于香叶基香叶基二磷酸水平降低所致。本研究确定法呢基二磷酸合酶是双膦酸盐抑制的甲羟戊酸途径酶。对肝脏胞质提取物产物的HPLC分析将阿仑膦酸钠抑制的潜在靶点（IC(50)=1700 nM）缩小为异戊烯基二磷酸异构酶和法呢基二磷酸合酶。重组人法呢基二磷酸合酶被阿仑膦酸钠抑制，IC(50)为460 nM（预孵育15分钟后）。阿仑膦酸钠不抑制从肝脏胞质溶胶中部分纯化的异戊烯基二磷酸异构酶或香叶基香叶基二磷酸合酶。重组法呢基二磷酸合酶也被帕米膦酸钠（IC(50)=500 nM）和利塞膦酸钠（IC(50)=\(3.9 nM\)）抑制，依替膦酸钠（IC50=\(80 μM\)）的抑制作用可忽略不计，氯膦酸盐则完全不抑制。在破骨细胞中，阿仑膦酸钠抑制[(3)H]甲羟戊酸内酯掺入18 - 25 kDa的蛋白质以及非皂化脂质（包括固醇）中。这些发现（i）确定法呢基二磷酸合酶是阿仑膦酸钠在甲羟戊酸途径中的选择性靶点，（ii）表明该酶被其他含氮双膦酸盐（如利塞膦酸钠）抑制，但不被氯膦酸盐抑制，这支持了不同双膦酸盐的不同作用机制，（iii）记录了在纯化的破骨细胞中阿仑膦酸钠对异戊二烯化和固醇生物合成的抑制作用。
