NE10790 (3-PEHPC)

FPP/farnesyl pyrophosphate synthase

[14C]甲羟戊酸掺入 Rab6 会被 NE 10790 抑制，但不会抑制 H-Ras 或 Rap1（分别被 FTase 和 GGTase I 改变的蛋白质）。当暴露于 NE 10790 时，J774 细胞的活力较低。未被 FTase 或 GGTase I 改变的 22-26 kDa 蛋白质的异戊二烯化会被 NE 10790 抑制 [1]。

NE10790是含氮BP RIS的类似物，其中一个膦酸酯基团被羧酸酯基团取代。NE10790在体内保留了抑制骨吸收的能力，尽管与RIS相比，其在啮齿动物中的抗吸收效力显著降低。这种效力的丧失至少部分是由于NE10790对骨的亲和力降低，因为其中一个膦酸酯基团的丧失只允许结合一个钙离子。然而，目前尚不清楚该化合物在细胞水平上影响破骨细胞功能的效果是否也较差，或者它是否通过与含氮BP相同的分子机制（即通过抑制FPP合酶）抑制骨吸收。NE10485是NE10790的类似物，其中杂环基团的氮被甲基化，连接到中心碳的羟基被氢取代。该化合物的抗吸收效力尚未得到表征[1]。

FPP合酶测定[1]
如前所述测定FPP合酶。简而言之，将含有2 nmol[1-14C]异戊烯基二磷酸（4μCi/mmol）和2 nmol GPP的40μl测定缓冲液（50 mm Tris，pH 7.7，10 mm NaF，2 mmMgCl2，1 mg/ml牛血清白蛋白，0.5 mm二硫苏糖醇）预热至37°C。通过加入1μl用测定缓冲液稀释至10μl的重组人FPP合酶（活性为8 pmol FPP/min）来启动该测定。允许该测定进行30分钟，并通过加入200μl饱和NaCl终止。然后用1ml水饱和丁-1-醇提取样品，通过将0.5ml丁醇与4ml通用闪烁剂混合来测定上相中的放射性量。然后使用Packard Tricarb 1900CA闪烁计数器进行计数。为了确定NE10790、RIS和NE10485对FPP合酶活性的影响，将化合物在测定缓冲液中稀释至5倍终浓度，并在反应开始前与酶制剂预孵育10分钟。

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蛋白质：法尼烷基转移酶测定[1]
通过评估从[3H]FPP转移到重组K-Ras的[3H]法呢酰的量来确定重组人法呢酰转移酶的活性（22）。标准反应混合物中试剂的最终浓度为50 mm Tris-Cl、pH 7.2、150 mm KCl、10 mm ZnCl2、3 mm MgCl2、0.2%（v/v）辛基-d-吡喃葡萄糖苷（NOGA洗涤剂）、1 mm二硫苏糖醇、0.9μm FPP、0.3μm[3H]FPP（15-30Ci/mmol）、40 nm FTase和15μm K-RasNE10790、NE10485和RIS在4°C下等分到硅化微量离心管中，并加入反应混合物中。通过将试管转移到37°C并孵育15分钟来引发反应，然后通过加入400μl乙醇：HCl（9:1 v/v）来停止反应。使蛋白质在室温下沉淀30分钟，然后在玻璃微纤维过滤器（GF/C）上过滤。用1ml乙醇洗涤试管三次，然后在加入5ml闪烁混合物后，通过液体闪烁计数定量每个过滤器上的放射性量。试验一式两份，独立重复三次，以验证再现性。

MTT法评估活细胞数[1]
如前所述，通过MTT法测定活J774巨噬细胞的数量。将J774细胞以104个细胞/孔的密度接种到96孔板中，然后在第二天用RIS、NE10790或NE10485处理，重复6孔。48小时后测定MTT试剂的减少量。

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[14C]甲羟戊酸和[3H]GGOH掺入完整细胞中的异戊二烯化蛋白质[1]
如前所述，检测J774巨噬细胞和纯化的兔破骨细胞中的异戊二烯化蛋白（16，24）。简而言之，通过与5μm美伐他汀孵育4小时，细胞中的美伐他汀被耗尽，然后转移到含有5μm美伐他汀和7.5μCi/ml[14C]美伐他汀内酯或30μCi/ml[3H]GGOH的新鲜培养基中，再加上RIS、NE10790、NE10485、FTI-277或GGTI-298。18小时后，将细胞在RIPA缓冲液中裂解（1%（v/v）Nonidet P-40、0.1%（w/v）十二烷基硫酸钠、0.5%（w/v。电泳后，将凝胶固定在10%（v/v）乙酸、40%（v/v”甲醇、50%（v/v“蒸馏水中，然后干燥14C标记的凝胶，并在暴露于柯达荧光屏后，在Bio-Rad Personal FX Imager上观察标记的蛋白质。在干燥之前，将3H标记的凝胶在Enhance中孵育30分钟。然后通过将凝胶在-70°C下暴露于预搅拌的Hyperfilm MP 6天来观察3H标记的蛋白质。

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破骨细胞极化和吸收分析[1]
如前所述，对体外成熟兔破骨细胞的破骨细胞数量、F-actin“环”和再吸收活性进行了评估。简而言之，让兔破骨细胞在96孔板中直径5mm的象牙牙本质盘上粘附2小时，然后在有或没有RIS、NE10790或NE10485的情况下用新鲜的α-最低必需培养基培养。48小时后，通过异硫氰酸四甲基罗丹明（TRITC）-鬼笔环素染色观察细胞内F-actin，然后计算每个椎间盘的肌动蛋白环数量，肌动蛋白环被定义为一个独特而完整的足小体环（肌动蛋白环是破骨细胞极化的指示，它们的存在与主动吸收高度相关）。然后，在37°C的醋酸盐缓冲液（pH 5.5）中，用萘酚ASBI磷酸盐、六唑化副玫瑰苯胺和50 mm酒石酸盐孵育30分钟，对椎间盘进行抗酒石酸酸性磷酸酶染色。为了检测培养物中破骨细胞的总数，然后计数多核（>2个核/细胞）、抗酒石酸性磷酸酶阳性细胞/椎间盘的数量。为了评估破骨细胞的再吸收，将椎间盘浸入20%（w/v）次氯酸钠中以去除所有细胞，然后通过反射光显微镜观察矿物表面的再吸收坑。然后使用蔡司Axiolab反射式光学显微镜检查凹坑/圆盘的面积，并使用基于Aphelion ActiveX组件内部开发的软件进行定量。

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通过免疫染色和共聚焦显微镜评估破骨细胞形态[1]
对于形态学研究，如上所述，在不含试剂或含有10-100μm RIS或500-1000μmNE10790的牙本质切片上培养兔破骨细胞48小时。将培养物在最低必需培养基和固定缓冲液（3.5%（w/v）多聚甲醛，2%（w/v，PBS中的蔗糖）的1:1（v:v）混合物中固定10分钟。然后在4°C下将细胞在Triton缓冲液（20 mm Hepes、300 mm蔗糖、50 mm NaCl、3 mmMgCl2、0.5%（v/v）Triton X-100、0.5%（w/v）叠氮化钠，在pH 7.0的PBS中）中渗透，然后使用小鼠单克隆抗体（10μg/ml）和异硫氰酸荧光素偶联的抗小鼠Ig多克隆抗体（1:40稀释）对paxillin进行免疫染色。用5单位/ml的TRITC鬼笔环肽缀合物（分子探针）染色后，通过其特征性的F-actin环结构（如上所述）鉴定再吸收破骨细胞。通过同时共聚焦反射显微镜检查牙本质表面，通过其减少的反射可以识别再吸收区域。扫描激光共聚焦显微镜在Leica TCS NT系统上进行。通过破骨细胞以1μm的连续步骤收集异硫氰酸荧光素和TRITC荧光染料的荧光图像，并分别在488nm、568nm和647nm发射波长下进行反射，并在xy和zx平面上显示。

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破骨细胞形态的透射电镜分析[1]
为了进行电子显微镜分析，在牙本质切片上用或不用1 mmNE10790处理兔破骨细胞48小时。细胞在2.5%（v/v）戊二醛磷酸盐缓冲液（0.1 m，pH 7.4）中固定至少24小时，然后在相同的固定剂中用2.5%（w/v）EDTA脱盐约48小时。然后在缓冲液中洗涤样品，在四氧化锇中后固定，在乙醇中脱水，并包埋在Epon中。使用乙酸铀酰和柠檬酸铅对超薄切片进行染色，然后使用飞利浦EM201显微镜进行检查。

[1]. Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages. J Biol Chem. 2001 Dec 21;276(51):48213-22.

Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass approximately 22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [(3)H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [(14)C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts.[1]

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To our knowledge, NE10790 is the first specific inhibitor of Rab prenylation to be identified. Other inhibitors of Rab GGTase, such as the monoterpene perillyl alcohol, are less specific and also affect GGTase I and FTase. NE10790 will therefore be a useful tool for further characterization of the role that Rab proteins play in cellular processes necessary for resorption by osteoclasts. Furthermore, the ability of NE10790 to specifically inhibit Rab prenylation in J774 macrophages, osteoclasts, and MC3T3 osteoblast-like cells suggests that this compound will be useful for examining the role of Rab proteins in a variety of cell types in vitro. Until now, the only insights into the consequences of loss of Rab prenylation have come from studies on the effect of genetic mutations in the Rab prenylation machinery. For example, a mutation in REP1 leads to a retinal degenerative disease called choroideremia. The loss of REP1 is partially compensated by the homologous REP2 protein but results in a selective defect in prenylation of a subset of Rab proteins. In addition, mutations in the yeast REP (mrs6-2) results in reduced prenylation of Rab proteins, causing failure of the polarized transport of vesicles toward the bud. Finally, mice with a mutation known asgunmetal have a 4-fold decrease in Rab GGTase activity in platelets as a result of a single base substitution that disrupts the splicing of the Rabggta mRNA. These mice exhibit prolonged bleeding, thrombocytopaenia, and reduced platelet contents, suggesting that Rab GGTase could be a novel target for the treatment of clotting disorders such as myocardial infarction and stroke.
The mechanism by which NE10790 inhibits Rab GGTase remains to be determined. Because the closely related analogue NE10485 (which differs only in that the nitrogen in the side chain is methylated and the hydroxyl group attached to the geminal carbon atom is replaced by a hydrogen) did not inhibit Rab GGTase and had no effect on protein prenylation, the use of other phosphonocarboxylate analogues similar to NE10790 may help to shed light on the molecular structures necessary for interaction with Rab GGTase. Furthermore, although the low potency of NE10790 could limit its potential therapeutic use, future studies addressing the inhibitory mechanism of NE10790 will ultimately help in the rational design of new, more potent inhibitors of this Rab GGTase that have potential for the treatment of thrombotic disorders in addition to diseases of excessive bone resorption such as post-menopausal osteoporosis.
In summary, we demonstrate in this study that NE10790, a phosphonocarboxylate analogue of the potent anti-resorptive drug RIS, inhibits Rab GGTase in vitro and specifically prevents the prenylation of Rab GTPases. NE10790 is therefore a useful new tool that provides a novel approach for investigating the function of Rab proteins in osteoclasts and other cell types.[1]

C8H10NO6P

247.1419

247.025

C, 38.88; H, 4.08; N, 5.67; O, 38.84; P, 12.53

152831-36-2

10014835

White to off-white solid powder

-1.8

137.76

4

7

4

16

316

0

FJVYPXVLXQXDHM-UHFFFAOYSA-N

InChI=1S/C8H10NO6P/c10-7(11)8(12,16(13,14)15)4-6-2-1-3-9-5-6/h1-3,5,12H,4H2,(H,10,11)(H2,13,14,15)

2-hydroxy-2-phosphono-3-(pyridin-3-yl)propanoic acid

3-PEHPC; 3 PEHPC; 3PEHPC; NE10790; 2-hydroxy-2-phosphono-3-(pyridin-3-yl)propanoic acid; 3Pehpc; 2-hydroxy-2-phosphono-3-pyridin-3-ylpropanoic acid; NE 10790; NE-10790.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

0.1 M NaOH : ~20 mg/mL (~80.93 mM)
H2O : ~5 mg/mL (~20.23 mM)

配方 1 中的溶解度: 2 mg/mL (8.09 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。 (<60°C).

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。