Manne V, Yan N, Carboni J M, Tuomari A V, Ricca C S, Brown J G, Andahazy M L, Schmidt R J, Patel D, Zahler R
Division of Discovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
Oncogene. 1995 May 4;10(9):1763-79.
We describe the biological properties of a new class of potent farnesyltransferase (FT) inhibitors designed as bisubstrate analog inhibitors. These inhibitors incorporate the structural motifs of both farnesyl pyrophosphate and the CAAX tetrapeptide, the two substrates of the reaction catalyzed by FT. Both the phosphinate inhibitor, BMS-185878, and the phosphonate inhibitor, BMS-184467, exhibited higher in vitro FT selectivity than some of the previously reported CVFM peptidomimentics and benzodiazepine analogs. Xenopus oocyte maturation induced by microinjected oncogenic Ras proteins was blocked by coinjected BMS-184467 and BMS-185878. However, both inhibitors showed poor cell activity presumably because of the doubly charged nature of the compounds. Thus, masking the charge on the carboxylate ion markedly improved the cell permeability of BMS-185878, leading to BMS-186511, the methyl carboxyl ester prodrug. BMS-186511 inhibited FT activity in whole cells as determined by inhibition of p21 Ras protein processing, inhibition of farnesylation of proteins including Ras and the accumulation of unfarnesylated Ras proteins in the cytosolic fraction. While the cellular effects of these bisubstrate analog inhibitors had no significant effect on growth of untransformed NIH3T3 cells, they produced pronounced inhibition of Ras transformed cell growth. Both the anchorage dependent and independent growth of ras transformed cells were severely curtailed by micromolar concentrations of BMS-186511. We also found that both H-ras and K-ras transformed cells are affected by this bisubstrate inhibitor. However, K-ras transformed cells appear to be less sensitive. The inhibition of FT activity in cells and the ensuing inhibition of ras transformed cell growth is further manifested in distinct morphological changes in cells. Cells flattened, became less refractile and grew in contact inhibited monolayer. Moreover, the highly diffused character of the actin cytoskeleton in the ras transformed cells was dramatically reverted to an organized network of stress cables crisscrossing the entire cells upon treatment with BMS-186511. All of these effects of BMS-186511 are limited to ras transformed cells that utilize farnesylated Ras, but are not seen in transformed cells that use geranylgeranyl Ras or myristoyl Ras. Significantly, these FT inhibitors did not produce any signs of gross cytotoxicity in untransformed, ras transformed cells or other oncogene transformed cells.
我们描述了一类新型强效法尼基转移酶（FT）抑制剂的生物学特性，这类抑制剂被设计成双底物类似物抑制剂。这些抑制剂融合了法尼基焦磷酸和CAAX四肽的结构基序，这两者是FT催化反应的两种底物。膦酸酯抑制剂BMS - 185878和膦酸酯抑制剂BMS - 184467在体外均表现出比一些先前报道的CVFM拟肽和苯二氮䓬类似物更高的FT选择性。显微注射致癌性Ras蛋白诱导的非洲爪蟾卵母细胞成熟被共注射的BMS - 184467和BMS - 185878阻断。然而，这两种抑制剂的细胞活性都很差，可能是由于化合物的双电荷性质。因此，掩盖羧酸根离子上的电荷显著提高了BMS - 185878的细胞通透性，从而得到甲基羧酸酯前药BMS - 186511。通过抑制p21 Ras蛋白加工、抑制包括Ras在内的蛋白质的法尼基化以及未法尼基化的Ras蛋白在胞质部分的积累来测定，BMS - 186511在全细胞中抑制FT活性。虽然这些双底物类似物抑制剂的细胞效应对未转化的NIH3T3细胞的生长没有显著影响，但它们对Ras转化细胞的生长产生了明显的抑制作用。微摩尔浓度的BMS - 186511严重抑制了ras转化细胞的贴壁依赖性和非贴壁依赖性生长。我们还发现H - ras和K - ras转化细胞均受这种双底物抑制剂的影响。然而，K - ras转化细胞似乎不太敏感。细胞中FT活性的抑制以及随之而来的ras转化细胞生长的抑制在细胞的明显形态变化中进一步体现。细胞变平，折光性降低，并在接触抑制的单层中生长。此外，在用BMS - 186511处理后，ras转化细胞中肌动蛋白细胞骨架高度分散的特征显著恢复为贯穿整个细胞的有组织的应力纤维网络。BMS - 186511的所有这些作用都仅限于利用法尼基化Ras的ras转化细胞，而在使用香叶基香叶基化Ras或肉豆蔻酰化Ras的转化细胞中未观察到。值得注意的是，这些FT抑制剂在未转化的、ras转化的细胞或其他癌基因转化的细胞中均未产生任何明显的细胞毒性迹象。
