Suramin analogues as subtype-selective G protein inhibitors.
Suramin analogues as subtype-selective G protein inhibitors.
复制标题
苏拉明类似物作为亚型选择性 G 蛋白抑制剂。
作者：
Michael Freissmuth;Stefan Boehm;W. Beindl;Peter Nickel;A. P. IJzerman;Martin Hohenegger;C. Nanoff
G蛋白α亚基暴露特异性结合位点，其允许蛋白质反应伴侣的顺序的、构象依赖性结合，例如，G蛋白β γ二聚体、受体和效应物。这些结构域代表了低分子量抑制剂结合的潜在位点。我们测试了以下苏拉明类似物作为G蛋白拮抗剂：8-（3-硝基苯甲酰氨基）-1，3，5-萘三磺酸（NF 007），8-（3-（3-硝基苯甲酰氨基）苯甲酰氨基）-1，3，5-萘三磺酸+酸NF 018），8，8 '-（羰基双（亚氨基-3，1-亚苯基））双-（1，3，5-萘三磺酸）（NF 023），8，8 '-（羰基双（亚氨基-3，1-亚苯基）羰基亚氨基-（3，1-亚苯基））双-（1，3，5-萘三磺酸）（NF 037）和苏拉明。这些化合物抑制[35 S] GTP γ S与纯化的重组G蛋白α亚基的结合，这种作用是由于抑制GDP释放所致。苏拉明对重组G α-s具有选择性（EC 50值约为240 nM;效力等级顺序为苏拉明> NF 037> NF 023> NF 018> NF 007），而NF 023对重组Gi α-l和重组Go α具有选择性（EC 50值约为300 nM;效力等级顺序为NF 023> / = NF 037>苏拉明> NF 018> NF 007）。还在细胞水平上证明了选择性。在大鼠交感神经元中，电压敏感性钙电流的α-2-肾上腺素能和毒蕈碱受体依赖性抑制由Gi/Go介导，而血管活性肠肽（VIP）的抑制由Gs介导。当细胞内应用100 μ M NF 023时，α 2-肾上腺素能和毒蕈碱受体的钙电流抑制大大降低，而对VIP的反应不受影响;相反，在记录移液管中仅用100 μ M苏拉明减弱对VIP的反应。苏拉明类似物不干扰α亚基和G蛋白β γ二聚体之间的相互作用，但与效应物的结合竞争。添加纯化的腺苷酸环化酶逆转苏拉明对[35 S] GTP γ S与重组Galpha-s结合速率的抑制作用，表明对共同位点的直接竞争;类似地，针对效应结合位点的表位的抗体的免疫沉淀被苏拉明抑制。我们的研究结果表明，它是可能的G蛋白抑制剂的设计目标上的α亚基的效应器结合位点。
G protein alpha subunits expose specific binding sites that allow for the sequential, conformation-dependent binding of protein reaction partners, e.g., G protein beta gamma dimers, receptors, and effectors. These domains represent potential sites for binding of low-molecular-weight inhibitors. We tested the following suramin analogues as G protein antagonists: 8-(3-nitrobenzamido)-1,3,5-naphtalenetrisulfonic acid (NF007), 8-(3-(3-nitrobenzamido)benzamido)-1,3,5-naphtalenetrisulfonic++ + acid NF018), 8,8'-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphtalenetri sulfonic acid) (NF023), 8,8'-(carbonylbis(imino-3,1-phenylene)carbonylimino-(3,1-phe nylene))bis-(1,3, 5-naphtalenetrisulfonic acid) (NF037), and suramin. The compounds suppressed [35S]GTPgammaS binding to purified, recombinant G protein alpha subunits, an effect that is due to inhibition of GDP release. Suramin is selective for recombinant Gsalpha-s (EC50 values o f approximately 240 nM; rank order of potency, suramin > NF037 > NF023 > NF018 > NF007), whereas NF023 is selective for recombinant Gi alpha-1 and recombinant Go alpha (EC50 value of approximately 300 nM; rank order of potency, NF023 > / = NF037 > suramin >0 NF018 > NF007). Selectivity was also demonstrated on a cellular level. In rat sympathetic neurons, alpha-2-adrenergic and muscarinic receptor-dependent inhibition of the voltage-sensitive calcium current is mediated by Gi/Go, whereas inhibition by vasoactive intestinal peptide (VIP) is mediated by Gs. Calcium current inhibition by alpha2-adrenergic and muscarinic receptors was greatly reduced when 100 microM NF023 was applied intracellularly, whereas the response to VIP was unaffected; in contrast, the response to VIP was blunted only with 100 microM suramin in the recording pipette. The suramin analogues do not interfere with the interaction between alpha subunits and G protein beta gamma dimer but compete with binding of the effector. The addition of purified adenylyl cyclase reverses the inhibitory effect of suramin on the rate of [35S]GTPgammaS binding to recombinant Gsalpha-s, indicating direct competition for a common site; similarly, immunoprecipitation by an antibody directed against an epitope of the effector binding site is inhibited by suramin. Our results show that it is possible to design G protein inhibitors that target the effector binding site on the alpha subunits.