MOLECULAR POLYPHARMACY: Modulation of GABAA Receptors

许多药物受体是变构蛋白，其功效反映了药物引起结构改变的程度。这项研究的长期目标是了解药物功效的分子性质。中心假设是受体具有有限数量的结构特征，变构剂通过这些结构特征影响其功能。正如药物相互作用研究导致了饱和结合位点的鉴定，本研究将通过研究通过不同结合位点作用于同一受体的药物之间的相互作用来寻找“变构饱和”的证据。如果一种足够有效的试剂影响到共同的结构特征，那么第二种试剂的作用就会减弱。研究将对GABAA受体进行，这是一个配体门控氯离子通道，至少有五个独立的变构调节剂结合位点。四种结构不同的调节剂（苯巴比妥，5 -孕-3 α - 1-20- 1，H+和La3+）将被研究。短暂转染GABAA受体基因的HEK 293细胞的外部斑块将在存在或不存在单一调节剂的情况下暴露于不同浓度的GABA的快速应用中。将确定每种调节剂对受体激活、脱敏、失活和脱敏后恢复的动力学的影响。然后将调制剂组合应用，以确定一种调制剂的存在如何影响另一种调制剂的效力和功效。这项研究的结果最终可以用于指导突变受体的研究，以确定负责变构调节的结构特征。GABAA受体是中枢神经系统中主要的抑制性神经递质受体，也是许多毒性药物和治疗剂的作用部位，本研究也将增加对GABAA受体的认识。

Many drug receptors are allosteric proteins and efficacy reflects the extent to which agents cause structural changes. The long term goal of this study is to understand the molecular nature of drug efficacy. The central hypothesis is that receptors posses a limited number of structural features by which allosteric agents influence their function. Just as drug interaction studies led to the identification of saturable binding sites, this study will look for evidence of "allosteric saturation" by studying the interactions between agents which act on the same receptor through distinct binding sites. The presence of a sufficiently efficacious agent should reduce the observed effect of a second agent if both affect a common structural feature. Studies will be conducted on the GABAA receptor which is a ligand-gated chloride channel with at least five separate allosteric modulator binding sites. Four structurally distinct modulators (phenobarbital, 5beta-pregnan-3alpha-o1-20-one,H+ and La3+) will be studied. Outside-out patches from HEK 293 cells transiently transfected with GABAA receptor genes will be exposed to rapid application of various concentrations of GABA in the presence or absence of a single modulator. The effect of each modulator on the kinetics of receptor activation, desensitization, deactivation and recovery from desensitization will be determined. Modulators will then be applied in combination to determine how the presence of one modulator affects the potency and efficacy of another. Results from this study can eventually be used to guide studies on mutated receptors in order to identify the structural features responsible for allosteric modulation. This study will also add to the understanding of the GABAA receptor which is the major inhibitory neurotransmitter receptor in the central nervous system and the site of action of many toxic and therapeutic agents.