Pestka S, Lemahieu R A
Antimicrob Agents Chemother. 1974 Oct;6(4):479-88. doi: 10.1128/AAC.6.4.479.
The relative ability of 44 erythromycin analogues to bind to ribosomes was determined by their effect on [(14)C]erythromycin binding to Escherichia coli ribosomes. The association and dissociation constants of each of these erythromycin derivatives were determined as well as their interaction coefficient for their binding to ribosomes. Substitutions were made on various portions of the erythromycin molecule with retention of substantial activity as measured by inhibition of [(14)C]erythromycin binding to ribosomes. Since the effect of erythromycin analogues on [(14)C]erythromycin binding to ribosomes provides a relatively sensitive assay for these compounds, erythromycin analogues with relatively little affinity for ribosomes could be detected. Compounds with association constants of 10(4) M(-1) were detectable; the association constant for erythromycin binding to ribosomes was approximately 10(8) M(-1). Thus, compounds with 0.0001 the association constant of erythromycin were detectable. This assay could be used alone or in conjunction with microbiological assays for primary screening of active analogues or other compounds which interfere with [(14)C]erythromycin binding to ribosomes. It permits an estimate of the general activity of compounds rapidly and directly. Variables such as metabolic modifications of the compounds and permeability are excluded. The present assay reflects the ability of the compounds to interact directly with their target organelle and may serve as a useful adjunct in developing new compounds.
通过44种红霉素类似物对[¹⁴C]红霉素与大肠杆菌核糖体结合的影响，测定了它们与核糖体结合的相对能力。确定了每种红霉素衍生物的缔合常数和解离常数，以及它们与核糖体结合的相互作用系数。在红霉素分子的不同部位进行了取代，通过抑制[¹⁴C]红霉素与核糖体的结合来衡量，保留了相当的活性。由于红霉素类似物对[¹⁴C]红霉素与核糖体结合的影响为这些化合物提供了一种相对灵敏的测定方法，因此可以检测到对核糖体亲和力相对较低的红霉素类似物。缔合常数为10⁴ M⁻¹的化合物是可检测的；红霉素与核糖体结合的缔合常数约为10⁸ M⁻¹。因此，缔合常数为红霉素0.0001的化合物是可检测的。该测定方法可单独使用，也可与微生物测定法结合使用，用于对活性类似物或其他干扰[¹⁴C]红霉素与核糖体结合的化合物进行初步筛选。它能快速、直接地估计化合物的一般活性。排除了诸如化合物的代谢修饰和通透性等变量。目前的测定方法反映了化合物与靶细胞器直接相互作用的能力，可作为开发新化合物的有用辅助手段。
