Szczepanska-Konkel M, Yusufi A N, Lin J T, Dousa T P
Department of Medicine, May Medical School, Rochester, MN 55905.
Biochem Pharmacol. 1989 Dec 1;38(23):4191-7. doi: 10.1016/0006-2952(89)90514-5.
Using the chemical structural analogs of phosphonoacetic acid (PAA) and related phosphonate compounds, we investigated which structural features are required for competitive inhibition of Na+-Pi cotransport in rat renal cortical brush border membrane (BBM) vesicles (BBMV). The effects of compounds on [Nao+ greater than Nai+]-gradient-dependent 32Pi uptake by BBMV were examined using various inhibitor-to-32Pi concentration ratios in the transport assay medium. The replacement of a phosphono-group with an arsono-group in PAA, or the substitution of a carboxylic group in PAA by an amino or hydroxyl group, totally abolished the inhibitory action on Na+-Pi cotransport. Decreased electronegativity of carboxyl in PAA by coupling with hydrazine or hydroxylamine lowered the inhibitory potenty of PAA. Substitution of H at the alpha-carbon of PAA with ethyl or p-Cl-phenyl groups completely abolished the inhibitory activity, whereas alpha-halogenation with Br greatly increased the inhibitory potency of PAA, close to that of phosphonoformic acid (PFA). The inhibition by all the active tested monophosphates was strictly competitive. The tested compounds displaced [14C]PFA pre-bound onto BBMV in the presence of 100 mM NaCl. The ability of monophosphates to inhibit Na+-Pi cotransport across BBM and the binding of [14C]PFA were closely correlated (r = 0.925; P greater than 0.001). These results show that: (a) strong electronegativity at both ends of the PAA molecule is needed for inhibitory action, (b) an alpha-aliphatic or aromatic substituent at the alpha-carbon probably hinders the access of the inhibitor to the Pi-binding site of the Na+-Pi cotransporter in BBM, whereas (c) an alpha-electrophilic substituent--Br--enhances the inhibitory potency of PAA. The tested compounds inhibited Na+-Pi cotransport by binding, in the presence of Na+, on the same site on the luminal surface of BBM as did PFA and, by extension, Pi.
利用膦酰基乙酸（PAA）的化学结构类似物及相关膦酸盐化合物，我们研究了竞争性抑制大鼠肾皮质刷状缘膜（BBM）囊泡（BBMV）中Na⁺-Pi共转运所需的结构特征。在转运测定培养基中使用各种抑制剂与³²Pi浓度比，检测化合物对BBMV中依赖于[Na⁺＞Na⁺]梯度的³²Pi摄取的影响。PAA中膦酰基被胂基取代，或PAA中的羧基被氨基或羟基取代，完全消除了对Na⁺-Pi共转运的抑制作用。通过与肼或羟胺偶联降低PAA中羧基的电负性，降低了PAA的抑制效力。PAA的α-碳上的氢被乙基或对氯苯基取代完全消除了抑制活性，而用溴进行α-卤化大大提高了PAA的抑制效力，接近膦甲酸（PFA）的抑制效力。所有测试的活性单磷酸盐的抑制作用均为严格竞争性。在100 mM NaCl存在下，测试化合物取代预先结合在BBMV上的[¹⁴C]PFA。单磷酸盐抑制Na⁺-Pi跨BBM转运的能力与[¹⁴C]PFA的结合密切相关（r = 0.925；P＞0.001）。这些结果表明：（a）PAA分子两端的强电负性是抑制作用所必需的；（b）α-碳上的α-脂肪族或芳香族取代基可能阻碍抑制剂进入BBM中Na⁺-Pi共转运体与Pi结合的位点；而（c）α-亲电取代基——Br——增强了PAA的抑制效力。测试化合物在Na⁺存在下通过与PFA以及Pi一样结合在BBM腔表面的同一部位来抑制Na⁺-Pi共转运。
