Björquist P, Ehnebom J, Inghardt T, Hansson L, Lindberg M, Linschoten M, Strömqvist M, Deinum J
Preclinical R&D, Astra Hässle, Mölndal, Sweden.
Biochemistry. 1998 Feb 3;37(5):1227-34. doi: 10.1021/bi971554q.
A novel low-molecular-weight inhibitor, AR-H029953XX, was developed from a known fibrinolytic compound, flufenamic acid, which prevented complex formation of human plasminogen activator inhibitor type 1 (PAI-1) with tissue plasminogen activator (tPA) by inhibition of PAI-1. To explore the binding site for AR-H029953XX, mutants of human PAI-1 were constructed by site-directed mutagenesis and were then expressed in CHO cells, purified, activated, and characterized. (1) PAI-1 with mutations in the reactive center loop: L1-PAI-1 (P10, Ser337Glu) had stability and activity similar to those of wild-type PAI-1 (wt-PAI-1), and L2-PAI-1 (P12, Ala335Glu) was highly stable but was a substrate for tPA. (2) PAI-1 with mutations near the binding epitope for the strongly inhibiting monoclonal antibody CLB-2C8: C1-PAI-1 (Phe114Glu), C2-PAI-1 (Val121Phe), C3-PAI-1 (Arg76Glu/Arg115Glu/Arg118Glu), and C4-PAI-1 (Arg115Glu) were all comparable in activity and stability to wt-PAI-1. AR-H029953XX (Ki = 25 microM) prevented complex formation between tPA and active wt-PAI-1 as well as that with mutants L1-, L2-, C1-, C2-, and C4-PAI-1. AR-H029953XX also inhibited binding of these PAI-1 variants to the antibody CLB-2C8, as measured by surface plasmon resonance. In contrast, AR-H029953XX had almost no inhibitory effect on the complex formation of tPA with C3-PAI-1. Moreover, AR-H029953XX had no effect on the binding rate of CLB-2C8 to C3-PAI-1, or on the binding to latent PAI-1 or to cleaved L2-PAI-1. The binding site of AR-H029953XX thus appears to be located in the neighborhood of the postulated epitope for CLB-2C8, near residues Arg76 and/or Arg118. This specific domain of the PAI-1 molecule might thus also be important for the mechanism of inhibitory activity toward tPA. Moreover, the structure of this region in active PAI-1 has to be different from the corresponding regions in latent and cleaved PAI-1.
一种新型低分子量抑制剂AR-H029953XX是从已知的纤溶化合物氟芬那酸开发而来的，它通过抑制1型人纤溶酶原激活物抑制剂（PAI-1）来阻止其与组织纤溶酶原激活物（tPA）形成复合物。为了探究AR-H029953XX的结合位点，通过定点诱变构建了人PAI-1的突变体，然后在CHO细胞中表达、纯化、激活并进行表征。（1）活性中心环发生突变的PAI-1：L1-PAI-1（P10，Ser337Glu）具有与野生型PAI-1（wt-PAI-1）相似的稳定性和活性，L2-PAI-1（P12，Ala335Glu）高度稳定，但却是tPA的底物。（2）在强抑制性单克隆抗体CLB-2C8的结合表位附近发生突变的PAI-1：C1-PAI-1（Phe114Glu）、C2-PAI-1（Val121Phe）、C3-PAI-1（Arg76Glu/Arg115Glu/Arg118Glu）和C4-PAI-1（Arg115Glu）在活性和稳定性方面均与wt-PAI-1相当。AR-H029953XX（Ki = 25 microM）可阻止tPA与活性wt-PAI-1以及与突变体L1-、L2-、C1-、C2-和C4-PAI-1形成复合物。通过表面等离子体共振测量，AR-H029953XX还可抑制这些PAI-1变体与抗体CLB-2C8的结合。相比之下，AR-H029953XX对tPA与C3-PAI-1形成复合物几乎没有抑制作用。此外，AR-H029953XX对CLB-2C8与C3-PAI-1的结合速率、与潜伏型PAI-1或裂解型L2-PAI-1的结合均无影响。因此，AR-H029953XX的结合位点似乎位于CLB-2C8假定表位附近，靠近Arg76和/或Arg118残基。PAI-1分子的这一特定结构域可能对其抑制tPA活性的机制也很重要。此外，活性PAI-1中该区域的结构必须与潜伏型和裂解型PAI-1中的相应区域不同。
