Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

Abstract

Plasminogen activator inhibitor type-1 (PAI-1) is a member of the serine protease inhibitor (serpin) family. Excessive PAI-1 activity is associated with human disease, making it an attractive pharmaceutical target. However, like other serpins, PAI-1 has a labile structure, making it a difficult target for the development of small molecule inhibitors, and to date, there are no US Food and Drug Administration-approved small molecule inactivators of any serpins. Here we describe the mechanistic and structural characterization of a high affinity inactivator of PAI-1. This molecule binds to PAI-1 reversibly and acts through an allosteric mechanism that inhibits PAI-1 binding to proteases and to its cofactor vitronectin. The binding site is identified by X-ray crystallography and mutagenesis as a pocket at the interface of β-sheets B and C and α-helix H. A similar pocket is present on other serpins, suggesting that this site could be a common target in this structurally conserved protein family.fibrinolysis | thrombolysis | fibrosis | cancer P lasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (serpin) implicated in numerous pathological processes, including coronary heart disease, chronic fibrotic and inflammatory diseases, and tumor invasion and metastasis (1-6). These associations have made PAI-1 an attractive pharmaceutical target. However, despite extensive studies, only a few small molecule inhibitors have been identified thus far (7-16), and the majority of these are poor pharmaceutical candidates as they have relatively low affinity for PAI-1 and are unable to inactivate PAI-1 bound to its plasma cofactor vitronectin.PAI-1 is the most potent physiologic inhibitor of tissue-type and urokinase-type plasminogen activators (tPA and uPA, respectively) (17). Like other serpins, PAI-1 has a solvent-exposed, reactive center loop (RCL) that contains an amino acid sequence that confers protease target specificity. The serpin inhibitory mechanism is a multistep process of coordinated conformational changes that are necessary to trap a target protease (18). The first step is the formation of a noncovalent Michaelis complex, followed by the initial steps of a typical serine protease catalytic attack leading to the covalent acyl-enzyme complex (19). However, before the protease can dissociate from the serpin, a dramatic conformational change occurs [termed the stressed to relaxed (S to R) transition], in which the cleaved RCL inserts into the central β-sheet A, translocating the covalently-bound protease 70 Å to the base of β-sheet A (20). This conformational change results in distortion of the protease active site (21,22) and thereby prevents the deacylation reaction, inactivating the protease. Should this conformational change be interrupted, the protease can complete its cleavage of the serpin RCL, remaining active and leaving the serpin in a cleaved, inactive, loop-inserted state (23).PAI-1 is unique among serpins in that it readily autoinactivates into a so-called latent form, where the PAI-1 ...

Small molecules inhibitors of plasminogen activator inhibitor-1 – An overview

“…Based on this study, in 2013, the same group [93] published the crystallographic structure of PAI-1 with the related polyphenolic compound CDE-096 (49) (Fig. 15), a novel synthetic molecule.…”

Plasminogen Activator Inhibitor-1 in Cancer: Rationale and Insight for Future Therapeutic Testing

“…These efforts, however, have been limited by a lack of crystal structure information on vitronectin-bound PAI-1 (47). Polyphenolic inhibitors of PAI-1 (CDE-066 and 096) synthesized on the basis of the structure of small molecules identified by a high throughput screen were found to bind to the sB/sC pocket of PAI-1 with an IC 50 of 32 μM and 25 nM, respectively (Figure 1) (48,49). CDE-096 induces allosteric conformational changes affecting the flexibility of PAI-1 and preventing it from binding to PA and decreasing vitronectin binding (49).…”

“…Polyphenolic inhibitors of PAI-1 (CDE-066 and 096) synthesized on the basis of the structure of small molecules identified by a high throughput screen were found to bind to the sB/sC pocket of PAI-1 with an IC 50 of 32 μM and 25 nM, respectively (Figure 1) (48,49). CDE-096 induces allosteric conformational changes affecting the flexibility of PAI-1 and preventing it from binding to PA and decreasing vitronectin binding (49). CDE-096 was still able to interact with vitronectin-bound PAI-1, although the magnitude of polarization was decreased 7.3-fold compared to binding free PAI-1.…”

Serpin latency transition at atomic resolution

“…This correspondence suggests that our simulations have identified residues that play important roles in mediating the latency transition. We note that Li et al (34) recently identified a pocket at the interface of sheets B and C that appears to be important for PAI-1 function, and a number of this subset of energetically important residues (K176, F207, E226, G264, and T267) are clustered in this region.…”