Identification of the Binding Site for a Low-Molecular-Weight Inhibitor of Plasminogen Activator Inhibitor Type 1 by Site-Directed Mutagenesis

Björquist, Petter; Ehnebom, J.; Inghardt, Tord; Hansson, Lennart; Lindberg, Maria; Linschoten, Marcel; Strömqvist, Mats; Deinum, Johanna

doi:10.1021/bi971554q

Cited by 68 publications

(70 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Preparation of Native, Latent, and Cleaved PAI-1-Glycosylated recombinant PAI-1 spontaneously converts to the latent conformation with a half-life of 3 h under physiological conditions (4,11), but this may be increased to 100 h if it is stored at high salt concentration, low pH, and room temperature (41). Our purification protocol of PAI-1 was a modification of existing methods (42,43), and all the steps were carried out at room temperature.…”

Section: Resultsmentioning

confidence: 99%

Polymerization of Plasminogen Activator Inhibitor-1

Zhou¹,

Faint²,

Charlton³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

The activity of the serine proteinase inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is controlled by the intramolecular incorporation of the reactive loop into ␤-sheet A with the generation of an inactive latent species. Other members of the serpin superfamily can be pathologically inactivated by intermolecular linkage between the reactive loop of one molecule and ␤-sheet A of a second to form chains of polymers associated with diverse diseases. It has long been believed that PAI-1 is unique among active serpins in that it does not form polymers. We show here that recombinant native and latent PAI-1 spontaneously form polymers in vitro at low pH although with distinctly different electrophoretic patterns of polymerization. The polymers of both the native and latent species differ from the typical loop-Asheet polymers of other serpins in that they readily dissociate back to their original monomeric form. The findings with PAI-1 are compatible with different mechanisms of linkage, each involving ␤-strand addition of the reactive loop to s7A in native PAI-1 and to s1C in latent PAI-1. Glycosylated native and latent PAI-1 can also form polymers under similar conditions, which may be of in vivo importance in the low pH environment of the platelet. Plasminogen activator inhibitor type 1 (PAI-1)1 is a member of the serine proteinase inhibitor or serpin superfamily (1, 2). Serpins play an important role in the control of proteinases involved in blood coagulation, complement activation, and inflammation and are distinguished functionally from other types of protein inhibitors by their ability to form SDS stable complexes with target proteinases. Crystal structures have shown that members of the family share a highly conserved tertiary fold consisting of three large ␤-sheets surrounded by nine ␣-helices. This scaffold presents the reactive loop as a pseudosubstrate that binds to and inhibits the target proteinase (3). The target proteinases of PAI-1 are urokinase-type plasminogen activator and tissue-type plasminogen activator (tPA) (4) and as such it is an important modulator of events of extracellular proteolysis in fibrinolysis and in the turnover of extracellular matrix (5).One of the most striking features of serpins is their ability to undergo a dramatic conformational rearrangement with the N-terminal portion of the reactive loop inserting into ␤-sheet A (6). This transition with cleavage of the loop is central to the formation of a stable inhibitory complex (7-10), but it can also occur spontaneously in vivo, without cleavage of the loop, to form an inactive latent conformation (11-14). Moreover, antithrombin and ␣ 1 -antitrypsin can be induced to adopt a latent conformation by heating in stabilizing concentrations of sodium citrate (15-17). Serpins are also able to link their reactive loop to a ␤-sheet of another molecule to form loop-sheet polymers, one form of which (18) has recently been crystallized (19,20). These polymers are of considerable importance because they underlie the deficiency of ␣...

show abstract

Section: Resultsmentioning

confidence: 99%

Polymerization of Plasminogen Activator Inhibitor-1

Zhou¹,

Faint²,

Charlton³

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Notably, these agents appear to act via several different mechanisms. For example, the small molecule AR-H029953xx has been suggested to bind near residues Arg-76 in ␣-hD and Arg-118 in the loop between ␣-hF and ␤-s1A, and it inhibited PAI-1 by preventing complex formation between PAI-1 and tPA (52). For another small molecule, PAI-039, e.g.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation

Fjellström

Deinum

Sjögren

et al. 2013

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: Inhibition of PAI-1 may yield beneficial effects in e.g. cardiovascular diseases and cancer. Results: The small molecule PAI-1 inhibitor, AZ3976, binds latent but not active PAI-1. The structure of the AZ3976⅐latent PAI-1 complex is presented. Conclusion: AZ3976 inhibits PAI-1 by accelerating latency transition, presumably by binding a prelatent form of PAI-1. Significance: This study provides new drug design opportunities for PAI-1 inhibitors.

show abstract

“…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)(8)(9)(10)(11)(12)(13)(14)(15)(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.…”

mentioning

confidence: 99%

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

Reinke

Sanders

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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 ...

show abstract

Identification of the Binding Site for a Low-Molecular-Weight Inhibitor of Plasminogen Activator Inhibitor Type 1 by Site-Directed Mutagenesis

Cited by 68 publications

References 26 publications

Polymerization of Plasminogen Activator Inhibitor-1

Polymerization of Plasminogen Activator Inhibitor-1

Characterization of a Small Molecule Inhibitor of Plasminogen Activator Inhibitor Type 1 That Accelerates the Transition into the Latent Conformation

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

Contact Info

Product

Resources

About