Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide

Xue, Yafeng; Björquist, Petter; Inghardt, Tord; Linschoten, Marcel; Müsil, Djordje; Sjölin, L.; Deinum, Johanna

doi:10.1016/s0969-2126(98)00064-1

Cited by 92 publications

(90 citation statements)

References 33 publications

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“…In contrast, the RCL of latent PAI-1 is fully inserted into β-sheet A (24), stabilizing β-sheet A in a state known as the relaxed conformation (40) with a reported T M of 67.5°C (41). PAI-1 pept is a complex of PAI-1 and an RCL mimicking octapeptide that is bound to β-sheet A in place of an inserted RCL (42), thereby exhibiting structural features of both active and latent PAI-1 (43). Specifically, PAI-1 pept adopts a structurally stable relaxed serpin conformation with a T M of 87.6°C, but contains an exposed RCL.…”

Section: Resultsmentioning

confidence: 99%

“…These movements of the central sheet are similarly required for the insertion of RCL mimicking peptides into sA and for conversion to the latent conformation (24,43,50), and CDE-096 strongly inhibits both peptide insertion and the latency transition. This effect is consistent with a mechanism where CDE-096 binding to the sB/sC pocket stabilizes PAI-1 in a conformation with sA closed at the top.…”

Section: Discussionmentioning

confidence: 99%

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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.

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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 ...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

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.

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“…Data presented in chapter 2 extends these studies using PAI-2 RCL analogues. Recently, the crystal structures of both antithrombin and PAI-1 complexed with synthetic RCL peptides confirmed that the peptides bind as strand 4 of p-sheet A , homologous to the position occupied by the R C L in the relaxed and latent forms of the molecules (Skinner et al 1998, Xue et al 1998.…”

Section: -The P14 Residue (Thr 367 ) Is Critical For R C L Insertion mentioning

confidence: 96%

“…Similarly, detection of RCL binding to PAI-2 using real-time biomolecular interaction analysis (BIAcore) was unsuccessful. Xue et al (1998) have previously demonstrated binding of a short RCL pentapeptide to PAI-1 using BIAcore, however the rate of binding of 14mer RCL peptide to PAI-2 under identical conditions appears to be much slower, making the reaction difficult to interpret by this technique. This interaction is also apparent in the structure of relaxed LEI, where the sidechai (Figure 3.7).…”

Section: Structural Changes In the Hinge Region Of Relaxed Pai-2mentioning

confidence: 99%

39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

1998

Fibrinolysis and Proteolysis

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“…Three structures of cleaved, human PAI-1 variants have been reported (Aertgeerts et al, 1995 (PDB entry 9PAI); Jensen and Gettins, 2008 (PDB entry 3CVM); Dewilde et al, 2009 (PDB entry 3EOX)). In addition, crystal structures of human PAI-1 variants were elucidated in which PAI-1 is complexed with an inhibiting pentapeptide (corresponding to P14-P10) (Xue et al, 1998) (PDB entry 1A7C) or with the somatomedin B domain of vitronectin (Zhou et al, 2003) (PDB entry 1OC0).…”

Section: Introductionmentioning

confidence: 99%

Subtle structural differences between human and mouse PAI-1 reveal the basis for biochemical differences

Dewilde

Craen

Compernolle

et al. 2010

Journal of Structural Biology

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Keywords:Plasminogen activator inhibitor-1 PAI-1 Serpin Crystal structure Rational drug design a b s t r a c t Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that plays an important role in cardiovascular disorders and tumor development. The potential role of PAI-1 as a drug target has been evaluated in various animal models (e.g. mouse and rat). Sensitivity to PAI-1 inhibitory agents varied in different species. To date, absence of PAI-1 structures from species other than human hampers efforts to reveal the molecular basis for the observed species differences.Here we describe the structure of latent mouse PAI-1. Comparison with available structures of human PAI-1 reveals (1) a differential positioning of a-helix A; (2) differences in the gate region; and (3) differences in the reactive center loop position. We demonstrate that the optimal binding site of inhibitors may be dependent on the orthologs, and our results affect strategies in the rational design of a pharmacologically active PAI-1 inhibitor.

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Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide

Cited by 92 publications

References 33 publications

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

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

39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

Subtle structural differences between human and mouse PAI-1 reveal the basis for biochemical differences

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