Human target validation of phosphoinositide 3-kinase (PI3K)ß: effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kß inhibitor.

BACKGROUND: Based on in vitro and animal data, PI3Kß is given an important role in platelet adhesion and aggregation but its role in insulin signaling is unclear. OBJECTIVE: To strengthen the PI3Kß target validation using the novel, short-acting inhibitor AZD6482. METHODS AND RESULTS: AZD6482 is a potent, selective and ATP competitive PI3Kß inhibitor (IC(50) 0.01 µm). A maximal anti-platelet effect was achieved at 1 µm in the in vitro and ex vivo tests both in dog and in man. In dog, in vivo AZD6482 produced a complete anti-thrombotic effect without an increased bleeding time or blood loss. AZD6482 was well tolerated in healthy volunteers during a 3-h infusion. The ex vivo anti-platelet effect and minimal bleeding time prolongation in the dog model translated well to data obtained in healthy volunteers. AZD6482 inhibited insulin-induced human adipocyte glucose uptake in vitro (IC(50) of 4.4 µm). In the euglycemic hyperinsulinemic clamp model, in rats, glucose infusion rate was not affected at 2.3 µm but reduced by about 60% at a plasma exposure of 27 µm. In man, the homeostasis model analysis (HOMA) index increased by about 10-20% at the highest plasma concentration of 5.3 µm. CONCLUSIONS: This is the first human target validation for PI3Kß inhibition as anti-platelet therapy showing a mild and generalized antiplatelet effect attenuating but not completely inhibiting multiple signaling pathways with an impressive separation towards primary hemostasis. AZD6482 at 'supratherapeutic' plasma concentrations may attenuate insulin signaling, most likely through PI3Kα inhibition.

Sideroxylonal C, a new inhibitor of human plasminogen activator inhibitor type-1, from the flowers of Eucalyptus albens.

Sideroxylonal C (3), a new phloroglucinol dimer, was isolated from the flowers of Eucalyptus albens through bioassay-guided fractionation. The structure elucidation was based on 1D and 2D NMR experiments, MS analysis, and comparison with sideroxylonals A (1) and B (2). Sideroxylonal C inhibited human plasminogen activator inhibitor type-1 at 4.7 microM without any significant effect on human tissue plasminogen activator.

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.

BACKGROUND: Plasminogen activator inhibitor type 1 (PAI-1) is an important endogenous regulator of the fibrinolytic system. Reduction of PAI-1 activity has been shown to enhance dissolution of blood clots. Like other serpins, PAI-1 binds covalently to a target serine protease, thereby irreversibly inactivating the enzyme. During this process the exposed reactive-centre loop of PAI-1 is believed to undergo a conformational change becoming inserted into beta sheet A of the serpin. Incubation with peptides from the reactive-centre loop transform serpins into a substrate for their target protease. It has been hypothesised that these peptides bind to beta sheet A, thereby hindering the conformational rearrangement leading to loop insertion and formation of the stable serpin-protease complex. RESULTS: We report here the 1.95 A X-ray crystal structure of a complex of a glycosylated mutant of PAI-1, PAI-1-ala335Glu, with two molecules of the inhibitory reactive-centre loop peptide N-Ac-TVASS-NH2. Both bound peptide molecules are located between beta strands 3A and 5A of the serpin. The binding kinetics of the peptide inhibitor to immobilised PAI-1-Ala335Glu, as monitored by surface plasmon resonance, is consistent with there being two different binding sites. CONCLUSIONS: This is the first reported crystal structure of a complex formed between a serpin and a serpin inhibitor. The localisation of the inhibitory peptide in the complex strongly supports the theory that molecules binding in the space between beta strands 3A and 5A of a serpin are able to prevent insertion of the reactive-centre loop into beta sheet A, thereby abolishing the ability of the serpin to irreversibly inactivate its target enzyme. The characterisation of the two binding sites for the peptide inhibitor provides a solid foundation for computer-aided design of novel, low molecular weight PAI-1 inhibitors.

Identification of the binding site for a low-molecular-weight inhibitor of plasminogen activator inhibitor type 1 by site-directed mutagenesis.

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.

Epitopes on plasminogen activator inhibitor type-1 important for binding to tissue plasminogen activator.

The molecular details of the rapid complex formation between tissue plasminogen activator (tPA, E.C. 3.4.21.68) and plasminogen activator inhibitor type-1 (PAI-1) are still not fully elucidated. We have used surface plasmon resonance (SPR), the BIAcore, to characterize the binding of a large panel of monoclonal antibodies to four forms of recombinant human PAI-1, including active and latent PAI-1 as well as the complex between PAI-1 and recombinant human tc tPA or the protease part of tPA, the B-chain. Antibodies that discriminate between these different forms of PAI-1 have been identified, which is reflected by differences in k(a), k(d) as well as in Kd. In addition, in a chromogenic assay with PAI-1 and tPA we determined the IC50-values for these antibodies, i.e., studied their ability to inhibit the decrease in tPA-activity caused by PAI-1. In a competition assay using SPR, we have also been able to study whether concurrent binding of these antibodies to PAI-1 was possible. We could thereby assign the antibodies to five groups according to their binding areas. Furthermore, by using this technique, we have for the first time been able to identify three distinct epitopes on PAI-1, which are all of importance for the interaction and complex-formation with tPA. Since the antibodies that bind to one of these areas all have very poor affinity for the complex between PAI-1 and tPA, we suggest that this not previously described epitope must be located near the final binding site for tPA in this complex. Altogether, this also supports the theory of a multistep reaction between PAI-1 and tPA, in which tPA interacts with different parts of the PAI-1-molecule.