Upon cerebral hypoxia-ischemia (HI), apoptosis-inducing factor (AIF) can move from mitochondria to nuclei, participate in chromatinolysis, and contribute to the execution of cell death. Previous work (Cande, C., N. Vahsen, I. Kouranti, E. Schmitt, E. Daugas, C. Spahr, J. Luban, R.T. Kroemer, F. Giordanetto, C. Garrido, et al. 2004. Oncogene. 23:1514–1521) performed in vitro suggests that AIF must interact with cyclophilin A (CypA) to form a proapoptotic DNA degradation complex. We addressed the question as to whether elimination of CypA may afford neuroprotection in vivo. 9-d-old wild-type (WT), CypA+/−, or CypA−/− mice were subjected to unilateral cerebral HI. The infarct volume after HI was reduced by 47% (P = 0.0089) in CypA−/− mice compared with their WT littermates. Importantly, CypA−/− neurons failed to manifest the HI-induced nuclear translocation of AIF that was observed in WT neurons. Conversely, CypA accumulated within the nuclei of damaged neurons after HI, and this nuclear translocation of CypA was suppressed in AIF-deficient harlequin mice. Immunoprecipitation of AIF revealed coprecipitation of CypA, but only in injured, ischemic tissue. Surface plasmon resonance revealed direct molecular interactions between recombinant AIF and CypA. These data indicate that the lethal translocation of AIF to the nucleus requires interaction with CypA, suggesting a model in which two proteins that normally reside in separate cytoplasmic compartments acquire novel properties when moving together to the nucleus.

Fragment-based lead generation was applied to find novel small-molecule inhibitors of beta-secretase (BACE-1), a key target for the treatment of Alzheimer's disease. Fragment hits coming from a 1D NMR screen were characterized by BIAcore, and the most promising compounds were soaked into protein crystals to help the rational design of more potent hit analogues. Problems arising due to our inability to grow BACE-1 crystals at the biologically relevant pH at which the screen was run were overcome by using endothiapepsin as a surrogate aspartyl protease. Among others, we identified 6-substituted isocytosines as a novel warhead against BACE-1, and the accompanying paper in this journal describes how these were optimized to a lead series of nanomolar inhibitors.1.

We have characterized the neutralization of the inhibitory activity of the serpin plasminogen activator inhibitor-1 (PAI-1) by a number of structurally distinct organochemicals, including compounds with environmentsensitive spectroscopic properties. In contrast to latent and reactive center-cleaved PAI-1 and PAI-1 in complex with urokinase-type plasminogen activator (uPA), active PAI-1 strongly increased the fluorescence of the PAI-1-neutralizing compounds 1-anilinonaphthalene-8-sulfonic acid and 4,4-dianilino-1,1-bisnaphthyl-5,5-disulfonic acid. The fluorescence increase could be competed by all tested nonfluorescent neutralizers, indicating that all neutralizers bind to a common hydrophobic area preferentially accessible in active PAI-1. Activity neutralization proceeded through two consecutive steps as follows: first step is conversion to forms displaying substrate behavior toward uPA, and second step is to forms inert to uPA. With some neutralizers, the second step was associated with PAI-1 polymerization. Vitronectin reduced the susceptibility to the neutralizers. Changes in sensitivity to activity neutralization by point mutations were compatible with the various neutralizers having overlapping, but not identical, binding sites in the region around ␣-helices D and E and ␤-strand 1A, known to act as a flexible joint when ␤-sheet A opens and the reactive center loop inserts as ␤-strand 4A during reaction with target proteinases. The defined binding area may be a target for development of compounds for neutralizing PAI-1 in cancer and cardiovascular diseases.Plasminogen activator inhibitor-1 (PAI-1) 1 is a fast and specific inhibitor of the serine proteinases urokinase-type (uPA) and tissuetype plasminogen activator (tPA) and, as such, an important regulator of extracellular proteolysis in turn over of extracellular matrix and in fibrinolysis (for reviews see Refs. 1 and 2). PAI-1 binds with high affinity to vitronectin (for reviews see Refs. 3 and 4) and may regulate cell migration and adhesion by inhibition of vitronectin binding of integrins and the uPA receptor (5-10). The PAI-1 level in malignant tumors is one of the most informative biochemical markers of a poor prognosis (for reviews see Refs. 11 and 12), and PAI-1 seems to be causally involved in tumor invasion and angiogenesis (13). A high PAI-1 level in blood plasma is a risk factor for ischemic cardiovascular disease and venous thromboembolism (for review see Ref. 14). PAI-1 is therefore a potential target for both anti-cancer and anti-thrombotic therapy.PAI-1 belongs to the serpin superfamily. Serpins are composed of 3 ␤-sheets and 9 ␣-helices. Serpins and their target proteinases form stable complexes by interaction of the active site of the proteinases with the reactive center peptide bond (P 1 -P 1 Ј) in the solvent-exposed, ϳ20-amino acid long peptide loop, the reactive center loop (RCL) (for reviews see Refs. 2 and 15-17). There is both structural and biochemical evidence that complex formation is associated with the P 1 -P 1 Ј bond being cle...

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.

Bioassay-guided fractionation of a CH2Cl2/MeOH extract of the sponge Suberea clavata using the serine protease factor XIa to detect antithrombotic activity led to the isolation of the new marine natural products, clavatadines A and B. Clavatadines A and B inhibited factor XIa with IC50's of 1.3 and 27 microM, respectively. A crystal structure of protein-inhibitor (clavatadine A) complex was obtained and revealed interesting selective binding and irreversible inhibition of factor XIa. The cocrystal structure provides guidance for the design and synthesis of future factor XIa inhibitors as antithrombotic agents.

SummaryThe relation between the antithrombotic effect in vivo, and the inhibition constant (K i) and the association rate constant (k on) in vitro was investigated for eight different thrombin inhibitors. The carotid arteries of anaesthetized rats were exposed to FeCl3 for 1 h, and the thrombus size was determined from the amount of incorporated 125I- fibrinogen. The thrombin inhibitors were given intravenously, and complete concentration- and/or dose-response curves were constructed. Despite a 50,000-fold difference between the k i-values comparable plasma concentrations of hirudin and melagatran were needed (0.14 and 0.12 μmol 1-1, respectively) to obtain a 50% antithrombotic effect (IC50) in vivo. In contrast, there was a comparable in vitro (k i-value) and in vivo (IC50) potency ratio for melagatran and inogatran, respectively. These results can be explained by the concentration of thrombin in the thrombus and improved inhibition by the low-molecular-weight compounds. For all eight thrombin inhibitors tested, there was an inverse relationship between k on-values in vitro and the slope of the dose response curves in vivo. Inhibitors with k on-values of <1 X 107 M-1 s-1 gave steep dose response curves with a Hill coefficient >1. The association time for inhibition of thrombin for slow-binding inhibitors will be too long to give effective antithrombotic effects at low plasma concentrations, but at increasing concentrations the association time will decrease, resulting in a steeper dose-response curve and thereby a more narrow therapeutic interval.

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.

Ovalbumin is a noninhibitory member of the serpin superfamily that does not spontaneously undergo the loop-to-sheet conformational change upon cleavage of its reactive center that is characteristic of inhibitory serpins. We tested the hypothesis that ovalbumin could be turned into a proteinase inhibitor by increasing the rate of loop insertion through hinge region mutations alone. We found that none of the three variants examined showed any detectable proteinase inhibitory properties. However, replacement of the P14 arginine residue of ovalbumin by serine, either alone or in combination with changes of P12-P10 to alanine, resulted in a large increase in the rate of loop insertion into beta-sheet A following cleavage at the P1-P1' bond by porcine pancreatic elastase (PPE), as shown by the spontaneous formation of a loop-inserted form upon cleavage that has increased the thermal stability. From the magnitude of the increase in stability of the cleaved, loop-inserted forms of the P14 ovalbumin variants, as well as the accessibility of the P1-P1'-cleaved reactive center loop to further proteolysis at P8-P7, we concluded that the reactive center loop can only partially insert into beta-sheet A and therefore that ovalbumin is also defective in the ability of beta-sheet A to expand to fully accommodate the whole of the reactive center loop. This defect, through its effect on the extent and/or rate of loop insertion, is likely to be a principal reason for ovalbumin not being a proteinase inhibitor.