Metabolism and distribution of two highly potent and selective peptidomimetic inhibitors of matriptase.

Matriptase is a serine protease expressed by several types of cancer cells and it participates in tumour growth and progression through the activation of hepatocyte growth factor (HGF) and urokinase-type plasminogen activator (uPA). The metabolism of two potent and selective peptidomimetic inhibitors of matriptase (CJ-1737 and CJ-672) was examined in vitro with enzyme preparations (9000g supernatants, microsomes, and plasma) from dog, pig, rat, and human. It was found that both compounds displayed interesting species-dependent differences. Though CJ-1737 was not metabolized by microsomes, by 9000g supernatants from all species, or by human or rat plasma, canine and porcine plasma enzymes rapidly hydrolysed this compound. In contrast, CJ-672 was metabolized exclusively by enzymes from human liver (microsomes and 9000g supernatants) via a two-step metabolic pathway. Additionally, the distribution of both compounds was investigated in mice. The highest amounts were measured in the kidney and liver, followed by the spleen, lung, and heart. In contrast to CJ-1737, high concentrations of CJ-672 were detected in the colon, indicating an additional biliary excretion. In summary, this work clarifies both the metabolism and distribution of two new matriptase inhibitors and demonstrates important metabolic differences between human enzymes and those from commonly used laboratory animals.

Quantitation of minimal residual disease in Philadelphia chromosome positive chronic myeloid leukaemia patients using real-time quantitative RT-PCR.

We used a recently developed system for real-time quantitative polymerase chain reaction (PCR) to determine residual disease in patients with chronic myeloid leukaemia. The expression of the Bcr-Abl hybrid oncogene was determined and normalized by using the PBGD housekeeping gene product as endogenous reference. The sensitivity and reproducibility of the assay was tested on cell line K562. A dilution of Bcr-Abl-positive cell line K562 remained positive at up to 250 fg of RNA. 10 copies of Bcr-Abl DNA in water could still be detected. The dynamic range of the method spanned six orders of magnitude. Analysis of 10 identical assays on K562 RNA resulted in a variation of 15%. To test the feasibility of normalization of Bcr-Abl dosage by the PBGD product, we compared the efficiencies of the RT-PCRs in 150 patient analyses. We concluded that PBGD was a suitable and stringent quality control standard. Three patients who were treated with donor leucocyte infusions for chronic myeloid leukaemia who had relapsed after bone marrow transplantation were followed over time. The normalized Bcr-Abl dosage was compared to the results of cytogenetics. Cytogenetic analysis was negative below a normalized Bcr-Abl dose of about 3 x 10(-2). This semi-automated method is fast, sensitive and accurate and enables a high throughput of samples.

Structure-based design of a potent chimeric thrombin inhibitor.

Using the three-dimensional structures of thrombin and the leech-derived tryptase inhibitor (LDTI), which does not inhibit thrombin, we were able to construct three LDTI variants inhibiting thrombin. Trimming of the inhibitor reactive site loop to fit thrombin's narrow active site cleft resulted in inhibition constants (Ki) in the 10 nM concentration range; similar values were obtained by the addition of an acidic C-terminal peptide corresponding to hirudin's tail to LDTI. Combination of both modifications is additive, resulting in very strong inhibition of thrombin (Ki in the picomolar range). On the one hand, these results confirm the significance of the restricted active site cleft of thrombin in determining its high cleavage specificity; on the other, they demonstrate that sufficient binding energy at the fibrinogen recognition exosite can force thrombin to accept otherwise unfavorable residues in the active site cleft. The best inhibitor thus obtained is as effective as hirudin in plasma-based clotting assays.

The thrombin E192Q-BPTI complex reveals gross structural rearrangements: implications for the interaction with antithrombin and thrombomodulin.

Previous crystal structures of thrombin indicate that the 60-insertion loop is a rigid moiety that partially occludes the active site, suggesting that this structural feature plays a decisive role in restricting thrombin's specificity. This restricted specificity is typified by the experimental observation that thrombin is not inhibited by micromolar concentrations of basic pancreatic trypsin inhibitor (BPTI). Surprisingly, a single atom mutation in thrombin (E192Q) results in a 10(-8) M affinity for BPTI. The crystal structure of human thrombin mutant E192Q has been solved in complex with BPTI at 2.3 A resolution. Binding of the Kunitz inhibitor is accompanied by gross structural rearrangements in thrombin. In particular, thrombin's 60-loop is found in a significantly different conformation. Concomitant reorganization of other surface loops that surround the active site, i.e. the 37-loop, the 148-loop and the 99-loop, is observed. Thrombin can therefore undergo major structural reorganization upon strong ligand binding. Implications for the interaction of thrombin with antithrombin and thrombomodulin are discussed.

The ornithodorin-thrombin crystal structure, a key to the TAP enigma?

Ornithodorin, isolated from the blood sucking soft tick Ornithodoros moubata, is a potent (Ki = 10(-12) M) and highly selective thrombin inhibitor. Internal sequence homology indicates a two domain protein. Each domain resembles the Kunitz inhibitor basic pancreatic trypsin inhibitor (BPTI) and also the tick anticoagulant peptide (TAP) isolated from the same organism. The 3.1 A crystal structure of the ornithodorin-thrombin complex confirms that both domains of ornithodorin exhibit a distorted BPTI-like fold. The N-terminal portion and the C-terminal helix of each domain are structurally very similar to BPTI, whereas the regions corresponding to the binding loop of BPTI adopt different conformations. Neither of the two 'reactive site loops' of ornithodorin contacts the protease in the ornithodorin-thrombin complex. Instead, the N-terminal residues of ornithodorin bind to the active site of thrombin, reminiscent of the thrombin-hirudin interaction. The C-terminal domain binds at the fibrinogen recognition exosite. Molecular recognition of its target protease by this double-headed Kunitz-type inhibitor diverges considerably from other members of this intensely studied superfamily. The complex structure provides a model to explain the perplexing results of mutagenesis studies on the TAP-factor Xa interaction.

Crystallographic evidence that the F2 kringle catalytic domain linker of prothrombin does not cover the fibrinogen recognition exosite.

The 2.6-A x-ray crystal structure of bovine alpha-thrombin in complex with rhodniin, a protein inhibitor isolated from the bug Rhodnius prolixus, has been solved and refined. The structure has enabled us to trace the N-terminal part of the 49-residue A-chain of bovine alpha-thrombin for the first time, which is fixed in a U-shaped loop on the molecular surface opposite the active site canyon. Model building shows that the 25 amino acid residues that link the A-chain and F2 kringle cannot run through the fibrinogen recognition exosite. This demonstrates that this fibrinogen recognition exosite is available in prothrombin and meizothrombin.

Two heads are better than one: crystal structure of the insect derived double domain Kazal inhibitor rhodniin in complex with thrombin.

Rhodniin is a highly specific inhibitor of thrombin isolated from the assassin bug Rhodnius prolixus. The 2.6 Angstrum crystal structure of the non-covalent complex between recombinant rhodniin and bovine alpha-thrombin reveals that the two Kazal-type domains of rhodniin bind to different sites of thrombin. The amino-terminal domain binds in a substrate-like manner to the narrow active-site cleft of thrombin; the imidazole group of the P1 His residue extends into the S1 pocket to form favourable hydrogen/ionic bonds with Asp189 at its bottom, and additionally with Glu192 at its entrance. The carboxy-terminal domain, whose distorted reactive-site loop cannot adopt the canonical conformation, docks to the fibrinogen recognition exosite via extensive electrostatic interactions. The rather acidic polypeptide linking the two domains is displaced from the thrombin surface, with none of its residues involved in direct salt bridges with thrombin. The tight (Ki = 2 x 10(-13) M) binding of rhodniin to thrombin is the result of the sum of steric and charge complementarity of the amino-terminal domain towards the active-site cleft, and of the electrostatic interactions between the carboxy-terminal domain and the exosite.