Design and synthesis of potent, orally active, inhibitors of carboxypeptidase U (TAFIa)

A series of 3-mercapto-propionic acid derivatives that function as reversible inhibitors of carboxypeptidase U have been prepared. We present a successful design strategy using cyclic, low basicity guanidine mimetics resulting in potent, selective and bioavailable inhibitors of carboxypeptidase U (TAFIa).     

Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B

This paper presents the crystal structure of porcine pancreatic carboxypeptidase B (pp-CpB) in complex with a variety of thiol-based inhibitors that were developed as antagonists of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recent studies have indicated that a selective inhibitor of TAFIa could enhance the efficacy of existing thrombolytic agents for the treatment of acute myocardial infarction, one of the most prevalent forms of heart attacks. Unfortunately, activated TAFIa rapidly degrades in solution and cannot be used for crystallographic studies. In contrast, porcine pancreatic CpB is stable at room temperature and is available from commercial sources. Both pancreatic CpB and TAFIa are zinc-based exopeptidases, and the proteins share a 47% sequence identity. The homology improves considerably in the active site where nearly all of the residues are conserved. The inhibitors used in this study were designed to mimic a C-terminal arginine residue, one of the natural substrates of TAFIa. The X-ray structures show that the thiol group chelates the active site zinc, the carboxylic acid forms a salt bridge to Arg145, and the guanidine group forms two hydrogen bonds to Asp255. A meta-substituted phenyl was introduced into our inhibitors to reduce conformational freedom. This modification vastly improved the selectivity of compounds against other exopeptidases that cleave basic residues. Comparisons between structures indicate that selectivity derives from the interaction between the guanidine group in the inhibitors and an acidic active site residue. The location of this acidic residue is not conserved in the various carboxypeptidases.     

Electrochemiluminescence assay for basic carboxypeptidases: inhibition of basic carboxypeptidases and activation of thrombin-activatable fibrinolysis inhibitor

Carboxypeptidases catalyze the removal of the C-terminal amino acid residues in peptides and proteins and exert important biological functions. Assays for carboxypeptidase activity that rely on change of absorbance generally suffer from low sensitivity and are difficult to adapt to high-throughput screening. We have developed a sensitive, robust assay for basic carboxypeptidase activity that makes use of electrochemiluminescent (ECL) detection of reaction product. In this assay, a peptide substrate contains the epitope for antibody (G2-10) binding which is masked by a C-terminal arginine. Carboxypeptidase activity exposes the epitope, allowing the binding of ruthenylated G2-10 which is then detected using ECL. High sensitivity allowed detection limits of 1-2 pM enzyme for carboxypeptidase B and activated thrombin-activatable fibrinolysis inhibitor (TAFIa). The inhibition of several basic carboxypeptidases by commercially available inhibitors was studied. This antibody-based method can be extended to other sensitive detection techniques such as amplified luminescent proximity homogeneous assay. The high sensitivity of the assay allowed the determination of the activatable levels of TAFI in human and other animal plasma in the presence of epsilon -aminocaproic acid, an active-site inhibitor that stabilizes TAFIa. A method to isolate in situ activated TAFIa from human serum in the presence of epsilon -aminocaproic acid was also developed.     

Stabilization versus inhibition of TAFIa by competitive inhibitors in vitro

Two competitive inhibitors of TAFIa (activated thrombin-activable fibrinolysis inhibitor), 2-guanidinoethylmercaptosuccinic acid and potato tuber carboxypeptidase inhibitor, variably affect fibrinolysis of clotted human plasma. Depending on their concentration, the inhibitors shortened, prolonged, or had no effect on lysis in vitro. The inhibitor-induced effects were both tissue-type plasminogen activator (tPA) and TAFIa concentration-dependent. Inhibitor-dependent prolongation was favored at lower tPA concentrations. The magnitude of the prolongation increased with TAFIa concentration, and the maximal prolongation observed at each TAFIa concentration increased saturably with respect to TAFIa. A theoretical maximal prolongation of 20-fold was derived from a plot of the maximum prolongation versus TAFIa. This represents, for the first time, a measurement of the maximal antifibrinolytic potential of TAFIa in vitro. Because TAFIa spontaneously decays, the stabilization of TAFIa was investigated as a mechanism explaining the inhibitor-dependent prolongation of lysis. Both inhibitors stabilized TAFIa in a concentration-dependent, non-saturable manner. Although their KI values differed by three orders of magnitude, TAFIa was identically stabilized when the fraction of inhibitor-bound TAFIa was the same. The data fit a model whereby only free TAFIa decays. Therefore, the variable effects of competitive inhibitors of TAFIa on fibrinolysis can be rationalized in terms of free TAFIa and lysis time relative to the half-life of TAFIa.     

Imidazole acetic acid TAFIa inhibitors: SAR studies centered around the basic P(1)(') group

Structural modifications of the aminopyridine P(1)(') group of imidazole acetic acid based TAFIa inhibitors led to the discovery of the aminocyclopentyl analog 28, a 1 nM TAFIa inhibitor with CLT(50) functional activity of 14 nM but without selectivity against CPB. While not as active, aminobutyl derivative 27 provided an improved 6.7-fold selectivity for TAFIa versus CPB.     

The intrinsic threshold of the fibrinolytic system is modulated by basic carboxypeptidases, but the magnitude of the antifibrinolytic effect of activated thrombin-activable fibrinolysis inhibitor is masked by its instability

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is intrinsically unstable, a property that complicates the study of its role in regulating fibrinolysis. To investigate the effect of basic carboxypeptidases on fibrinolysis under conditions of constant carboxypeptidase activity, we employed pancreatic carboxypeptidase B (CPB), a homologous, stable basic carboxypeptidase, as a surrogate for TAFIa. Clots formed from TAFI-depleted plasma or from purified components were supplemented with tissue-type plasminogen activator and either CPB or TAFIa. The clot lysis data indicate that the down-regulation of fibrinolysis mediated by basic carboxypeptidases involves a threshold mechanism. At carboxypeptidase concentrations above the threshold, plasminogen activation is maintained in a fully down-regulated state; experiments in plasma showed that fibrinolysis is essentially halted by saturating concentrations of TAFIa and that fibrinolysis can be prolonged more than 45-fold by a stable carboxypeptidase. The threshold carboxypeptidase concentration was dependent on tissue-type plasminogen activator and antiplasmin concentrations, indicating that the threshold is determined by the steady-state plasmin concentration. Although obvious with CPB, the threshold was masked by the intrinsic instability of TAFIa and became apparent only when the effect of TAFIa was investigated over the picomolar concentration range. Because of the threshold effect and the instability of TAFIa, exponential increases in TAFIa concentration generate linear increases in lysis time. A model relating lysis time to TAFIa concentration, TAFIa half-life, and the threshold concentration of TAFIa is provided. The threshold effect has potentially important implications regarding the role of TAFIa and the regulation of clot lysis in vivo.     

Mutations in the substrate binding site of thrombin-activatable fibrinolysis inhibitor (TAFI) alter its substrate specificity

Thrombin-activable fibrinolysis inhibitor (TAFI) is a zymogen that inhibits the amplification of plasmin production when converted to its active form (TAFIa). TAFI is structurally very similar to pancreatic procarboxypeptidase B. TAFI also shares high homology in zinc binding and catalytic sites with the second basic carboxypeptidase present in plasma, carboxypeptidase N. We investigated the effects of altering residues involved in substrate specificity to understand how they contribute to the enzymatic differences between TAFI and carboxypeptidase N. We expressed wild type TAFI and binding site mutants in 293 cells. Recombinant proteins were purified and characterized for their activation and enzymatic activity as well as functional activity. Although the thrombin/thrombomodulin complex activated all the mutants, carboxypeptidase B activity of the activated mutants against hippuryl-arginine was reduced. Potato carboxypeptidase inhibitor inhibited the residual activity of the mutants. The functional activity of the mutants in a plasma clot lysis assay correlated with their chromogenic activity. The effect of the mutations on other substrates depended on the particular mutation, with some of the mutants possessing more activity against hippuryl-His-leucine than wild type TAFIa. Thus mutations in residues around the substrate binding site of TAFI resulted in altered C-terminal substrate specificity.     

Thrombin-activable fibrinolysis inhibitor (TAFI) zymogen is an active carboxypeptidase

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase found in human plasma, presumably as an inactive zymogen. The current dogma is that proteolytic activation by thrombin/thrombomodulin generates the active enzyme (TAFIa), which down-regulates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. In this study, we have shown that the zymogen exhibits continuous and stable carboxypeptidase activity against large peptide substrates, and we suggest that the activity down-regulates fibrinolysis in vivo.     

Structural basis for the selective inhibition of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) by a selenium-containing inhibitor with chloro-aminopyridine as a basic group

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) is a target molecule for treating thromboembolic disorders. We previously reported that design and synthesis of compound 1 containing a selenol group and chloloaminopyridine. Compound 1 showed high inhibitory activity towards TAFIa, with a high degree of selectivity for TAFIa over carboxypeptidase N (CPN). Here we report investigation of this selectivity. To obtain co-crystal of 1/pp-CPB (a surrogate of TAFIa), we synthesized protected compound 5 as a stabilized precursor of 1. The X-ray crystal structure and docking study indicated that the Cl substituent is accommodated in the pp-CPB specific pocket whereas CPN has no identical pocket. This is important information for the design of drugs targeting TAFIa with high selectivity.     

Generation and characterization of a highly stable form of activated thrombin-activable fibrinolysis inhibitor

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase B that can down-regulate fibrinolysis. TAFIa is a labile enzyme that can be inactivated by conformational instability or proteolysis. TAFI is approximately 40% identical to pancreatic carboxypeptidase B (CPB). In contrast to TAFIa, pancreatic CPB is a stable protease. We hypothesized that regions or residues that are not conserved in TAFIa compared with pancreatic CPB play a role in the conformational instability of TAFIa and that replacement of these non-conserved residues with residues of pancreatic CPB would lead to a TAFIa molecule with an increased stability. Therefore, we have expressed, purified, and characterized two TAFI-CPB chimeras: TAFI-CPB-(293-333) and TAFI-CPB-(293-401). TAFI-CPB-(293-333) could be activated by thrombin-thrombomodulin, but not as efficiently as wild-type TAFI. After activation, this mutant was unstable and was hardly able to prolong clot lysis of TAFI-deficient plasma. Binding of TAFI-CPB-(293-333) to both plasminogen and fibrinogen was normal compared with wild-type TAFI. TAFI-CPB-(293-401) could be activated by thrombin-thrombomodulin, although at a lower rate compared with wild-type TAFI. The activated mutant displayed a markedly prolonged half-life of 1.5 h. Plasmin could both activate and inactivate this chimera. Interestingly, this chimera did not bind to plasminogen or fibrinogen. TAFI-CPB-(293-401) could prolong the clot lysis time in TAFI-deficient plasma, although not as efficiently as wild-type TAFI. In conclusion, by replacing a region in TAFI with the corresponding region in pancreatic CPB, we were able to generate a TAFIa form with a highly stable activity.     

The p11 subunit of annexin II heterotetramer is regulated by basic carboxypeptidase

The Ca(2+)-dependent phospholipid-binding protein annexin II heterotetramer (AIIt) is composed of two copies of annexin II and a p11 dimer. The interaction of the carboxyl-terminal lysine residues of the p11 subunit of AIIt with the lysine-binding kringle domains of plasminogen is believed to play a key role in plasminogen binding and stimulation of the tPA-catalyzed cleavage of plasminogen to plasmin. In the current report, we show that AIIt-stimulated plasminogen activation is regulated by basic carboxypeptidases, in vitro. The incubation of AIIt with a 1/400 molar ratio of carboxypeptidase B for periods as short as 2 min resulted in a significant loss in AIIt-stimulated plasminogen activation. Carboxypeptidase B (CpB) as well as thrombin-activated fibrinolysis inhibitor (TAFIa) and carboxypeptidase N (CpN) rapidly reduced AIIt-stimulated plasminogen activation by 80%. The molar ratio of carboxypeptidase/AIIt for half-maximal inhibition of AIIt was 1/4700, 1/700, and 1/500 for CpB, TAFIa, and CpN, respectively. Treatment of AIIt with carboxypeptidase resulted in loss of both carboxyl-terminal lysine residues from the p11 subunit, which correlated with a decrease in the k(cat) and an increase in the K(m) for plasminogen activation. The data reveal a novel mechanism for the regulation of AIIt-stimulated plasminogen activation.     

Peptidomimetic nitrile inhibitors of malarial protease falcipain-2 with high selectivity against human cathepsins

Falcipain-2 (FP2) is an essential enzyme in the lifecycle of malaria parasites such as Plasmodium falciparum, and its inhibition is viewed as an attractive mechanism of action for new anti-malarial agents. Selective inhibition of FP2 with respect to a family of human cysteine proteases (that include cathepsins B, K, L and S) is likely to be required for the development of agents targeting FP2. Here we describe a series of P2-modified aminonitrile based inhibitors of FP2 that provide a clear strategy toward addressing selectivity for the P. falciparum and show that it can provide potent FP2 inhibitors with strong selectivity against all four of these human cathepsin isoforms.     

Thrombin-activable fibrinolysis inhibitor zymogen does not play a significant role in the attenuation of fibrinolysis

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) plays a significant role in the prolongation of fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which lyses a clot by cleaving fibrin after selected arginine and lysine residues. TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues. It was recently reported that TAFI zymogen possesses sufficient carboxypeptidase activity to attenuate fibrinolysis through a mechanism similar to TAFIa. Here, we show with a recently developed TAFIa assay that when thrombin is used to clot TAFI-deficient plasma supplemented with TAFI, there is some TAFI activation. The extent of activation was dependent upon the concentration of zymogen present in the plasma, and lysis times were prolonged by TAFIa in a concentration-dependent manner. Potato tuber carboxypeptidase inhibitor, an inhibitor of TAFIa but not TAFI, abolished the prolongation of lysis in TAFI-deficient plasma supplemented with TAFI zymogen. In addition, TAFIa but not TAFI catalyzed release of plasminogen bound to soluble fibrin degradation products. The data presented confirm that TAFI zymogen is effective in cleaving a small substrate but does not play a role in the attenuation of fibrinolysis because of its inability to cleave plasmin-modified fibrin degradation products.     

β-Branched acyclic nucleoside analogues as inhibitors of Plasmodium falciparum dUTPase

We report a series of β-branched acyclic tritylated deoxyuridine analogues as inhibitors of Plasmodium falciparum deoxyuridine-5'-triphosphate nucleotidohydrolase (PfdUTPase), an enzyme involved in nucleotide metabolism that acts as first line of defence against uracil incorporation into DNA. Compounds were assayed against both PfdUTPase and intact parasites showing a correlation between enzyme inhibition and cellular assays. β-Branched acyclic uridine analogues described here showed equal or slightly better potency and selectivity compared with previously reported analogues. The best inhibitor gave a K(i) of 0.5 μM against PfdUTPase with selectivity greater than 200-fold compared to the corresponding human enzyme and sub-micromolar growth inhibition of P. falciparum (EC(50) 0.6 μM). A crystal structure of the complex of PfdUTPase with one of the inhibitors shows that this acyclic derivative binds to the active site in a similar manner to that previously reported for a tritylated cyclic deoxyuridine derivative.     

Role of zinc ions in activation and inactivation of thrombin-activatable fibrinolysis inhibitor.

Thrombin-activatable fibrinolysis inhibitor (TAFI) circulates as an inactive proenzyme of a carboxypeptidase B-like enzyme (TAFIa). It functions by removing C-terminal lysine residues from partially degraded fibrin that are important in tissue-type plasminogen activator mediated plasmin formation. TAFI was classified as a metallocarboxypeptidase, which contains a Zn(2+), since its amino acid sequence shows approximately 40% identity with pancreatic carboxypeptidases, the Zn(2+) pocket is conserved, and the Zn(2+) chelator o-phenanthroline inhibited TAFIa activity. In this study we showed that TAFI contained Zn(2+) in a 1:1 molar ratio. o-Phenanthroline inhibited TAFIa activity and increased the susceptibility of TAFI to trypsin digestion. TAFIa is spontaneously inactivated (TAFIai) by a temperature-dependent intrinsic mechanism. The lysine analogue epsilon-ACA, which stabilizes TAFIa, delayed the o-phenanthroline mediated inhibition of TAFIa. We investigated if inactivation of TAFIa involves the release of Zn(2+). However, the zinc ion was still incorporated in TAFIai, indicating that inactivation is not caused by Zn(2+) release. After TAFIa was converted to TAFIai, it was more susceptible to proteolytic degradation by thrombin, which cleaved TAFIai at Arg(302). Proteolysis may make the process of inactivation by a conformational change irreversible. Although epsilon-ACA stabilizes TAFIa, it was unable to reverse inactivation of TAFIa or R302Q-rTAFIa, in which Arg(302) was changed into a glutamine residue and could therefore not be inactivated by proteolysis, suggesting that conversion to TAFIai is irreversible.     

3,4-disubstituted azetidinones as selective inhibitors of the cysteine protease cathepsin K. Exploring P2 elements for selectivity

A novel series of 3,4-disubstituted azetidinones based inhibitors of the cysteine protease cathepsin K (Cat K) has been identified. Although not optimized, some of these compounds show at least 100-fold selectivity against other cathepsins. The use of cyclic moieties as P2 elements has proven to be crucial to achieve a high degree of selectivity.     

Thrombin activatable fibrinolysis inhibitor, a potential regulator of vascular inflammation

The latent plasma carboxypeptidase thrombin-activable fibrinolysis inhibitor (TAFI) is activated by thrombin/thrombomodulin on the endothelial cell surface, and functions in dampening fibrinolysis. In this study, we examined the effect of activated TAFI (TAFIa) in modulating the proinflammatory functions of bradykinin, complement C5a, and thrombin-cleaved osteopontin. Hydrolysis of bradykinin and C5a and thrombin-cleaved osteopontin peptides by TAFIa was as efficient as that of plasmin-cleaved fibrin peptides, indicating that these are also good substrates for TAFIa. Plasma carboxypeptidase N, generally regarded as the physiological regulator of kinins, was much less efficient than TAFIa. TAFIa abrogated C5a-induced neutrophil activation in vitro. Jurkat cell adhesion to osteopontin was markedly enhanced by thrombin cleavage of osteopontin. This was abolished by TAFIa treatment due to the removal of the C-terminal Arg168 by TAFIa from the exposed SVVYGLR alpha 4 beta 1 integrin-binding site in thrombin-cleaved osteopontin. Thus, thrombin cleavage of osteopontin followed by TAFIa treatment may sequentially up- and down-modulate the pro-inflammatory properties of osteopontin. An engineered anticoagulant thrombin, E229K, was able to activate endogenous plasma TAFI in mice, and E229K thrombin infusion effectively blocked bradykinin-induced hypotension in wild-type, but not in TAFI-deficient, mice in vivo. Our data suggest that TAFIa may have a broad anti-inflammatory role, and its function is not restricted to fibrinolysis.     

Substrate optimization for monitoring cathepsin C activity in live cells

A series of peptidic fluorogenic substrates were synthesized to develop a flow cytometry assay (FACS) to monitor the proteolytic activity of cathepsin C in live cells. Of the 16 substrates tested, (NH(2)-aminobutyric-homophenylalanine)(2)-rhodamine demonstrated the best reactivity and selectivity profile in the FACS assay using the B721 human B-lymphoblastoid cell line. The resulting FACS assay was validated through correlation of the IC(50) values with a competitive radiolabeling assay against a series of small molecule inhibitors of cathepsin C.     

Chlamydocin analogs bearing carbonyl group as possible ligand toward zinc atom in histone deacetylases

A series of chlamydocin analogs with various carbonyl functionalities were designed and synthesized as histone deacetylase (HDAC) inhibitors. Chlamydocin is a cyclic tetrapeptide containing an epoxyketone surrogate in the side chain which makes it irreversible inhibitor of HDACs, whereas apicidins are a class of cyclic tetrapeptides that contain an ethylketone moiety as zinc ligand. We replaced the epoxyketone moiety of chlamydocin with several ketones and aldehyde to synthesize potent reversible and selective HDAC inhibitors. The inhibitory activity of the cyclic tetrapeptides against histone deacetylase enzymes were evaluated and the result showed most of them are potent inhibitors. Some of them have remarkable selectivity among the HDACs.     

Inactivation of active thrombin-activable fibrinolysis inhibitor takes place by a process that involves conformational instability rather than proteolytic cleavage

Thrombin-activable fibrinolysis inhibitor (TAFI) is present in the circulation as an inactive zymogen. Thrombin converts TAFI to a carboxypeptidase B-like enzyme (TAFIa) by cleaving at Arg(92) in a process accelerated by the cofactor, thrombomodulin. TAFIa attenuates fibrinolysis. TAFIa can be inactivated by both proteolysis by thrombin and spontaneous temperature-dependent loss of activity. The identity of the thrombin cleavage site responsible for loss of TAFIa activity was suggested to be Arg(330), but site-directed mutagenesis of this residue did not prevent inactivation of TAFIa by thrombin. In this study we followed TAFI activation and TAFIa inactivation by thrombin/thrombomodulin in time and characterized the cleavage pattern of TAFI using matrix-assisted laser desorption ionization mass spectrometry. Mass matching of the fragments revealed that TAFIa was cleaved at Arg(302). Studies of a mutant R302Q-TAFI confirmed identification of this thrombin cleavage site and, furthermore, suggested that inactivation of TAFIa is based on its conformational instability rather than proteolytic cleavage at Arg(302).