Inhibition of human beta-tryptase by Bowman-Birk inhibitor derived peptides.

Four 11-residue peptides based on the Bowman-Birk inhibitor (BBI) structure were synthesized. These were tested for their ability to inhibit human beta-tryptase. Peptides with a basic residue at P1 inhibited tryptase even though the intact BBI protein is inactive. This result is interpreted in terms of the unique structural arrangement of active sites in tryptase which prevent access by large protein inhibitors.     

The B12 anti-tryptase monoclonal antibody disrupts the tetrameric structure of heparin-stabilized beta-tryptase to form monomers that are inactive at neutral pH and active at acidic pH

The novel tetrameric structure of human beta-tryptase faces each active site into the central pore, thereby restricting access of most biologic protease inhibitors. The mechanism by which the anti-tryptase mAb B12 inhibits human beta-tryptase peptidase and proteolytic activities at neutral pH, but augments proteolytic activity at acidic pH, was examined. At neutral pH, B12-beta-tryptase complexes are inactive. At acidic pH, B12 (intact and Fab) minimally affects peptidase activity when added to beta-tryptase tetramers, but does induce susceptibility to inhibition by soybean trypsin inhibitor and antithrombin III. Surprisingly, B12 Fab-beta-tryptase complexes formed at both neutral and acidic pH exhibit the apparent molecular mass of a complex with 1 beta-tryptase monomer and 1 Fab by gel filtration. B12 does not compete with heparin for binding to tryptase at either neutral or acidic pH. Thus, B12 directly disrupts beta-tryptase tetramers to monomers that are inactive at neutral pH, whereas at acidic pH, are active and more accessible to protein inhibitors and substrates.     

Design of novel, potent, and selective human beta-tryptase inhibitors based on alpha-keto-[1,2,4]-oxadiazoles

A series of novel alpha-keto-[1,2,4]-oxadiazoles has been synthesized as human tryptase inhibitors for evaluation as a new class of anti-asthmatic agent. The inhibitor design is focused on using a prime-side hydrophobic pocket and the S2 pocket of beta-tryptase to achieve inhibition potency and selectivity over other serine proteases.     

Human beta-tryptase: detection and characterization of the active monomer and prevention of tetramer reconstitution by protease inhibitors

beta-Tryptase is a trypsin-like serine protease stored in mast cell secretory granules primarily as an enzymatically active tetramer. The current study aims to determine whether monomeric beta-tryptase also can exhibit enzyme activity, as suggested previously. At neutral pH beta-tryptase tetramers in the absence of heparin or dextran sulfate spontaneously convert to inactive monomers. Addition of a polyanion to these monomers at neutral pH fails to convert them back to a tetramer or to an enzymatically active state. In contrast, at acidic pH addition of a polyanion resurrects enzyme activity. Whether this activity is associated with tetramers or monomers depends on the concentration of beta-tryptase. Under the experimental conditions employed at pH 6 in the presence of heparin, the monomer concentration at which 50% conversion to tetramers occurs is 193 ng/mL. Activity against tripeptide substrates by monomers is detected at pH 6 but not at pH 7.4, whereas tetramer activity is greater at pH 7.4 than pH 6.0. Active monomers are inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, antithrombin III, and alpha2-macroglobulin, whereas active tetramers are resistant to these inhibitors. Active monomers form complexes with these inhibitors and cleave both antithrombin III and alpha2-macroglobulin. These inhibitors also prevent reconstitution of monomers to tetramers, indicating that inactive monomers become active monomers before becoming active tetramers. The ability of tryptase monomers to become active at acidic pH raises the possibilities of expanded substrate specificities as well as inhibitor susceptibilities where the low-pH environments associated with inflammation or poor vascularity are encountered in vivo.     

Bivalent inhibition of beta-tryptase: distance scan of neighboring subunits by dibasic inhibitors

Based on bifunctional diketopiperazines as templates and m-aminomethyl-phenylalanine as arginine mimetic, we have synthesized a new class of structurally related dibasic tryptase inhibitors with systematically increasing spacer length. These compounds were used to scan the distance between the active sites of two neighboring subunits of the beta-tryptase tetramer. The K(i)-values obtained are a function of the distance between the terminal amino groups and indicate optimal binding of inhibitors with 29-31 bonds between the amino groups. These experimental data are in full agreement with predictions derived from a novel modeling program that allows the docking of bivalent ligands.     

Synthesis and evaluation of 4-substituted benzylamine derivatives as beta-tryptase inhibitors

Since beta-tryptase is considered a critical mediator of asthma, potent tryptase inhibitors may be useful as new agents for the treatment of asthma. We investigated 4-substituted benzylamine derivatives and obtained M58539 (15h) as a potent inhibitor of beta-tryptase (IC50 = 5.0 nM) with high selectivity against other serine proteases, low molecular weight, clog P value less than 5, lack of amidino and guanidino groups, and independence of Zn2+ ion.     

A tick protein with a modified Kunitz fold inhibits human tryptase

TdPI, a tick salivary gland product related to Kunitz/BPTI proteins is a potent inhibitor of human beta-tryptase. Kinetic assays suggest that three of the four catalytic sites of tryptase are blocked by TdPI, and that the inhibition of one of these involves a peptide flanking the Kunitz head. In the course of the inhibition, tryptase cleaves TdPI at several positions. Crystal structures of the TdPI head, on its own and in complex with trypsin, reveal features that are not found in classical Kunitz/BPTI proteins and suggest the mode of interaction with tryptase. The loop of TdPI connecting the beta-sheet with the C-terminal alpha-helix is shortened, the disulphide-bridge pattern altered and N and C termini separated to produce a highly pointed molecule capable of penetrating the cramped active sites of tryptase. TdPI accumulates in the cytosolic granules of mast cells, presumably suppressing inflammation in the host animal's skin by tryptase inhibition.     

N-terminal extension of the yeast IA3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity

Yeast IA(3) aspartic proteinase inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme, saccharopepsin. Even aspartic proteinases that are very closely similar to saccharopepsin (e.g. the vacuolar enzyme from Pichia pastoris) are not susceptible to significant inhibition. The Pichia proteinase was selected as the target for initial attempts to engineer IA(3) to re-design the specificity. The IA(3) polypeptides from Saccharomyces cerevisiae and Saccharomyces castellii differ considerably in sequence. Alterations made by deletion or exchange of the residues in the C-terminal segment of these polypeptides had only minor effects. By contrast, extension of each of these wild-type and chimaeric polypeptides at its N-terminus by an MK(H)(7)MQ sequence generated inhibitors that displayed subnanomolar potency towards the Pichia enzyme. This gain-in-function was completely reversed upon removal of the extension sequence by exopeptidase trimming. Capture of the potentially positively charged aromatic histidine residues of the extension by remote, negatively charged side-chains, which were identified in the Pichia enzyme by modelling, may increase the local IA(3) concentration and create an anchor that enables the N-terminal segment residues to be harboured in closer proximity to the enzyme active site, thus promoting their interaction. In saccharopepsin, some of the counterpart residues are different and, consistent with this, the N-terminal extension of each IA(3) polypeptide was without major effect on the potency of interaction with saccharopepsin. In this way, it is possible to convert IA(3) polypeptides that display little affinity for the Pichia enzyme into potent inhibitors of this proteinase and thus broaden the target selectivity of this remarkable small protein.     

A Nucleotide-dependent molecular switch controls ATP binding at the C-terminal domain of Hsp90. N-terminal nucleotide binding unmasks a C-terminal binding pocket.

In vivo function of the molecular chaperone Hsp90 is ATP-dependent and requires the full-length protein. Our earlier studies predicted a second C-terminal ATP-binding site in Hsp90. By applying direct biochemical approaches, we mapped two ATP-binding sites and unveiled the C-terminal ATP-binding site as the first example of a cryptic chaperone nucleotide-binding site, which is opened by occupancy of the N-terminal site. We identified an N-terminal gamma-phosphate-binding motif in the middle domain of Hsp90 similar to other GHKL family members. This motif is adjacent to the phosphate-binding region of the C-terminal ATP-binding site. Whereas novobiocin disrupts both C- and N-terminal nucleotide binding, we found a selective C-terminal nucleotide competitor, cisplatin, that strengthens the Hsp90-Hsp70 complex leaving the Hsp90-p23 complex intact. Cisplatin may provide a pharmacological tool to dissect C- and N-terminal nucleotide binding of Hsp90. A model is proposed on the interactions of the two nucleotide-binding domains and the charged region of Hsp90.     

Structural and functional consequences of the presence of a fourth disulfide bridge in the scorpion short toxins: solution structure of the potassium channel inhibitor HsTX1

We have determined the three-dimensional structure of the potassium channel inhibitor HsTX1, using nuclear magnetic resonance and molecular modeling. This protein belongs to the scorpion short toxin family, which essentially contains potassium channel blockers of 29 to 39 amino acids and three disulfide bridges. It is highly active on voltage-gated Kv1.3 potassium channels. Furthermore, it has the particularity to possess a fourth disulfide bridge. We show that HsTX1 has a fold similar to that of the three-disulfide-bridged toxins and conserves the hydrophobic core found in the scorpion short toxins. Thus, the fourth bridge has no influence on the global conformation of HsTX1. Most residues spatially analogous to those interacting with voltage-gated potassium channels in the three-disulfide-bridged toxins are conserved in HsTX1. Thus, we propose that Tyr21, Lys23, Met25, and Asn26 are involved in the biological activity of HsTX1. As an additional positively charged residue is always spatially close to the aromatic residue in toxins blocking the voltage-gated potassium channels, and as previous mutagenesis experiments have shown the critical role played by the C-terminus in HsTX1, we suggest that Arg33 is also important for the activity of the four disulfide-bridged toxin. Docking calculations confirm that, if Lys23 and Met25 interact with the GYGDMH motif of Kv1.3, Arg33 can contact Asp386 and, thus, play the role of the additional positively charged residue of the toxin functional site. This original configuration of the binding site of HsTX1 for Kv1.3, if confirmed experimentally, offers new structural possibilities for the construction of a molecule blocking the voltage-gated potassium channels.     

Bivalent inhibitor of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Ubr1p, the recognition (E3) component of the Saccharomyces cerevisiae N-end rule pathway, contains at least two substrate-binding sites. The type 1 site is specific for N-terminal basic residues Arg, Lys, and His. The type 2 site is specific for N-terminal bulky hydrophobic residues Phe, Leu, Trp, Tyr, and Ile. Previous work has shown that dipeptides bearing either type 1 or type 2 N-terminal residues act as weak but specific inhibitors of the N-end rule pathway. We took advantage of the two-site architecture of Ubr1p to explore the feasibility of bivalent N-end rule inhibitors, whose expected higher efficacy would result from higher affinity of the cooperative (bivalent) binding to Ubr1p. The inhibitor comprised mixed tetramers of beta-galactosidase that bore both N-terminal Arg (type 1 residue) and N-terminal Leu (type 2 residue) but that were resistant to proteolysis in vivo. Expression of these constructs in S. cerevisiae inhibited the N-end rule pathway much more strongly than the expression of otherwise identical beta-galactosidase tetramers whose N-terminal residues were exclusively Arg or exclusively Leu. In addition to demonstrating spatial proximity between the type 1 and type 2 substrate-binding sites of Ubr1p, these results provide a route to high affinity inhibitors of the N-end rule pathway.     

Subsite specificity of anthrax lethal factor and its implications for inhibitor development

The lethal factor of Bacillus anthracis is a major factor for lethality of anthrax infection by this bacterium. With the aid of the protective antigen, lethal factor gains excess to the cell cytosol where it manifests toxicity as a metalloprotease. For better understanding of its specificity, we have determined its residue preferences of 19 amino acids in six subsites (from P3 to P3′) as relative k_cat/K_m values (specificity constants). These results showed that lethal factor has a broad specificity with preference toward hydrophobic residues, but not charged or branched residues. The most preferred residues in these six subsites are, from P1 to P3′, Trp, Leu, Met, Tyr, Pro, and Leu. The result of residue preference was used to design new substrates with superior hydrolytic characteristics and inhibitors with high potency. For better use of the new findings for inhibitor design, we have modeled the most preferred residues in the active site of lethal factor. The observed interactions provide new insights to future inhibitor designs.     

Conserved N-Terminal Negative Charges Support Optimally Efficient N-type Inactivation of Kv1 Channels

N-type inactivation is produced by the binding of a potassium channel''s N-terminus within the open pore, blocking conductance. Previous studies have found that introduction of negative charges into N-terminal inactivation domains disrupts inactivation; however, the Aplysia AKv1 N-type inactivation domain contains two negatively charged residues, E2 and E9. Rather than being unusual, sequence analysis shows that this N-terminal motif is highly conserved among Kv1 sequences across many phyla. Conservation analysis shows some tolerance at position 9 for other charged residues, like D9 and K9, whereas position 2 is highly conserved as E2. To examine the functional importance of these residues, site directed mutagenesis was performed and effects on inactivation were recorded by two electrode voltage clamp in Xenopus oocytes. We find that inclusion of charged residues at positions 2 and 9 prevents interactions with non-polar sites along the inactivation pathway increasing the efficiency of pore block. In addition, E2 appears to have additional specific electrostatic interactions that stabilize the inactivated state likely explaining its high level of conservation. One possible explanation for E2''s unique importance, consistent with our data, is that E2 interacts electrostatically with a positive charge on the N-terminal amino group to stabilize the inactivation domain at the block site deep within the pore. Simple electrostatic modeling suggests that due to the non-polar environment in the pore in the blocked state, even a 1 Å larger separation between these charges, produced by the E2D substitution, would be sufficient to explain the 65× reduced affinity of the E2D N-terminus for the pore. Finally, our studies support a multi-step, multi-site N-type inactivation model where the N-terminus interacts deep within the pore in an extended like structure placing the most N-terminal residues 35% of the way across the electric field in the pore blocked state.     

Human inter-alpha-trypsin inhibitor: localization of the Kunitz-type domains in the N-terminal part of the molecule and their release by a trypsin-like proteinase

The N-terminal amino-acid sequence of human ITI has been found to be identical with that of the acid-stable human 30-kDa inhibitors (HI-30) from urine, serum, and those released from inter-alpha-trypsin inhibitor by trypsin or chymotrypsin. Serum HI-30 and HI-30 released by trypsin differ from the urinary inhibitor by an additional C-terminal arginine residue. Compared to these two inhibitors the inhibitor released by chymotryptic proteolysis is elongated C-terminally by an additional phenylalanine residue. These results strongly favour HI-30 as the N-terminus of the inter-alpha-trypsin inhibitor and its release from this inhibitor in vivo by cleavage of the Arg123-Phe124 peptide bond by trypsin-like proteinases.     

Different modes of dipeptidyl peptidase IV (CD26) inhibition by oligopeptides derived from the N-terminus of HIV-1 Tat indicate at least two inhibitor binding sites.

Dipeptidyl peptidase IV (DP IV, CD26) plays an essential role in the activation and proliferation of lymphocytes, which is shown by the immunosuppressive effects of synthetic DP IV inhibitors. Similarly, both human immunodeficiency virus-1 (HIV-1) Tat protein and the N-terminal peptide Tat(1-9) inhibit DP IV activity and T cell proliferation. Therefore, the N-terminal amino acid sequence of HIV-1 Tat is important for the inhibition of DP IV. Recently, we characterized the thromboxane A2 receptor peptide TXA2-R(1-9), bearing the N-terminal MWP sequence motif, as a potent DP IV inhibitor possibly playing a functional role during antigen presentation by inhibiting T cell-expressed DP IV [Wrenger, S., Faust, J., Mrestani-Klaus, C., Fengler, A., St?ckel-Maschek, A., Lorey, S., K?hne, T., Brandt, W., Neubert, K., Ansorge, S. & Reinhold, D. (2000) J. Biol. Chem.275, 22180-22186]. Here, we demonstrate that amino acid substitutions at different positions of Tat(1-9) can result in a change of the inhibition type. Certain Tat(1-9)-related peptides are found to be competitive, and others linear mixed-type or parabolic mixed-type inhibitors indicating different inhibitor binding sites on DP IV, at the active site and out of the active site. The parabolic mixed-type mechanism, attributed to both non-mutually exclusive inhibitor binding sites of the enzyme, is described in detail. From the kinetic investigations and molecular modeling experiments, possible interactions of the oligopeptides with specified amino acids of DP IV are suggested. These findings give new insights for the development of more potent and specific peptide-based DP IV inhibitors. Such inhibitors could be useful for the treatment of autoimmune and inflammatory diseases.     

Conserved salt bridge between the N- and C-terminal heptad repeat regions of the human immunodeficiency virus type 1 gp41 core structure is critical for virus entry and inhibition

The fusogenic human immunodeficiency virus type 1 (HIV-1) gp41 core structure is a stable six-helix bundle formed by its N- and C-terminal heptad repeat sequences. Notably, the negatively charged residue Asp⁶³² located at the pocket-binding motif in the C-terminal heptad repeat interacts with the positively charged residue Lys⁵⁷⁴ in the pocket formation region of the N-terminal heptad repeat to form a salt bridge. We previously demonstrated that the residue Lys⁵⁷⁴ plays an essential role in six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this study, the functionality of residue Asp⁶³² has been specifically characterized by mutational analysis and biophysical approaches. We show that Asp⁶³² substitutions with positively charged residues (D632K and D632R) or a hydrophobic residue (D632V) could completely abolish Env-mediated viral entry, while a protein with a conserved substitution (D632E) retained its activity. Similar to the Lys⁵⁷⁴ mutations, nonconserved substitutions of Asp⁶³² also severely impaired the α-helicity, stability, and conformation of six-helix bundles as shown by N36 and C34 peptides as a model system. Furthermore, nonconserved substitutions of Asp⁶³² significantly reduced the potency of C34 to sequestrate six-helix bundle formation and to inhibit HIV-1-mediated cell-cell fusion and infection, suggesting its importance for designing antiviral fusion inhibitors. Taken together, these data suggest that the salt bridge between the N- and C-terminal heptad repeat regions of the fusion-active HIV-1 gp41 core structure is critical for viral entry and inhibition.     

The caspase-like sites of proteasomes,their substrate specificity,new inhibitors and substrates,and allosteric interactions with the trypsin-like sites

Proteasomes are the primary sites for protein degradation in mammalian cells. Each proteasome particle contains two chymotrypsin-like, two trypsin-like, and two caspase-like proteolytic sites. Previous studies suggest a complex network of allosteric interactions between these catalytic and multiple regulatory sites. We used positional scanning combinatorial substrate libraries to determine the extended substrate specificity of the caspase-like sites. Based on this analysis, several new substrates were synthesized, the use of which confirmed earlier observations that caspase-like sites (often termed postglutamyl peptide hydrolase) cleave after aspartates better than after glutamates. Highly selective inhibitors of the caspase-like sites were also generated. They stimulated trypsin-like activity of yeast 20 S proteasomes up to 3-fold but not when binding of the inhibitor to the caspase-like sites was prevented in a mutant carrying an uncleaved propeptide. Although substrates of the caspase-like sites allosterically inhibit the chymotrypsin-like activity, inhibitors of the caspase-like sites do not affect the chymotrypsin-like sites. Furthermore, when caspase-like sites were occupied by the uncleaved propeptide or inhibitor, their substrates still inhibited the chymotrypsin-like activity. Thus, occupancy of the caspase-like sites stimulates the trypsin-like activity of proteasomes, but substrates of the caspase-like sites inhibit the chymotrypsin-like activity by binding to a distinct noncatalytic site.     

SSR182289A, a selective and potent orally active thrombin inhibitor

SSR182289A 1 is the result of a rational optimisation process leading to an orally active thrombin inhibitor. The structure incorporates an original 2-(acetylamino)-[1,1'-biphenyl]-3-sulfonyl N-terminal motif, a central l-Arg surrogate carrying a weakly basic 3-amino-pyridine, and an unusual 4-difluoropiperidine at the C-terminus. Its synthesis is convergent and palladium catalysis has been employed for the construction of the key C-C bonds: Suzuki coupling for the bis-aryl fragment and Sonogashira reaction for the delta- bond of the central amino-acid chain. The compound is a potent inhibitor of thrombin's activities in vitro and demonstrates potent oral anti-thrombotic potencies in three rat models of thrombosis. The observed in vitro potency could be rationalized through the examination of the interactions within the SSR182289A 1 - thrombin crystal structure. SSR182289A 1, has been therefore selected for further development.     

Elastase inhibition by the inter-alpha-trypsin inhibitor and derived inhibitors of man and cattle

The inhibitory properties of HI-14 and BI-14, the active 14-kDa parts released from the corresponding human and bovine inter-alpha-trypsin inhibitors, are compared. The structurally homologous inhibitors composed of two tandem Kunitz-type domains differ in their inhibitory specificity, although the reactive site residue in position P1 is occupied by identical (arginine in the C-terminal domain II) or similar (methionine and leucine in the N-terminal domain I of HI-14 and BI-14, respectively) amino-acid residues. The N-terminal domain I of HI-14 is completely inactive against chymotrypsin and pancreatic elastase, whereas BI-14 is a strong inhibitor of these enzymes. Elastase from polymorphonuclear granulocytes interacts with both inhibitors but with different affinities. Compared with the bovine inhibitor, the human inhibitor shows a much lower affinity from this enzyme. Human ITI and its physiological 30-kDa derivative (HI-30) show the same inhibitory properties as HI-14. The differences between human and bovine inhibitors might be explained by a preceding oxidation of Met in vivo of the reactive site residue in position P1 and/or by the influence of the environmental parts connected with this antielastase reactive site region in human ITI or in the active domains thereof.     

Human mast cell beta-tryptase is a gelatinase

Remodeling of extracellular matrix is an important component in a variety of inflammatory disorders as well as in normal physiological processes such as wound healing and angiogenesis. Previous investigations have identified the various matrix metalloproteases, e.g., gelatinases A and B, as key players in the degradation of extracellular matrix under such conditions. Here we show that an additional enzyme, human mast cell beta-tryptase, has potent gelatin-degrading properties, indicating a potential contribution of this protease to matrix degradation. Human beta-tryptase was shown to degrade gelatin both in solution and during gelatin zymographic analysis. Further, beta-tryptase was shown to degrade partially denatured collagen type I. beta-Tryptase bound strongly to gelatin, forming high molecular weight complexes that were stable during SDS-PAGE. Mast cells store large amounts of preformed, active tryptase in their secretory granules. Considering the location of mast cells in connective tissues and the recently recognized role of mast cells in disorders in which connective tissue degradation is a key event, e.g., rheumatoid arthritis, it is thus likely that tryptase may contribute to extracellular matrix-degrading processes in vivo.