Discovery of benzothiazole guanidines as novel inhibitors of thrombin and trypsin IV

In a project to find novel neutral P1 fragments for the synthesis of thrombin inhibitors with improved pharmacokinetic properties, fragments containing a benzothiazole guanidine scaffold were identified as weak thrombin inhibitors. WaterLOGSY (Water-Ligand Observed via Gradient SpectroscopY) NMR was used to detect fragments binding to thrombin and these fragments were followed up by Biacore A100 affinity measurements and enzyme assays. A crystal structure of the most potent compound with thrombin was obtained and revealed an unexpected binding mode as well as the key interactions of the fragment with the protein. Based on these results, the structure-based design and synthesis of a small series of optimized novel substituted benzothiazole guanidines with comparatively low pK(a) values was accomplished. Testing of these compounds against human trypsin I and human trypsin IV revealed unexpected inhibitory activity and selectivity of some of the compounds, making them attractive starting points for selective trypsin inhibitors.     

Double-headed protease inhibitors from black-eyed peas. IV. Half-site reactivity in the formation of complexes with trypsin and chymotrypsin.

Complex formation between two new double-headed protease inhibitors from black-eyed peas, trypsin-chymotrypsin inhibitor (BEPCI) and a trypsin inhibitor (BEPTI), and trypsin and chymotrypsin was investigated in the concentration range from 10-8 to 10-4 M by titration experiments and gel filtration chromatography. Dissociation equilibrium constants measured for complexes detected in the titration experiments range from as large as 10-8 M for trypsin bound nonspecifically to the chymotrypsin site of BEPCI to as small as 10-18 M2 for the interaction of BEPCI with chymotrypsin. The identity and stoichiometry of complexes detected during titration experiments were confirmed by gel filtration of mixtures of native and fluorescently labeled proteases and inhibitors. Half-site reactivity is observed in the formation of complexes between BEPCI or BEPTI and trypsin and chymotrypsin at all experimentally practical concentrations. The double-headed complex contains 1 molecule each of trypsin, chymotrypsin, and BEPCI dimer. The bimolecular rate constants of complex formation between trypsin or chymotrypsin and isolated BEPCI oligomers range from 1.8 X 10(5) M-1 S-1 for chymotrypsin and BEPCI monomer to 4.4 X 10(7) M-1 S-1 for trypsin and the rapidly equilibrating BEPCI dimer. The estimated rate constants for the dissociation of half-site-liganded dimer complexes and liganded monomer complexes range from 7.5 X 10-3 S-1 for the trypsin-liganded BEPCI monomer complex to 1.6 X 10-6 S-1 for the chymotrypsin-liganded BEPCI dimer complex.     

Serpin protease inhibitors in plant biology

Protease inhibitors of the serpin family are ubiquitous in the plant kingdom but relatively little is known about their biological functions in comparison with their counterparts in animals. X-ray crystal structures have provided crucial insights into animal serpin functions. The recently solved structure of AtSerpin1 from Arabidopsis thaliana, which has the highly conserved reactive center P2-P1' Leu-Arg-Xaa (Xaa = small residue), displays both conserved and plant-specific serpin features. Sequence homology suggests that AtSerpin1 belongs to serpin Clade B, composed of intracellular mammalian serpins, which is consistent with the lack of strong evidence for secretion of serpins from plant cells. The major in vivo target protease for AtSerpin1 is the papain-like cysteine RD21 protease, a match reminiscent of the inhibition of cathepsins K, L and S by the Clade-B mammalian serpin, SCCA-1 (SERPINB3). The function of AtSerpin1 and other serpins that contain P2-P1' Leu-Arg-Xaa (the 'LR' serpins) in plants remains unknown. However, based on its homology and interactive partners, AtSerpin1 and perhaps other serpins are likely to be involved in regulating programmed cell death or associated processes such as senescence. Abundant accumulation of serpins in seeds and their presence in phloem sap suggest additional functions in plant defense by irreversible inhibition of digestive proteases from pests or pathogens. Here we review the most recent findings in plant serpin biology, focusing on advances in describing the structure and inhibitory specificity of the LR serpins.     

Three high molecular weight protease inhibitors of rat plasma. Reactions with trypsin

We have compared the reactions of trypsin with human alpha 2-macroglobulin (alpha 2M), and three rat plasma protease inhibitors, alpha 1-macroglobulin (alpha 1M), alpha 1-inhibitor III (alpha 1I3), and alpha 2M. All four of these proteins appear to contain reactive thiol esters. The electrophoretic mobility in agarose gels of human and rat alpha 2M is increased by 1 mol of trypsin, while the mobility of alpha 1M and alpha 1I3 is decreased. Treatment with methylamine causes similar mobility changes, except in the case of rat alpha 2M. Titration of human and rat macroglobulins by repeated small additions of trypsin and by assay of liberated SH groups or enhanced ligand fluorescence revealed a stoichiometry of about 1 mol of trypsin/mol of inhibitor. In contrast, addition of macroglobulin to a fixed amount of trypsin and detection of residual amidase or protease activity revealed a stoichiometry of about 2 mol of trypsin for 1 mol of human alpha 2M, about 1.4 mol for rat alpha 1M, and about 1 mol for rat alpha 2M. One mol of trypsin reacted with 2 or more mol of alpha 1I3 by the criteria of SH groups liberated or protease inhibition. Methylamine-treated rat alpha 2M binds a significant amount of trypsin releasing about 2 mol of SH. Radioactive beta-trypsin was covalently bound to subunits of the purified plasma inhibitors. The Mr of the labeled products with rat and human alpha 2M had molecular weights which suggested trypsin was bound to intact as well as cleaved subunit chains and also to multiple chains via cross-linking. Rat alpha 1M also produced a product which may be an intact subunit alpha chain plus trypsin. Greater than 80% of the trypsin was bound covalently to these inhibitors at low molar ratios.     

The adaptation of insects to plant protease inhibitors

Plants and herbivores have been co-evolving for thousands of years, and as a result, plants have defence mechanisms that offer protection against many herbivores such as nematodes, insects, birds and mammals. Only when a herbivore has managed to adapt to these defence mechanisms does it have the potential to become a pest. One such method of plant defence involves the production of protease inhibitors (PIs). These inhibitors are proteins that may be found constitutively in various parts of the plant, or may be induced in response to herbivore attack. PIs work at the gut level, by inhibiting the digestion of plant protein. This review focuses on insect herbivores and looks at the mechanisms involved in the role and function of PIs in plant defense against insects, as well as at the ability of well adapted species to overcome the effects of these plant PIs.     

Transition metals as protease inhibitors

An alternative approach to the development of clinically useful protease inhibitors was investigated. The approach utilized coordination chemistry of transition metal ions rather than substrate analogs to block active sites of these enzymes. In the case of serine proteases it was found that aqueous Ti(IV) is a potent inhibitor of the trypsin subclass, but not the chymotrypsin subclass. The direct binding of Ti(IV) to trypsin was made possible by the presence of a free carboxyl group at the bottom of the substrate binding pocket of the enzyme, and the five-coordinate geometry of TiO(SO₄)(H₂O). Although initial binding of Ti(IV) was reversible, it was followed in time by irreversible inhibition. Direct binding of octahedral or tetrahedral metal ion complexes was prevented by the inability of the enzyme active sites to promote formation of a five-coordinate transition state of the metal ion required for reaction. These studies demonstrate the ability of direct metal ion binding as a way to enhance blocking of enzyme active sites as compared with that of traditional organic inhibitors. Application of these findings was investigated by measuring the affect Ti(IV) had on growth ofEscherichia coli, Salmonella typhimurium, andPseudotnonas aeruginosa. Five-coordinate titanyl sulfate completely inhibited the growth of these organisms. This suggests that five-coordinate titanyl sulfate, which is easier and less expensive to manufacture than conventional antibiotics, may be useful in controlling endemic infections ofE. coli andS. typhimurium.     

Lipopeptides as dimerization inhibitors of HIV-1 protease.

In AIDS therapy, attempts have been made to inhibit the virus-encoded enzymes, e.g. HIV-1 protease, using active site-directed inhibitors. This approach is questionable, however, due to virus mutations and the high toxicity of the drugs. An alternative method to inhibit the dimeric HIV protease is the targeting of the interface region of the protease subunits in order to prevent subunit dimerization and enzyme activity. This approach should be less prone to inactivation by mutation. A list of improved 'dimerization inhibitors' of HIV-1 protease is presented. The main structural features are a short 'interface' peptide segment, including non-natural amino acids, and an aliphatic N-terminal blocking group. The high inhibitory power of some of the lipopeptides [e.g. palmitoyl-Tyr-Glu-Leu-OH, palmitoyl-Tyr-Glu-(L-thyronine)-OH, palmitoyl-Tyr-Glu-(L-biphenyl-alanine)-OH] with low nanomolar Ki values in the enzyme test suggests that mimetics with good bio-availability can be derived for AIDS therapy.     

Tellurium-based cysteine protease inhibitors: evaluation of novel organotellurium(IV) compounds as inhibitors of human cathepsin B

New organotellurium(IV) compounds with specific cysteine protease inhibitory activity were synthesized. Serine and aspartic protease activity were not affected by any of these compounds. All Te(IV) compounds tested exhibited high specific second-order constant for cathepsin B inactivation. Tellurium(IV) compound 6 was the best inhibitor of the series, showing a second-order constant of 36,000 M(-1)s(-1). This value is about 100-fold higher than the second-order rate for cysteine protease inactivation shown by the historic Te(IV) compound AS 101 (1). The inhibition was irreversible and time and concentration dependent; no saturation kinetics were observed, suggesting a direct bimolecular reaction. The results described in this paper show that the new organotellurium(IV) compounds are powerful inhibitors of cathepsin B, constituting promising potential anti-metastatic agents.     

Modified nucleotide polymers as inhibitors of DNA polymerases.

We have compared the relative inhibitory activity of poly (A) with its analogues poly N6-isopentenyl adenylic acid (poly(i6 A)) and poly N6-benzyl adenylic acid (poly(bzl6A)), and of poly (U) with its analogue poly 2'-fluoro-2'-deoxyuridylic acid (poly(dUfl)), against DNA polymerase, alpha, beta and gamma and terminal deoxynucleotidyl transferase from human cells and two oncorna virus DNA polymerases. Although poly (A) and its analogues were equally inhibitory against endogenous RNA-directed DNA polymerases of murine and feline leukemia viruses, the analogues in contrast to poly (A) were strongly inhibitory against all four cellular enzymes. Poly (dUfl), on the other hand, was up to 100-fold more potent than poly (U) against both viral and cellular enzymes. Since poly (U) at 100 mug/ml and poly (dUfl) at 1 mug/ml had no effect on terminal deoxynucleotidyl transferase while inhibiting other enzymes by 80--100 per cent these polymers could be useful in the characterization and assay of terminal deoxynucleotidyl transferase. In addition, the polymers such as poly (igA) and poly (bzl5A) which were strongly inhibitory to all cellular enzymes, could be useful in cancer chemotherapy if taken up preferentially by the malignant calls due to their high pinocytic activity. The results also demonstrate potential for large variation in inhibitory activity of polyribonucleotides as related to their chemical composition.