Kallikrein inhibitors.

So far the Cl inactivator, alpha 2-macroglobulin, antithrombin III (in the presence of heparin), and alpha 1-antitrypsin have been identified as inhibitors of plasma kallikrein; alpha 1-antitrypsin reacts slowly also with tissue kallikreins. Of the various naturally occurring kallikrein inhibitors the basic trypsin-kallikrein inhibitor of bovine organs, aprotinin (the active substance of Trasylol), has attained by far the most interest. This inhibitor, which is produced by mast cells, has unusual properties due to its compact tertiary structure. Additional topics of aprotinin and structurally related inhibitors discussed are the mechanism of enzyme-inhibitor complex formation, the production of chemical mutants of aprotinin, the structural basis of kallikrein inhibition, and selected aspects regarding aprotinin medication.     

Molecular mechanisms of band 3 inhibitors. 3. Translocation inhibitors

During the translocation of the band 3 transport site between the inward- and outward-facing orientations, the Cl- transport site complex passes through a transition state lying on the reaction pathway between the two extreme orientations. Niflumic acid, 2-[(7-nitrobenzofurazan-4-yl)amino]ethanesulfonate, and 2,4,6-trichlorobenzenesulfonate each are translocation blockers that can bind to both the inward- and outward-facing conformations of band 3. The principal mechanism of these inhibitors is a reduction in the translocation rate, since they have essentially no effect on the apparent KD for Cl- binding to the transport site and the migration of Cl- between the transport site and solution. Instead, these inhibitors raise the free energy of formation of the transition state during translocation and thereby can lock the transport site into either the inward- or outward-facing orientation. In contrast, 2,4-dinitrofluorobenzene (DNFB) appears to restrict the accessibility of the transport site to solution Cl-; also, the DNFB reaction rate is increased by Cl-, suggesting that DNFB modification may occur during translocation. Thus DNFB is proposed to trap the Cl--transport site complex site during translocation to yield a conformation intermediate to the inward- and outward-facing orientations. A model is presented for the molecular mechanism of transport across biological membranes. The transport machinery is proposed to contain greater than or equal to 6 transmembrane helices that surround a central channel containing a sliding hydrophobic barrier. The transport site lies between two of the channel-forming helices and remains stationary while the hydrophobic barrier slides from one end of the channel to the other, thereby exposing the transport site to the opposite solution compartment.     

Molecular mechanisms of band 3 inhibitors. 1. Transport site inhibitors

The band 3 protein of red cells is a transmembrane ion transport protein that catalyzes the one-for-one exchange of anions across the cell membrane. 35Cl NMR studies of Cl- binding to the transport sites of band 3 show that inhibitors of anion transport can be grouped into three classes: (1) transport site inhibitors (examined in this paper), (2) channel-blocking inhibitors (examined in the second of three papers in this issue), and (3) translocation inhibitors (examined in the third of three papers in this issue). Transport site inhibitors fully or partially reduce the affinity of Cl- for the transport site. The dianion 4,4'-di-nitrostilbene-2,2'-disulfonate (DNDS) and the arginine-specific reagent phenylglyoxal (PG) each completely eliminate the transport site 35Cl NMR line broadening, and each compete with Cl- for binding. These results indicate that DNDS and PG share a common inhibitory mechanism involving occupation of the transport site: one of the DNDS negative charges occupies the site, while PG covalently modifies one or more essential positive charges in the site. In contrast, 35Cl NMR line broadening experiments suggest that 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) leaves the transport site partially intact so that the affinity of Cl- for the site is reduced but not destroyed. This result is consistent with a picture in which DIDS binds near the transport site and partially occupies the site.     

Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.

The biosynthesis of the various types of N-linked oligosaccharide structures involves two series of reactions: 1) the formation of the lipid-linked saccharide precursor, Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol, by the stepwise addition of GlcNAc, mannose and glucose to dolichyl-P, and 2) the removal of glucose and mannose by membrane-bound glycosidases and the addition of GlcNAc, galactose, sialic acid, and fucose by Golgi-localized glycosyltransferases to produce different complex oligosaccharide structures. For most glycoproteins, the precise role of the carbohydrate is still not known, but specific N-linked oligosaccharide structures are key players in targeting of lysosomal hydrolases to the lysosomes, in the clearance of asialoglycoproteins from the serum, and in some cases of cell:cell adhesion. Furthermore, many glycoproteins have more than one N-linked oligosaccharide, and these oligosaccharides on the same protein frequently have different structures. Thus, one oligosaccharide may be of the high-mannose type whereas another may be a complex chain. One approach to determining the role of specific structures in glycoprotein function is to use inhibitors that block the modification reactions at different steps, causing the cell to produce glycoproteins with altered carbohydrate structures. The function of these glycoproteins can then be assessed. A number of alkaloid-like compounds have been identified that are specific inhibitors of the glucosidases and mannosidases involved in glycoprotein processing. These compounds cause the formation of glycoproteins with glucose-containing high mannose structures, or various high-mannose or hybrid chains, depending on the site of inhibition. These inhibitors have also been useful for studying the processing pathway and for comparing processing enzymes from different organisms.     

Natural and synthetic inhibitors of thrombin. II. Synthetic low-molecular-weight inhibitors]

The up-to-date problems, concerning the structure and properties of two types of inhibitors are reviewed. It is particularly considered properties of low-molecular weight thrombin inhibitors that have electrophilic groups capable to react with Ser-195 of thrombin (peptidyl-chloromethyl ketones, aldehydes, ketomethylene derivatives and derivatives of boric and phosphoric acids) and the competitive reversible thrombin inhibitors. The review focuses on methods of modification of the structure in the natural inhibitors and design of new peptidomimetics. The prospects for prophylaxis and treatment of diverse thromboembolic diseases are discussed.     

Natural plant enzyme inhibitors. VI. Studies on trypsin inhibitors of Colocasia antiquorum tubers.

A trypsin inhibitor was purified from the tubers of Colocasia antiquorum. The inhibitor acted on bovine trypsin, human trypsin and weakly on bovine chymotrypsin. The inhibitor, which had a molecular weight of 40 000, contained trace amounts of carbohydrates. The purified inhibitor was stable over a pH range of 2.0--12.0 and was more thermostable than the crude preparations. Trinitrobenzene sulphonate treatment resulted in the inactivation of the inhibitor. Chymotrypsin, pepsin and pronase digested the inhibitor. Pretreatment with trypsin at neutral pH resulted in the partial loss of antitryptic activity, whereas treatment at pH 3.7 led to complete inactivation. Evidence for the formation of a trypsin-inhibitor complex at pH 7.6 is provided. During the plant growth, in the early phase (0--40 days) there was a gradual increase in protein content and in antitryptic activity. The middle phase (40--55 days) was characterized by a rapid fall and abolition of the antitryptic activity and a diminution in protein content in the tubers. The immature tubers had low antitryptic activity compared to the mature ones. Mild heat treatment caused a sharp rise in antitryptic activity in the extracts of immature tubers but not with the mature tuber preparations.     

Reversible inhibitors of penicillinases.

Reversible competitive inhibitors of a penicillinase, beta-lactamase 1 from Bacillus cereus, were studied. These represent the first inhibitors of a penicillinase that lack the beta-lactam ring. The products of the enzymic reaction, namely penicilloic acids, are inhibitors; their decarboxylation products, the penilloic acids, are also inhibitors, and have somewhat lower Ki values. Inhibitors have been prepared from benzylpenicillin, phenoxymethyl-penicillin, methicillin (2,6-dimethoxybenzamidopenicillanic acid) and 3-hydroxy-4-nitrobenzamidopenicillanic acid. Decarboxylation of the penicilloic acids from benzyl-penicillin, or from phenoxymethylpenicillin, leads to epimerization (at C-5) of the penilloic acid. Nuclear-magnetic resonance spectroscopy at a frequency of 270 MHz can distinguish the epimers. Other competitive inhibitors studied were boric acid, benzene boronic acid and m-aminobenzeneboronic acid. Boric acid itself was the best inhibitor of beta-lactamase I so far found.