Herbert R. Wenzel

Semisynthetic engineering of proteinase inhibitor homologues.

A semisynthetic approach to modulate the inhibitory specificity of aprotinin, the Kunitz trypsin inhibitor from bovine mast cells, is described. By the use of peptide-chemical procedures a single amino acid of its reactive site can be replaced by any other coded or non-coded amino acid. Thus, a series of aprotinin homologues have been prepared which demonstrate the individual contribution of a single side chain to the inhibition of a particular target proteinase and enable specific inhibitors to be designed.

Hydroxamate derivatives of substrate‐analogous peptides containing aminomalonic acid are potent inhibitors of matrix metalloproteinases

Novel peptides containing the sequence ‐Pro‐Leu‐Ama(NHOH)‐ were synthesized and characterized by spectroscopic techniques. Their inhibitory properties towards the activated form of native human gelatinase B (MMP‐9) and the catalytic domain of neutrophil collagenase (cdMMP‐8) were determined. The most effective inhibitor synthesized exhibits K i values of 2×10−6 M (cdMMP‐8) and 5×10−9 M (MMP‐9) thus attaining interesting discrimination between the tested metalloproteinases. A most important feature of this type of inhibitor is its peptide nature making the compounds similar to natural substrates. In spite of the peptide character of the inhibitors synthesized, the P1‐P1′‐peptide bond shows a high resistance to cleavage by the proteinases.

Strong crossreaction of human anti-aprotinin antibodies from heart transplant patient with [Arg15] aprotinin ☆

We detected anti-aprotinin antibodies by an enzyme immunoassay in serum of a 33 year old man who showed anaphylactic reactions during heart transplantation under aprotinin reexposition. The antibodies were isolated by affinity chromatography by aprotinin immobilized on CNBr activated Sepharose. The crossreactivity was tested by a competitive enzyme immunoassay (50% inhibition) against different aprotinin homologues and two human Kunitz-type protease inhibitors, bikunin and TFPI. In comparison with native aprotinin (immunoreactivity = 100%) the crossreaction of the homologue [Arg15]aprotinin was 76%, of [Val15]aprotinin 15% and of isoaprotinin 1, [Ala14,38]aprotinin and [seco15/16]aprotinin less than 10%. An immunoreactivity with bikunin and TFPI was not detected. Similar results were obtained with polyclonal anti-aprotinin antibodies from rabbit. Our results show that human anti-aprotinin antibodies are mainly directed against the reactive site of aprotinin. From this we conclude that the reactive site exposes the major epitope resulting in a major target site for antibodies in a species independent way, and therefore, it is obvious that the recombinant aprotinin homologue [Arg15]aprotinin, which is scheduled for therapy in open-heart surgery, will have similar immunogenic effects as native aprotinin.

Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P1 lysine15 residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine15-alanine16 peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine15 methylester group was then selectively hydrolyzed. Afterward, lysine15 itself was split off. Arginine, glutamic acid, methionine, andl-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.

THE PH-DEPENDENCE OF THE EQUILIBRIUM-CONSTANT KHYD FOR THE HYDROLYSIS OF THE LYS15-ALA16 REACTIVE-SITE PEPTIDE-BOND IN BOVINE PANCREATIC TRYPSIN-INHIBITOR (APROTININ)

ThepH dependence of the equilibrium constant KHyd for the hydrolysis of the Lys15-Ala16 reactive-site peptide bond of the bovine pancreatic trypsin inhibitor (aprotinin) was investigated over thepH range 2.3–6.5. Solutions of aprotinin, modified aprotinin with the Lys15-Ala16 peptide bond cleaved and mixtures of both species were incubated with 10 mol% porcine β-trypsin. The state of equilibrium was determined by analytical cation-exchange HPLC. The KHyd values obtained did not exactly obey the simple equation of Dobry et al. (1952), which had to be used in an extended form with two additional parameters for a satisfactory fit. ThepH-independent equilibrium constant is 0.90 and thepK values of the Lys15 carboxyl group and of the Ala16 amino group are 3.10 and 8.22, respectively. ThepK of an additional group is apparently perturbed by the peptide-bond hydrolysis. It is 4.60 in the native and 4.40 in the modified aprotinin.

Enzymatic semisynthesis of aprotinin homologues mutated in P' positions.

The replacement of amino acids in the P′1 and P′2 position of aprotinin, the bovine pancreatic trypsin inhibitor, is described. Using the “modified” inhibitor as starting material, with the hydrolyzed reactive-site peptide bond Lys15-Ala16, the residues P′1 (Ala16) and P′2 (Arg17) were split off by the action of aminopeptidase K. Incorporation of suitable dipeptides containing a basic residue (Lys or Arg) in the C-terminal position was carried out in a “one pot” reaction involving trypsin-catalyzed coupling. In this way, the native fragment Ala16-Arg17 was reintroduced and also replaced by Gly-Arg, Ala-Lys, and Leu-Arg yielding intact inhibitor molecules. The mechanism for incorporation of dipeptides was investigated by treating the aprotinin derivative with the Arg17-Ile18 peptide bond hydrolyzed with trypsin under proteosynthetic conditions. We established that only inhibitor molecules cleaved between Lys15 and Xaa16 are intermediates leading to the desired products. The inhibitory properties of the new aprotinin homologues were tested, and the significance of the P′1 residue for the inhibition of trypsin, kallikrein, and chymotrypsin was deduced.

8 – Reversible Inhibitors of Serine Proteinases: Naturally Occurring Miniproteins, Semisynthetic Variants, Recombinant Homologs, and Synthetic Peptides

Proteinases play an important role in the normal functioning of biological systems. They are involved in crucial processes such as food digestion, blood coagulation and fibrinolysis, blood pressure regulation, and fertilization. Certain proteinases release peptide hormones and neuromodulators from inactive precursors or degrade message-transmitting peptides, thus initiating or terminating a variety of biological responses. It is clear that besides a pronounced cleavage specificity, a tight regulation of proteolytic activity by endogenous proteinase inhibitors is necessary. The number of proteinaceous inhibitors that have been isolated and characterized so far is large and growing steadily. The majority are directed toward members of the serine proteinases, one of the four classes of endopeptidases. Serine proteinase inhibitors can be grouped into at least sixteen different inhibitor families on the basis of sequence similarity, topological relationships between the disulfide bridges, and the location of the binding site for the cognate proteinase. Detailed knowledge of the structure and reactivity of the inhibitors is indispensable for a thorough understanding of the controlling functions they exercise in a variety of fundamental physiological proteolytic processes. This chapter discusses these inhibitors and some approaches that might lead to satisfactory therapeutic compounds.

New nitroxide reagents for the selective spin-labelling at the guanidino moiety of arginine residues in peptides and proteins

Abstract The synthesis of three glyoxal derivatives carrying a nitroxide moiety was achieved by the α-oxidation of the corresponding methyl ketones using selenium dioxide. These reagents appear to be useful for the specific spin-labelling at arginine side-chains. Sixteen peptides and proteins were successfully labelled and the rotational correlation times, as deduced from ESR spectra, were correlated with the molecular weights of the target macromolecules.

Complex formation of guanidinated bovine trypsin inhibitor (Kunitz) with trypsin, chymotrypsin and trypsinogen as studied by the spin-label technique

1. Introduction Studying the interaction between protein protein- ase inhibitors and their target enzymes by physico- chemical methods has often been hampered by the lack of suitable intrinsic spectroscopic signals, which change upon complex formation. specifically spin-labelled at the amino-terminus and its complexes with trypsin, chymotrypsin and trypsin- ogen were chosen as a first test system. 2. Experimental 2.1.

3D Structure and Drug Design

Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation. Their proteolytic activity must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Experimental evidence confirms that MMPs play a decisive role in a wide variety of pathologic conditions that involve connective tissue destruction such as arthritis, tumor growth and metastasis. Modulation of MMP regulation is possible at several biochemical sites, but direct inhibition of enzyme action provides a particularly attractive target for therapeutic intervention. To date the main focus of the therapeutic applications of MMP inhibitors (MMPIs) has been in the areas of cancer and arthritis. One key issue in their clinical development relates to whether broad spectrum inhibitors, active against a range of different enzymes, or selective inhibitors targeted against a particular subset of the MMPs, represents the optimal strategy. Some orally active compounds for the treatment of cancer and/or arthritis are currently under clinical investigations. Representative examples include succinamides (Marimastat), linear sulfonamides (CGS-27023A), heterocyclic sulfonamides (Prinomastat) and biphenylbutyric acid derivatives (BAY-129566). However, since their inception during the eighties, MMPIs have gone through several cycles of metamorphosis. From this, two important approaches to the design, synthesis, and biological evaluation of MMPIs are highlighted: 1. the invention of alternatives to hydroxamic acid zinc chelators and 2. the construction of nonpeptide scaffolds. One current example from our own work in each of these two approaches is described.