Peptidase inactivation

Biochemical characterization of a synthetic NPFF agonist with high affinity and resistance to brain peptidase inactivation... 

Several drugs in current medical use are mechanism-based enzyme inactivators. Eor example, the antibiotic penicillin exerts its effects by covalently reacting with an essential serine residue in the active site of glycoprotein peptidase, an enzyme that acts to cross-link the peptidoglycan chains during synthesis of bacterial cell walls (Eigure 14.17). Once cell wall synthesis is blocked, the bacterial cells are very susceptible to rupture by osmotic lysis, and bacterial growth is halted. 

Importantly, both incretins when secreted by the intestine are rapidly degraded by the dipeptidyl peptidase IV (DPPFV), which removed the two amino-terminus histidine-alanine residues, thereby, inactivating the incretins. This enzyme is present at the surface of the epithelial intestinal cells and capillaries in the vicinity of the K and L cells secreting GIP and GLP-1, respectively. It is also present in the... 

Proteases (proteinases, peptidases, or proteolytic enzymes) are enzymes that break peptide bonds between amino acids of proteins. The process is called peptide cleavage, a common mechanism of activation or inactivation of enzymes. They use a molecule of water for this, and are thus classified as hydrolases. 

Mentiein, R. (1999) Dipeptidyl-peptidase IV (CD26)-role in the inactivation of regulatory peptides. Regulatory Peptides, 85, 9-24. 

Fig. 2.3. Schematic representation of consecutive zymogen activation reactions (cascade). Following initiation by a physiological signal ( ), the zymogens X, Y, and Z are sequentially activated. The resulting peptidases (Xa and Ya) are inactivated by specific inhibitors (Ix and Iy) to limit their action (modified from [20a]).

Some drugs possessing amide bonds, such as prilocaine, and of course, peptides, can be hydrolyzed by peptidases and inactivated in this manner. Peptidases are also of pharmacological interest because they are responsible for the formation of highly reactive cleavage products (fibrin, p. 146) and potent mediators (angiotensin 11, p. 124 bradykinin, enkephalin, p. 210) from biologically inactive peptides. 

ACE is a rather nonspecific peptidase that can cleave C-terminal dipeptides from various peptides (dipeptidyl carboxypeptidase). As kininase 11, it contributes to the inactivation of kinins, such as bradykinin. ACE is also present in blood plasma however, enzyme localized in the luminal side of vascular endothelium is primarily responsible for the formation of angiotensin 11. The lung is rich in ACE, but kidneys, heart, and other organs also contain the enzyme. 

This enzyme [EC 3.4.17.3] (also referred to as lysine carboxypeptidase, arginine carboxypeptidase, kininase I, or anaphylatoxin inactivator) is a zinc-dependent member of peptidase family M14. The enzyme hydrolyzes the peptide bond at the C-terminus provided that the C-terminal amino acid is either arginine or lysine. The enzyme inactivates bradykinin and anaphylatoxins in blood plasma. 

This enzyme [EC 3.4.22.8], also referred to as clostridio-peptidase B, is an endopeptidase belonging to the peptidase family Cll that has been isolated from the bacterium Clostridium histolyticum. It catalyzes the hydrolysis of peptide bonds with a preference for the Arg—Xaa bond (including the Arg—Pro bond), but not the Lys— Xaa peptide bond. This calcium ion-activated enzyme is inactivated by EDTA. 

The hormone-hke peptide incretin stimulates the release of insuhn by a feedback process that involves cleaving the molecule to an inactive form. The protease enzyme dipeptidal peptidase (DPP) in turn cleaves incretin, in effect inactivating this enzyme. Inhibition of DPP consequently extends the action of incretin. This inhibition thus prevents the increased levels of blood glucose that mark diabetes. The protease inhibitor vidagliptin, which is modeled in part on the terminal sequence in DPP, has been found to sustain levels of insulin in Type II diabetics. The inhibition is apparently reversible in spite of the presence in the structure of the relatively reactive a-aminonitrile function. Construction of one intermediate in the convergent synthesis comprises the reaction of amino adamantamine (21-1) with a mixture of nitric and... 

Peptidase activity is prevented in the blanks by inactivating the enzyme prior to adding substrate. 

Pyrrolidone carboxylyl peptidase from B. subtilis 135) exhibits properties similar to the enzyme obtained from Pseudomonas. It is of interest that the enzyme is inactivated by shaking with air at room temperature for 5 min and that reactivation is readily achieved by brief incubation with 2-mercaptoethanol and EDTA. 

A recent report describes the presence of pyrrolidone carboxylyl peptidase in rat liver (46). A partial purification of the enzyme has been obtained by ammonium sulfate fractionation and gel filtration on Sepha-dex G-200. The rat liver enzyme is similar to that obtained from Pseudomonas thus, it is protected against inactivation by 2-pyrrolidone and exhibits similar substrate specificity. 

Ribonuclease Ti is fairly resistant to proteases. The threonine residue at the carboxyl terminal of the enzyme can be removed by carboxy-peptidase A without loss of activity (67). Leucine aminopeptidase does not release amino acids from the amino terminal (68). Ribonuclease Ti is not inactivated by trypsin or chymotrypsin in the presence of 0.2 M phosphate (69), which probably binds the enzyme and protects it from inactivation (67). Treatment of the enzyme with trypsin in the absence of phosphate inactivates it (67). Ribonuclease Tj is hydrolyzed by pepsin with progressive loss of activity (69). 

Various peptide Michael acceptors have been described as a new class of inactivators for cysteine proteases. 5-7 The carbonyl group of the scissile peptide bond in the substrate is replaced by a nucleophile trapping moiety such as a vinylogous structure. An amino acid vinyl sulfone, l-(methylsulfonyl)-4-phenylbut-l-en-3-amine [H2NCH(Bzl)CH=CHS02Me] and a dipeptide derivative, Gly-HNCH(Bzl)CH=CHS02Me have both been prepared as inhibitors of cysteine proteases, leucine aminopeptidase and dipeptidyl peptidase I, respectively.1 5 A series of peptide vinyl sulfones has been synthesized as potent inhibitors for different cysteine proteases. 1A8 ... 

The cellular effects of FTase inhibition with 3 were observed with concentrations 5000-50,000 higher than the in vitro IC50 for FTase inhibition by carboxylic acid Id. Incomplete hydrolysis of the lactone in vivo could be partially responsible for this discrepancy in activity. However, it was also found that the lactone prodrug used in the context of the doubly reduced peptide isostere, i.e. 3, was chemically unstable at physiological pH. Rapid cyclization to the diketopiperazine 5 significantly reduced FTase inhibitory activity.40 Simple N-alkylation of the reactive secondary amine to give 4 led to loss of activity vs. FTase. To simultaneously protect the compound from both metabolic inactivation (via peptidases) and chemical instability, isosteric replacements of the second amide bond other than methylene-amino were explored. Since the second amide bond in the tetrapeptide inhibitors could be reduced without loss of activity in vitro, peptide bond replacements which were both rigid (olefin) and flexible (alkyl, ether) were synthesized. 

Inactivation of DD-peptidases thus depends on a rapid and nearly quantitative accumulation of E-A intermediate resulting, jointly, from its stability (k2 is very low, of the order of 10 4-10 6s ), and its rapid formation. The factor governing the rate of acyl-enzyme formation is k2IK, where A- = (A j + k2)lki (value of the order of 103-106M-1 s 1). 

---