Elastase natural substrates

Further changes in the P1-P5 region, which resulted in improved efficacy for these synthetic substrates, were based on elastin, HLE s natural substrate [36]. This excellent substrate is an insoluble, structural protein, which is primarily composed of hydrophobic amino acid residues. However, it also contains a number of Lys-derived, cross-linked residues, such as desmosine and isodesmosine, that incorporate a positively charged pyridinium ring. In order to model this cross-linking feature, Lys or various amino-protected forms of Lys, were systematically incorporated into the substrate MeO-8uc-Ala-Ala-Pro-Val-NA (4-1). Replacement of any single residue with Lys led to decreased activity, for example, (4-2)-(4-4) Table 2.4). However, the use of side-chain protected Lys derivatives (e.g. the NHj terminus protected with benzyloxycarbonyl or picolinyl) led to increased reactivity to elastase with the optimal position for substitution being P4, see (4-5)-(4-8). 

As mentioned in Volume 13 of these Reports, 4-oxoazetidin-2-yl phosphonates and phosphinates (19) can be prepared by Arbusov-like reactions between P compounds and 4-acetoxyazetidin-2-one (20). Acid hydrolysis of (19) yields phosphono- and phosphino-aspartic acids (21) which can be converted into peptides with antibacterial activity. Diastereomeric mixtures of phosphono-dipeptides, which can be prepared from racemic dialkyl 1 -aminoalkylphosphonates, can be separated by ion-exchange chromatography. It appears that it is easier to synthesize phosphonodipeptides from these phosphonates as their P-dialkyl esters rather than as the free phosphonic acids. Phosphonic acid analogues of A-Cbz-alanine and -phenylalanine can be converted into ester and amide fluoridates, e.g., (22, R = OMe or NHCHMeg). These fluoridates are the most potent inhibitors of elastase and chymotrypsin yet reported and seem to mimic the natural substrates of these enzymes. ... 

Elastase (EC 3.4.21.11) an endopeptidase specific for the Elastic (see) in animal elastic fibers. Its inactive precursor, proelastase, is formed in the vertebrate pancreas and converted in the duodenum to elastase by the action of trypsin. The natural substrate of E. is elas-tin, an insoluble protein rich in valine, leucine and isoleucine. E. attacks the peptide bond adjacent to a nonaromatic, hydrophobic amino acid. The best synthetic substrates are therefore acetyl-Ala-Ala-Ala-OCHj and benzoylalanine methyl ester. Benzoylarginine ester (a trypsin substrate), and acetyltyrosine ester (a chymotrypsin substrate) are not attacked by E. 

Finally, coumarin derivatives may act as general inhibitors of serine proteases or as specific inhibitors of human leukocyte elastase, depending on the nature of the substituents, through two distinct mechanisms, suicide substrates (a-chymotrypsin)... 

A straightforward approach is to hunt for short polypeptides that meet the specificity requirement of an enzyme but which, because of peculiarities of the sequence, are acted upon very slowly. Such a peptide may contain unusual or chemically modified amino acids. For example, the peptide Thr-Pro-nVal-NMeLeu-Tyr-Thr (nVal=norvaline NMeLeu = N-methylleucine) is a very slow elastase substrate whose binding can be studied by X-ray diffraction and NMR spectroscopy.6 Thiol proteases are inhibited by succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide, which includes a sequence common to a number of naturally occurring peptide inhibitors called cystatins.f They are found in various animal tissues where they inhibit cysteine proteases. 

A final group of covalent small-molecule inhibitors of proteases are mechanism-based inhibitors. These inhibitors are enzyme-activated irreversible inhibitors, and they involve a two-hif mechanism that completely inhibits the protease. Some isocoumarins and -lactam derivatives have been shown to be mechanistic inhibitors of serine proteases. A classic example is the inhibition of elastase by several cephalosporin derivatives developed at Merck (Fig. 8). The catalytic serine attacks and opens the -lactam ring of the cephalosporin, which through various isomerization steps, allows for a Michael addition to the active site histidine and the formation of a stable enzyme-inhibitor complex (34). These mechanism-based inhibitors require an initial acylation event to take place before the irreversible inhibitory event. In this way, these small molecules have an analogous mechanism of inhibition to the naturally occurring serpins and a-2-macroglobin, which also act as suicide substrates. 

Even though serine proteinases share a common mechanism of peptide bond cleavage, they differ dramatically in their primary substrate specificity, exhibiting a preference for a certain type of amino acid residue. Hydrolysis studies using natural and synthetic substrates have demonstrated that NE preferentially cleaves peptide bonds after small aliphatic amino acid residues such as valine and alanine [21]. Elastase has been shown to have a pH optimum between 8.0 and 9.0 with most protein substrates and is strongly inhibited by the plasma inhibitor ai-PI. 

Studies with ab initio types of hybrid potential include the early work of Weiner et al. on the nature of catalysis in trypsin and the studies of the catalytic activity of phospholipase A2 by Hillier et al. Investigations with semiempirical hybrid potentials are more extensive and include calculations of the reactions in triosephosphate isomerase by Bash et al. and in chorismate mutase by Lyne et al. and a study of the proton jump in the catalytic triad of human neutrophil elastase. The study of the chorismate mutase reaction was especially interesting because the enzyme is the only known one that catalyzes a pericyclic reaction that also occurs readily in solution. The results of the hybrid study were particularly lucid in this case because the enzyme works, not by chemically catalyzing the reaction, but by preferentially binding a distorted form of the substrate and stabilizing the transition state. 

---