Papain aldehyde inhibitors

Westrik and Wolfenden (65) have recently reported that aldehydes, with side chains similar to those comprising the acyl portion of substrates which papain effectively hydrolyzes, were very potent inhibitors of this enzyme. Umezawa and his associates (66) have also recently reported that certain microorganisms (actinomyces) produced an aldehyde, acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin), which inhibits papain. The structures of some of the more effective aldehyde inhibitors of papain are shown in Figure 4. 

Figure 14, Analogy of formation of thiol adduct with aldehyde inhibitors to formation of tetrahedral intermediate in papain catalysis...

Shokhen et al. [56] analyzed possible mechanisms for the reversible formation of the complex between papain, a prototype enzyme of cysteine proteases, and pep-tidy 1 aldehyde inhibitors, using the quantum mechanical (DFT)/self consistent reaction field (virtual solvent) approach. 

Figure 17.3 Schematic representation of the design of the symmetric cathepsin K inhibitor diacylaminomethyl ketone (1,3-bis[[A/-[(phenylmethoxy)carbonyl]-L-leucyl]amino]-2-propanone), based on the crystal structures of papain bound to leupeptin (Leu-Leu-Arg-aldehyde) and to Cbz-Leu-Leu-Leu-aldehyde, and an example of its further optimization.

The cysteinyl proteases include papain calpains I and II cathepsins , H, and L proline endopeptidase and interleukin-converting enzyme (ICE) and its homologs. The most well-studied cysteinyl protease is likely papain, and the first x-ray crystallographic structures of papain [193] and a peptide chloromethylketone inhibitor-papain complex [194] provided the first high resolution molecular maps of the active site. Pioneering studies in the discovery of papain substrate peptide-based inhibitors having P, electrophilic moieties such as aldehydes [195], ketones (e.g., fluoromethylketone, which has been determined [196] to exhibit selectivity for cysteinyl proteases versus serinyl proteases), semicarbazones, and nitriles are noteworthy since 13C-NMR spectro-... 

Peptide nitriles are reversible inhibitors of cysteine proteases. 1,2 Peptide nitrile reacts with the active site thiol group to form an imidothioate, a dead-end product that does not undergo hydrolysis to an amide.134 This imidothioate derivative has been detected by NMR spectroscopic studies.P 5 The inhibition of papain, a cysteine protease, by a peptide nitrile proved to be reversible in a dialysis experiment. 3 Peptide nitriles are weaker inhibitors of cysteine proteases than the corresponding aldehydes. 61 Most peptide nitriles show poor inhibition toward serine proteases, however those nitriles with proper peptide sequences are potent inhibitors of serine proteases. 7-9 ... 

The simplest member of this family of inhibitors, the methoxy-methyl ketone (Fig. 5.8a), has been studied as a complex with papain [14]. This enzyme-inhibitor complex shows binding of the peptide portion of the inhibitor on the prime side of the active site in a manner similar to that seen for Cbz-Leu-Leu-Leu-aldehyde (Fig. 5.2d) bound to papain. In contrast to the thiohemi-acetal seen with the aldehyde, the carbonyl of the methoxy-methyl ketone (Fig. 5.8a) is quite distant from the active site thiol, with no possibility of covalent interaction. Therefore, this inhibitor meets the criteria for class I inhibition. The n-propyloxy ketone (Fig. 5.8b), which also binds on the prime side of the active site, has the ketone in close proximity to the active site thiol of cathepsin K, as seen in the structure of the inhibitor-enzyme complex [20]. Covalent attachment appears to have followed from... 

One of the key intermediates shown in this reaction scheme is the formation of a tetrahedral adduct during acylation and deacylation (84). Additional support for the formation of a tetrahedral intermedite comes from the observation already referred to— that aldehydes may act as potent inhibitors of papain. Westerik and Wolfenden (65) attribute the inhibitory eflFect of aldehydes to the formation of a stable thiol adduct (thiohemiacetal) analogous to the tetrahedral intermediate produced when papain acts on a substrate. This relationship is depicted in Figure 14. When the complete picture for the mechanism of catalysis by the thiol proteases finally emerges, it will no doubt be similar to the mechanism of action of the serine proteinases. 

As shown in Fig. 12-15, the side chain of Asn 175 provides papain with a third member of a catalytic triad analogous to that of serine pro teases.Glutamine 19, together with the pephde backbone NH of Cys 25, provides an oxyanion hole. Many studies, including structure determinahons on bound aldehyde and other inhibitors, and observahon by indicate that... 

The U-4CR of lOw, 6zb, 9d, and Ize provided the Ugi product 278, which under acidic conditions afforded the key intermediate A(-acylpyrrole 279 (Scheme 7.88). In the presence of various nucleophiles, 279 afforded amides (280 and 281) and methyl ester 282 under refluxing conditions in good yields. In the presence of LiOH, 279 afforded the acid 283 at room temperature in an excellent yield. Employment of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), NaBH4, and Homer-Wadsworth-Emmons conditions provided the aldehyde 284, alcohol 285, and the a,p-unsaturated ester 286, respectively. These synthetic transformations can be very useful in particular, compound 286 is a valuable intermediate for the synthesis of 3-aza-bicyclo[3.1,0]hexane ring system, constrained amino acids, papain inhibitors, ( )-isocynodine, and ( )-isocynometrine [102],... 

---