Aminopeptidase Mil

Aminopeptidases. These enzymes cleave the Tyr -Gly amide bond of the enkephalins releasing Tyr they include soluble aminopeptidase (EC 3.4.11.11 aminoacylpeptide hydrolase), aminopeptidase Mil (puromycin-sensitive aminopeptidase) and aminopeptidase M (EC 3.4.11.2 aminopeptidase N, puromycin-insensitive aminopeptidase). 

In washed brain slices, an in vitro model for the evaluation of effects of membrane-bound enzymes on enkephalin hydrolysis, the main hydrolysis products have been identified as Tyr and Tyr-Gly-Gly. The formation of Tyr-Gly-Gly is dependent on the action of enkephalinase and is reduced in the presence of thiorphan (an enkephalinase inhibitor). In the presence of bestatin (a general aminopeptidase inhibitor), the formation of Tyr is reduced Tyr is also reduced, to a lesser extent, in the presence of puromycin (an aminopeptidase Mil inhibitor). In the presence of thiorphan the formation of Tyr increases whereas in the presence of bestatin there is an increase in formation of Tyr-Gly-Gly. The level of Tyr-Gly in this model is low and is unaffected by either thiorphan or bestatin indicating that the action of DAP is unimportant. Recovery of endogenous enkephalins released by depolarisation of brain slices is enhanced in the presence of thiorphan or bestatin and is complete when both inhibitors are present. This does not occur in the case of puromycin or captopril (an ACE inhibitor) [31]. 

Both enkephalinase and membrane-bound aminopeptidases, particularly aminopeptidase M, are strong candidates as brain enkephalin-degrading neuropeptidases and fulfil the criteria laid down for this role [26]. The significance of aminopeptidase Mil has been insufficiently investigated for the enzyme to be disregarded as an enkephalin neuropeptidase. The role of DAP appears to be minor, at least in the CNS, although a peripheral role may be indicated [37], ACE is unlikely to be involved in deactivation of endogenously released enkephalin. 

A major part of the work on the structural requirements for binding of inhibitors to the active site of either aminopeptidase M or aminopeptidase Mil has been conducted using bestatin (see Figure 6.3A) as the model inhibitor and interpreted from the known structure-activity relationships of bestatin analogues towards leucine-aminopeptidase [43]. 

Length of peptide-type backbone. Aminopeptidase M11 inhibitory activity is enhanced markedly by addition of a phenylalanine residue to the chain (see Figure 6.3E). This indicates that aminopeptidase Mil contains an extended active site which favours the binding of a tripeptide or possibly a longer peptide. 

N-Terminal function. Removal of the A -terminal function and its associated benzyl group led to a marked decrease in aminopeptidase Mil... 

Carboxymethylamino-4-oxo-3-(4 -aminophenylamino)butanoic acid (3) and the corresponding unsubstituted analogue (6) are fairly potent inhibitors of enkephalinase with weak inhibitory potency towards aminopeptidase Mil. The compounds (4) and (5) represent the first combined inhibitors of enkephalinase and aminopeptidase Mil, however, aminopeptidase Ml 1 is not considered to be of primary importance in degradation of endogenous enkephalins [41],... 

Table 6.10. IN VITRO ENKEPHALINASE (EC 3.4.24.11, ENK) AND AMINOPEPTIDASE Mil (APMII) INHIBITORY ACTIVITY OF SOME BUTANDIOIC...

Carboxymethylamino-4-oxo-3-(4 -aminophenylamino)butanoic acid (3), its ethyl ester (4) and corresponding unsubstituted-aryl analogues (6) and (5) (see Table 6. JO) are fairly potent inhibitors of enkephalinase (K 0.14-0.39 / M) with inibitory potency (K, 15-75 /.iM ) towards aminopeptidase Mil [82]. In the mouse abdominal constriction test, the esters (4) and (5) showed systemic antinociceptive activity with ED50 values of 62 and 81 mg/kg, respectively. In the mouse tail immersion test, both (4) and (5) exhibited antinociceptive activity when administered icv. The results from the mouse abdominal constriction test for compounds (4) and (5) indicated the same rank order of potency as their in vitro inhibitory potency for enkephalinase and aminopeptidase Mil. Another notable observation is that these compounds also exhibited the same rank order in their antinociceptive effects when administered icv alone in the mouse tail immersion test. This direct effect has not been reported for other more potent enkephalindegrading enzyme inhibitors. Compound (4) uniquely exhibited antinociceptive activity when administered subcutaneously in the mouse tail immersion test, an effect which is only partially reversible by naltrexone. This result is in contrast to that for compound (5), which displayed only one third and one quarter of the potency of enkephalinase and aminopeptidase... 

---