Granzyme inhibitors

The chemistry of granzymes has been extensively studied in the laboratory of Dr James Powers (Atlanta, Georgia) in collaboration with several other groups (see ref. 58). For example, the BLT-esterase release method described in Section 2.4 is one of the applications of these studies. As discussed above, peptide inhibitors of serine proteases should be chloromethylketones. However, these compounds, because of their high reactivity, could inhibit other cellular proteases. For this reason, several other chemical inhibitors, mainly isocoumarin derivatives, have been developed.

• For granzyme B, the most specific peptide inhibitor described is Z-AAD-cmk, which does not inhibit granzyme A or related proteases (6). In the case of isocoumarins, 3,4-dichloroisocoumarin (DCI) has been described to inhibit granzyme B much more efficiently than granzyme A (around 50-fold difference in K^) (59).

• Granzyme A, unlike granzyme B, is a trypsin-like protease. In a cytotoxicity assay, it can be considered that the major targets for these inhibitors are trypsin-like granzymes, granzyme A being the predominant one. The most specific peptide inhibitor for trypsin-like proteases is d-FPR-cmk, which does not inhibit granzyme B (6). On the other hand, 7-( phenyl-ureido)-4-chloro-3-(2-isothioureidoethoxy)-isocoumarin, abbreviated as IGA, is the most potent isocoumarin described for granzyme A inhibition (60,61).

Z-AAD-cmk and d-FPR-cmk are sold by Enzyme System Products (Dublin, California) and DCI is sold by many chemical companies (e.g. Sigma).

Protocol 9. Use of granzyme inhibitors in whole-cell cytotoxicity assays


1. Stock solutions of peptide and iosocumarin granzyme inhibitors are usually prepared in DMSO and stored at-20°C.

2. Pre-incubation of inhibitors with target cells is not needed in this case since the inhibitors exhibit sufficient permeability through the cell membrane. The effective concentrations for DCI, IGA, and Z-AAD-cmk are between 20 and 50 |xM.

3. The considerations of effector to target ratios in Protocol 8 also apply here. Hence, E:T ratios from 1:1 to 5:1, but not greater, should be used in this type of experiment.

4. The possible increase in spontaneous release of the label from target cells induced by the inhibitor itself should be carefully controlled. If the spontaneous release induced by the inhibitor alone is greater than 30% of the total labelling, then this inhibitor cannot be used in such an assay. In our hands, Z-AAD-cmk, DCI and IGA do not induce an increase in the spontaneous release in 125IUdR release assays at the concentrations mentioned, even when using long incubation times (up to 16 h). However, these inhibitors do induce spontaneous 51Cr release in long-term assays. These inhibitors can only be used at the indicated concentrations in short-term assays (3-5 h), and the particular conditions of incubation with each target cell used need to be carefully optimized.

5. Bearing in mind these considerations, perform any of the cytotoxicity tests described above in the presence of the described granzyme inhibitors.

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