Specific targeting of human caspases using designed ankyrin repeat proteins

Abstract

Caspases play important roles in cell death, differentiation, and proliferation. Due to their high homology, especially of the active site, specific targeting of a particular caspase using substrate analogues is very difficult. Although commercially available small molecules based on peptides are lacking high specificity due to overlapping cleavage motives between different caspases, they are often used as specific tools. We have selected designed ankyrin repeat proteins (DARPins) against human caspases 1-9 and identified high-affinity binders for the targeted caspases, except for caspase 4. Besides previously reported caspase-specific DARPins, we generated novel DARPins (D1.73, D5.15, D6.11, D8.1, D8.4, and D9.2) and confirmed specificity for caspases 1, 5, 6, and 8 using a subset of caspase family members. In addition, we solved the crystal structure of caspase 8 in complex with DARPin D8.4. This binder interacts with non-conserved residues on the large subunit, thereby explaining its specificity. Structural analysis of this and other previously published crystal structures of caspase/DARPin complexes depicts two general binding areas either involving active site forming loops or a surface area laterally at the large subunit of the enzyme. Both surface areas involve non-conserved surface residues of caspases.

Non-invasive in vivo imaging of tumour-associated cathepsin B by a highly selective inhibitory DARPin

“…Although this approach has found substantial use in developing extremely selective binders to various receptors, including for potential therapeutic applications 57 , 58 , it has not been used in cellular or non-invasive diagnostic imaging of proteases. Moreover, the only protease-targeting DARPins were developed as selective inhibitors of a tobacco virus protease 59 or caspases 35 , 60 to help elucidate the molecular basis of their specificity.…”

Vaccinia Virus Encodes a Novel Inhibitor of Apoptosis That Associates with the Apoptosome

“…In contrast, proteins like human gankyrin, an ANK-containing oncoprotein often overexpressed in many hepatocellular carcinomas, inhibits apoptosis by binding to the Mdm2 E3 ubiquitin ligase, leading to ubiquitination and degradation of proapoptotic p53 (80,81). Because these proteins are critical to the survival of healthy versus damaged cells, synthetic ANKs (DARPins) have been engineered as a means to control apoptosis (30,31,82). New information concerning ANK proteins that alter apoptosis, such as the novel way that M1 inhibits intrinsic apoptosis at the level of the apoptosome, could potentially benefit the development of novel DARPins.…”

“…These are 33-residue motifs that form alpha-helical structures and provide platforms for protein-protein interactions (28). This property has led to the use of designed ANK repeat proteins (DARPins) as a drug development platform (30,31). VACV strain WR encodes at least eight known or predicted ANK proteins, including 005-008 and 211-214 (Copenhagen B25 homologs), 014-017 (variola virus strain Bangladesh D8 homologs), 019 (Copenhagen C9 homolog), 030 (M1), 031 (K1), 186 (B4), 188 (B6), and 199 and 202 (B18) (32,33).…”

“…The goal of this study was to identify a function for the VACV ANK-encoding M1L gene, a gene with no previously ascribed function that is located within a region of the WR genome that was deleted during the derivation of MVA. Because multiple natural and synthetic ANK proteins (e.g., DARPins) inhibit apoptosis (31,(43)(44)(45)(46), M1 may possess this same function. Interestingly, M1 inhibited intrinsic apoptosis under several conditions and in multiple cell lines.…”

Non-immunoglobulin scaffold proteins: Precision tools for studying protein-protein interactions in cancer

“…Evasion of apoptosis and cell death is a key hallmark of cancer and is usually the result of a change in the balance of anti-apoptotic and pro-apoptotic signals [1]. SBPs have been developed to target both of these forms of signal with Alphabodies selected against the anti-apoptotic Mcl-1 [20] and DARPins against the pro-apoptotic caspases [31]. Alphabodies, artificial SBPs of three anti-parallel helices [15], targeting Mcl-1, an anti-apoptotic member of the Bcl-2 family that sequesters the pro-apoptotic Bax [66], show pM binding affinities [20].…”