Recognition L/D-microproteins, reported in this study are small, compact, atactic, protein-like peptides that function as antibody mimics. These L/D-microproteins are designed to comprise all the key advantages of antibodies, such as high affinity and long serum half-life, while reducing size and immunogenicity. They are designed using mutational molecular dynamics (MMD) and synthesized using automated solid-phase peptide synthesis (SPPS) and by the CuAAC click reaction to enhance structural and metabolic stability. The recognition L/D-microproteins are designed to target the caspase 3/7 binding site (BIR2 domain) and the caspase 9 binding site (BIR3 domain) of the intracellular cancer protein X-linked inhibitor of apoptosis (XIAP). Overexpression of XIAP is observed in tumours leading to increased cell survival and resistance to chemotherapeutics.¹ Inhibition of XIAP’s interaction with caspases induces apoptosis, making this approach beneficial for targeting tumour cells. Five XIAP domains have been structurally determined, but the structure of full-sized XIAP is not available. Hence, the spatial organization of these domains is not known. This study reports the expression of the full-size protein XIAP. The XIAP protein was fused with maltose-binding protein (MBP) to stabilise it for expression in E. coli, the full-length XIAP-MBP was successfully isolated. MBP was subsequently cleaved from XIAP using TEV protease.² The complete structure of XIAP was further investigated using photo-crosslinking, which may enable more precise designs of inhibitors against XIAP.³ To assess the designed and synthesized recognition L/D-microproteins, binding and activity assays were performed. Preliminary results show binding to the target protein XIAP, validating our structural model and suggesting the potential for developing more potent inhibitors targeting XIAP and other proteins of interest for the treatment of various diseases.

Fig. 1: An overview of the project, highlighting the computational design of microproteins, SPPS, protein expression of XIAP-MBP, and affinity assays, aimed at inducing apoptosis in cancerous tumour cells.

References:

[1]  A. D. Schimmer, S. Dalili, R. A. Batey, S. J. Riedl, Cell Death Differ, 2006, 13.

[2] P. Polykretis, E. Luchinat, A. Bonucci, A. Giachetti, M. A. Graewert, D. I. Svergun, L. Banci, IUCrJ, 2019, 6, 948-957.

[3] F. J. O'Reilly, J. Rappsilber, Nat Struct Mol Biol, 2018, 25, 1000-1008.