Extended Data Fig. 1: Design and development of NNPIs targeting CTSS or CTSB.

a) FRET assay assessing cleavage of 3 different substrate peptides by CTSS (n = 3 independent replicates; mean and SD values are shown). b) Binding mode of the CD74 peptide LRMKLPKP calculated using the Attracting Cavities docking software. In the top panel, the CTSS surface is displayed and colored in brown. The peptide is shown in ball and stick representation. The central KL residues of the CD74 peptide are calculated to be in the vicinity of the CTSS Cys139 catalytic residue, labeled in bold (bottom). c) Results of FRET assay testing CTSS inhibition by NNPI-1, 2, 3 and 4 (n = 2 independent replicates). d) FRET assay assessing CTSS inhibition at increasing NNPI-3 concentrations (n = 3 independent replicates; mean and SD values are shown). e) Sequence and CTSS inhibition capacity of different NNPIs with single amino acid substitutions compared to NNPI-3 (n = 3 independent replicates; mean and SD values are shown). f) Sequence and CTSS inhibition capacity of different NNPIs combining 2 amino acid substitutions compared to NNPI-3 (n = 3 independent replicates; mean and SD values are shown). The most potent NNPIs are labeled in blue. g) FRET dose-response quantification of CTSS inhibition by NNPI-C2 (n = 3 independent replicates; mean and SD values are shown). h) Sequence and CTSS inhibition capacity of different NNPIs combining up to 2 new amino acid substitutions compared to NNPI-C2 (n = 3 independent replicates; mean and SD values are shown). The most potent NNPI is labeled in red, and its molecular structure is represented on the right. i) Sequence and CTSB inhibition capacity of different NNPIs with up to 3 amino acid substitutions compared to NNPI-C10 (n = 3 independent replicates; mean and SD values are shown). The most potent NNPI is labeled in red, and its molecular structure is represented on the right.