Volume 24, Issue 3, Pages 381-390 (March 2017) Rapid Discovery of Potent and Selective Glycosidase-Inhibiting De Novo Peptides  Seino A.K. Jongkees, Sami Caner, Christina Tysoe, Gary D. Brayer, Stephen G. Withers, Hiroaki Suga  Cell Chemical Biology  Volume 24, Issue 3, Pages 381-390 (March 2017) DOI: 10.1016/j.chembiol.2017.02.001 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 381-390DOI: (10. 1016/j. chembiol. 2017 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Overview of the Strategy Employed in This Work (A) Incorporation of post-translational modification with the RaPID system for discovery of non-standard peptides, emphasizing the incorporation of diverse phenolic groups. Illustrated is the workflow for one round of selection. eFx, enhanced flexizyme. (B) Two-step post-translational modification of a peptide library to incorporate resorcinol moieties, and comparison with the core inhibitory motif of montbretin (A). See also Figures S1 and S2; Scheme S1. Cell Chemical Biology 2017 24, 381-390DOI: (10.1016/j.chembiol.2017.02.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Sequence Logos Derived from Alignment of the Top 255 Most Abundant Sequences for the L- and D-Tyrosine-Initiated Libraries in Their Respective Sixth Rounds These alignments represent 82% and 80% of the total reads, respectively. Residues are colored blue for positively charged, red for negatively charged, yellow for cysteine, green for glycine and proline, and magenta for aromatic. See also Figure S3. Cell Chemical Biology 2017 24, 381-390DOI: (10.1016/j.chembiol.2017.02.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 X-Ray Crystal Structure of piHA-Dm Bound to HPA (A) Top view of the active-site surface of the HPA/piHA-Dm complex (PDB: 5KEZ). The structure of piHA-Dm (green stick model) as bound in the active-site cleft of HPA and fitted into its corresponding simulated annealing omit electron density map shown at the 3σ level (magenta). Blue surfaces represent the positions of active-site triad residues (Asp197, Glu233, and Asp300). (B) Magnified view of active-site residues (blue lines) involved in hydrogen-bond interactions with the peptide inhibitor (green stick model). (C) Schematic of the hydrogen-bond network observed within the HPA/piHA-Dm complex. Active-site residues of HPA are labeled, along with the residues forming the bound peptide (green numbers). The cyclic part of the peptide is shown as a ring from amino acids 1 through 5 and the peptide continues in a linear mode from amino acids 6 through 9. Each hydrogen-bond interaction is symbolized by a red dashed line that connects interacting atoms, which are highlighted with red circles, or blue circles in the case of water-mediated interactions. See also Table S2. Cell Chemical Biology 2017 24, 381-390DOI: (10.1016/j.chembiol.2017.02.001) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Structural Comparison of HPA Inhibition Motifs Montbretin A (PDB: 4W93) (A), tendamistat (PDB: 1BVN) (B), piHA-Dm (PDB: 5KEZ) (C), helianthamide (PDB: 4X0N) (D), and acarbose (PDB: 1CPU) (E). All diagrams are drawn in the same orientation, with active-site residues shown as blue sticks and hydrogen-bond interactions as black dashed lines. Subtitles beneath each figure indicate the respective inhibition motif. See also Figure S6. Cell Chemical Biology 2017 24, 381-390DOI: (10.1016/j.chembiol.2017.02.001) Copyright © 2017 Elsevier Ltd Terms and Conditions