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. 2024 Sep 11;4(9):3649-3656.
doi: 10.1021/jacsau.4c00633. eCollection 2024 Sep 23.

Rational Design of Dual-Domain Binding Inhibitors for N-Acetylgalactosamine Transferase 2 with Improved Selectivity over the T1 and T3 Isoforms

Ismael Compañón  1 , Collin J Ballard  2 , Erandi Lira-Navarrete  3 , Tanausú Santos  1 , Serena Monaco  4 , Juan C Muñoz-García  4   5 , Ignacio Delso  4 , Jesus Angulo  4   5 , Thomas A Gerken  2   6 , Katrine T Schjoldager  3 , Henrik Clausen  3 , Tomás Tejero  7   8 , Pedro Merino  7   9 , Francisco Corzana  1 , Ramon Hurtado-Guerrero  3   9   10 , Mattia Ghirardello  1
Affiliations

Rational Design of Dual-Domain Binding Inhibitors for N-Acetylgalactosamine Transferase 2 with Improved Selectivity over the T1 and T3 Isoforms

Ismael Compañón et al. JACS Au. .

Abstract

The GalNAc-transferase (GalNAc-T) family, consisting of 20 isoenzymes, regulates the O-glycosylation process of mucin glycopeptides by transferring GalNAc units to serine/threonine residues. Dysregulation of specific GalNAc-Ts is associated with various diseases, making these enzymes attractive targets for drug development. The development of inhibitors is key to understanding the implications of GalNAc-Ts in human diseases. However, developing selective inhibitors for individual GalNAc-Ts represents a major challenge due to shared structural similarities among the isoenzymes and some degree of redundancy among the natural substrates. Herein, we report the development of a GalNAc-T2 inhibitor with higher potency compared to those of the T1 and T3 isoforms. The most promising candidate features bivalent GalNAc and thiophene moieties on a peptide chain, enabling binding to both the lectin and catalytic domains of the enzyme. The binding mode was confirmed by competitive saturation transfer difference NMR experiments and validated through molecular dynamics simulations. The inhibitor demonstrated an IC50 of 21.4 μM for GalNAc-T2, with 8- and 32-fold higher selectivity over the T3 and T1 isoforms, respectively, representing a significant step forward in the synthesis of specific GalNAc-T inhibitors tailored to the unique structural features of the targeted isoform.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
(A) Structure of GalNAc-T2 in complex with glycopeptide MUC5AC-13 and UDP. (B) Schematic representation of the GalNAc-T2 regions and of the binding mode with MUC5AC-13 glycopeptide. (C) Binding mode of the MUC5AC-13 GalNAc moiety (yellow) with GalNAc-T2 lectin pocket (gray). (D) Binding mode of MUC5AC-13 PVP residues (green) with the hydrophobic region of GalNAc-T2 (gray). PDB ID: 5AJP. (E) Chemical structure of inhibitors 1–4. (F) Representative frame derived from 1.0 μs molecular dynamics (MD) simulation of compound 3 with GalNAc-T2 and expansion on the interactions between the thiophene moiety and residues W282, F280, and F361. (G) Average distance between the aromatic moieties of 1, 2, and 3 with the residues W282, F280, and F361 of GalNAc-Ts calculated through 1.0 μs MD simulations; data represent mean + SD (standard deviation).
Figure 2
Figure 2
(A) Synthetic approach for the synthesis of building blocks 8 and 12. Reagent and conditions: (i) HBr (33% in AcOH), Et2O, 0 °C to rt, 16 h, 96%; (ii) 13, NaHCO3, Bu4NBr, H2O, EtOAc, 24 h, rt, 82%; (iii) EDT, TFA, DCM, 0 °C to rt, 1 h, 81%; (iv) SOCl2, DCM, 0 °C to rt, 1 h, 85%; and (v) TFA, DCM, 0 °C to rt, 2 h, 87%. (B) Relative inhibitory activity % of compounds 2 and 3 against GalNAc-T2 using MUC1a as acceptor substrate. (C) Relative inhibitory activity % of compounds 3, 4, 5, 15, and 16 at a normalized concentration of 500 μM against GalNAc-T2 using MUC1a as acceptor substrate; data represent mean + SD. (D) Chemical structure of inhibitors 17–19. (E) Average distance between the thiophene moiety of 17, 18, and 19 with residue F361 of GalNAc-Ts calculated through 1.0 μs MD simulations; data represent mean + SD (standard deviation). (F) Relative inhibitory activity % of compounds 18 and 19 against GalNAc-T2 using MUC1a as acceptor substrate.
Figure 3
Figure 3
(A) STD NMR binding epitope map of 19 upon binding to GalNAc-T2, analyzed as an average per residue, due to significant proton signal overlapping. Colors represent different degrees of contact, from light blue to purple, as shown in the legend, and STD NMR competition experiments performed adding UDP-GalNAc to a sample containing 19 and GalNAc-T2. (B) Representative MD frame of 19 in complex with GalNAc-T2. Color code of 19 corresponds to the degree of contact with GalNAc-T2 as for Figure 3A. (C) Relative inhibitory activity % of compound 19 against GalNAc-T1, -T2, and -T3 using MUC1a as acceptor substrate.
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