Design, synthesis, and anticancer activity evaluation of irreversible allosteric inhibitors of the ubiquitin-conjugating enzyme Ube2g2

Abstract

The RING finger-dependent ubiquitin ligase (E3) gp78, known as the tumor autocrine motility factor receptor, contributes to tumor progression. The protein interacts with its cognate ubiquitin-conjugating enzyme (E2), Ube2g2, via its RING domain and a unique region called G2BR that strongly binds to E2. The binding of G2BR to Ube2g2 allosterically enhances the binding of RING to E2, and the binding of RING triggers the departure of G2BR from E2 also in an allosteric fashion. Targeting these allosteric events, we developed a family of inhibitors that irreversibly block E2-E3 interactions and thereby eliminate the tumorigenic effect of gp78. One among 19 compounds screened with the NCI 60 tumor cell lines exhibited outstanding anticancer activities. At 10 μM, it caused >50% growth inhibition to 40% of the cell lines; at 100 μM, it showed lethiferous activity against most cell lines.

Fig. 1. Allosteric regulation of a processive ubiquitination machine. Comparative analysis of the crystal structures of ligand-free Ube2g2 (PDB entry 2CYX), Ube2g2 in complex with the G2BR of gp78 (PDB entry ; 3H8K), and Ube2g2 in complex with both the G2BR and RING of gp78 (PDB entry ; 4LAD) reveals allosteric regulation events along the functional cycle of Ube2g2. (A) Schematic representation of the E2 enzyme Ube2g2 with the G2BR-binding, RING-binding, and Ub-conjugating sites indicated. The minus sign indicates the hidden carboxyl group of the Glu108 side chain. It forms a salt bridge with the Arg379 guanidinium group of RING. (B) In an ATP-dependent manner, a Ub molecule is activated by an E1 enzyme and conjugated to Ube2g2. (C) The E3 enzyme gp78 binds to Ube2g2 with the G2BR and RING motifs. G2BR is non-structured before binding to the E2 enzyme. The Arg379 guanidinium group of RING is indicated with a plus sign. (D) Upon binding to Ube2g2, G2BR becomes an α-helix. In an allosteric manner, the binding of G2BR triggers the exposure of the hidden carboxyl group of Glu108. (E) Readily, the Glu108^Ube2g2–Arg379^gp78 salt bridge forms, and the binding of RING promotes the ligation of Ub to the substrate and the release of G2BR from Ube2g2. (F) The departure of G2BR destroys the salt bridge, promoting the release of RING from Ube2g2. The ligand-free Ube2g2 will be loaded with another Ub for the next ubiquitination cycle.

Fig. 2. Design of the irreversible allosteric inhibitor CW19 targeting Ube2g2. (A) CC0651, the allosteric inhibitor for Cdc34. (B) Superimposed Cdc34 structures in its ligand-free (apo-Cdc34, PDB entry ; 2OB4) and CC0651-bound (Cdc34:CC0651, PDB entry ; 3RZ3) forms. (C) CW19, the irreversible allosteric inhibitor for Ube2g2 (this work). (D) The Ube2g2–CW19 model, created with the Phyre2 server using the Cdc34 structure in the Cdc34:CC0651 complex as a template, is superimposed with the apo-Ube2g2 structure (PDB entry ; 2CYX). In panels B and D, detailed interactions between the protein and inhibitor are highlighted with zoom-in boxes. In panel D, the G2BR-binding and RING-binding sites are indicated with molecular surfaces modelled based on the G2BR and RING motifs in the Ube2g2:RING–G2BR structure (PDB entry ; 4LAD).

Scheme 1. Chemical synthesis of type D inhibitors (CW1 to CW10). Starting reagent, Boc-4-iodo-d-phenylalanine (1) (5.00 g, 12.78 mmol). (a) 3–5-Dichlorophenylboronic acid, Na₂CO₃, Pd(PPh₃)₄, THF/H₂O (2 : 1), 70 °C, 48 h, yield 90%; (b) CH₃I, NaHCO₃, DMF, rt, 12 h, yield 92%; (c) DIBAL, CH₂Cl₂, rt, 12 h, yield 70%; (d) HCl, EtOH, rt, 6 h, yield 96%; (e) acrylic acid (13) or 3-butenoic acid (14), PyAOP, DIPEA, DMF, rt, 3 h, yield 62% (13, CW1) and 65% (14, CW2), respectively; (f) Dess–Martin periodinane, CH₂Cl₂, rt, 5 h, yield 97%; (g) triethyl phosphonoacetate, NaH, DMF, 0 °C, 1 h or rt, 2 h, yield 96%; (h) same as d, yield 98%; (i) same as e, yield 52% (8, CW3) and 55% (9, CW4), respectively; (j) NaOH, MeOH/H₂O (1 : 1), rt, 12 h, yield 77% (10, CW5) and 80% (11, CW6), respectively; (k) methanesulfonamide, K₃Fe(CN)₆, OsO₄, (DHQD)₂PHAL, K₂CO₃, t-BuOH/H₂O (1 : 1), rt, 12 h, yield 65%; (l) same as d, yield 95%; (m) same as e, yield 67% (17, CW7) and 64% (18, CW8), respectively; (n) NaOH, THF/MeOH/H₂O (1 : 1 : 1) (19, CW9) or MeOH/H₂O (1 : 1) (20, CW10), rt, 12 h, yield 70% (19) and 76% (20), respectively.

Scheme 2. Chemical synthesis of type L inhibitors (CW11 to CW20). Starting reagent, Boc-4-iodo-l-phenylalanine (21) (5.00 g, 12.78 mmol). (a) 3–5-Dichlorophenylboronic acid, Na₂CO₃, Pd(PPh₃)₄, THF/H₂O (2 : 1), 70 °C, 48 h, yield 98%; (b) CH₃I, NaHCO₃, DMF, rt, 12 h, yield 96%; (c) DIBAL, CH₂Cl₂, rt, 12 h, yield 78%; (d) HCl, EtOH, rt 6 h, yield 99%; (e) acrylic acid (33) or 3-butenoic acid (34), PyAOP, DIPEA, DMF, rt, 3 h, yield 70% (33, CW11) and 75% (34, CW12), respectively; (f) Dess–Martin periodinane, CH₂Cl₂, rt 5 h, yield 90%; (g) triethyl phosphonoacetate, NaH, DMF, 0 °C, 1 h or rt, 2 h, yield 97%; (h) same as d, yield 99%; (i) same as e, yield 59% (28, CW13) and 55% (29, CW14), respectively; (j) NaOH, MeOH/H₂O (1 : 1), rt, 12 h, yield 74% (30, CW15) and 80% (31, CW16), respectively; (k) methanesulfonamide, K3Fe(CN)₆, OsO4, (DHQD)₂PHAL, K₂CO₃, t-BuOH/H₂O (1 : 1), rt, 12 h, yield 67%; (l) same as d, yield 84%; (m) same as e, yield 59% (37, CW17) and 85% (38, CW18), respectively; (n) NaOH, THF/MeOH/H₂O (1 : 1 : 1) (39) or MeOH/H₂O (1 : 1) (40), rt, 12 h, yield 68% (39, CW19) and 64% (40, CW20), respectively.

Fig. 3. Mean growth percentages of tumor cell lines in the presence of CW2 through CW20 (one-dose screening).

Fig. 4. Anticancer activity of CW3. (A) In vitro anticancer activity of CW3 tested with the National Cancer Institute (NCI) 60 tumor cell lines by the NCI Developmental Therapeutics Program. (B) Covalent docking of CW3 into its binding site on Ube2g2 results in the Ube2g2–CW3 conjugate.