Screening of protein-protein interaction modulators via sulfo-click kinetic target-guided synthesis

Abstract

Kinetic target-guided synthesis (TGS) and in situ click chemistry are among unconventional discovery strategies having the potential to streamline the development of protein-protein interaction modulators (PPIMs). In kinetic TGS and in situ click chemistry, the target is directly involved in the assembly of its own potent, bidentate ligand from a pool of reactive fragments. Herein, we report the use and validation of kinetic TGS based on the sulfo-click reaction between thio acids and sulfonyl azides as a screening and synthesis platform for the identification of high-quality PPIMs. Starting from a randomly designed library consisting of 9 thio acids and 9 sulfonyl azides leading to 81 potential acylsulfonamides, the target protein, Bcl-X(L), selectively assembled four PPIMs, acylsulfonamides SZ4TA2, SZ7TA2, SZ9TA1, and SZ9TA5, which have been shown to modulate Bcl-X(L)/BH3 interactions. To further investigate the Bcl-X(L) templation effect, control experiments were carried out using two mutants of Bcl-X(L). In one mutant, phenylalanine Phe131 and aspartic acid Asp133, which are critical for the BH3 domain binding, were substituted by alanines, while arginine Arg139, a residue identified to play a crucial role in the binding of ABT-737, a BH3 mimetic, was replaced by an alanine in the other mutant. Incubation of these mutants with the reactive fragments and subsequent LC/MS-SIM analysis confirmed that these building block combinations yield the corresponding acylsulfonamides at the BH3 binding site, the actual "hot spot" of Bcl-X(L). These results validate kinetic TGS using the sulfo-click reaction as a valuable tool for the straightforward identification of high-quality PPIMs.

Figure 1

Kinetic TGS approach targeting PPIs. A) TGS approaches are based on the principle that multidentate interactions between a ligand and a biological target are collectively much stronger than the corresponding monovalent interactions of each of the fragments.(60) Thus, target-assembled compound most likely will have a stronger interaction with the biological target as compared to the individual building blocks.(60) In kinetic TGS, fragments decorated with complementary reactive groups are incubated with the target biomolecule. If two fragments reside simultaneously in close proximity in binding pockets of the target, the two reactive functionalities react with each other forming a covalent linkage between the two fragments. B) Acylsulfonamide 1, ABT-737 and ABT-263 compounds targeting Bcl-X_L. C) Proof-of-concept study to demonstrate that the amidation between thio acids and sulfonyl azides is suited for kinetic TGS targeting PPIs.

Figure 2

Synthesis of sulfonyl azides, thio acids and acylsulfonamides. Reaction conditions: (a) SOCl₂, DMF, reflux, 2 h (b) 2-(phenylthio)ethanamine (0.5 eq), K₂CO₃, CHCl₃, 12 h, RT (c) NaN₃, acetone, H₂O, 0 °C, 3 h, 70% (over 3 steps) (d) K₂CO₃, CH₃CN:H₂O (9:1), 12 h, RT, 87% (e) 2-(phenylthio)ethanamine (0.5 eq), K₂CO₃, CH₃CN:H₂O (9:1), 12 h, RT, 60% (f) ICH₂CN, K₂CO₃, CH₃CN:H₂O (10:1), 2 d, 60 °C, 79% (g) 12 N HCl, 90 °C, 3 h, 66% (h) i) (COCl)₂, CH₂Cl₂, 0 °C to RT, 8 h; ii) dimethylthioformamide, H₂S, 15 min, 25% (i) NaSH, acetone, H₂O, 2 h, RT (j) NaSH, neat, 0 °C to RT, 1 h (k) EDCI, DMAP, CH₂Cl₂, RT, 24 - 48 h (l) (CH₃)₂CHOCOCl, N-methyl piperidine, THF, 0 °C, 30 min (m) LiAlHSeH, THF, 0 °C, 30 min (n) RSO₂N₃, THF, 0 °C to RT, 3 h.

Figure 3

Kinetic TGS screening of Bcl-X_L via sulfo-click chemistry.

Figure 4

LC/MS-SIM analysis of kinetic TGS incubations with fragments SZ7 and TA2 targeting Bcl-X_L. The samples were incubated at 37 °C for 6 hours and subjected to LC/MS-SIM analysis with gradient system 1 (see supporting information). A) Incubation sample containing fragments SZ7 and TA2 in absence of Bcl-X_L; B) Incubation sample containing fragments SZ7 and TA2 in presence of 2 μM Bcl-X_L; C) Incubation sample containing fragments SZ7 and TA2 in presence of 2 μM Bcl-X_L and 20 μM Bim BH3 peptide; D) Incubation sample containing fragments SZ7 and TA2 in presence of 2 μM Bcl-X_L and 20 μM mutant Bim BH3 peptide; E) Synthetic SZ7TA2 as the reference compound.

Figure 5

LC/MS-SIM analysis of kinetic TGS incubations with fragments SZ4 and TA2 targeting the wildtype and mutant of Bcl-X_L. The samples were incubated at 37 °C for 6 hours and subjected to LC/MS-SIM analysis with gradient system 1 (see supporting information). A) Incubation sample containing fragments SZ4 and TA2 in absence of wildtype Bcl-X_L; B) Incubation sample containing fragments SZ4 and TA2 in presence of 2 μM wildtype Bcl-X_L; C) Incubation sample containing fragments SZ4 and TA2 in presence of 2 μM single mutant ^R139ABcl-X_L; D) Synthetic SZ4TA2 as the reference compound.

Figure 6

LC/MS-SIM analysis of kinetic TGS incubations with fragments SZ4 and TA2 targeting the wildtype and double mutant of Bcl-X_L. The samples were incubated at 37 °C for 6 hours and subjected to LC/MS-SIM analysis with gradient system 2 (see supporting information). A) Incubation sample containing fragments SZ4 and TA2 in absence of wildtype Bcl-X_L; B) Incubation sample containing fragments SZ4 and TA2 in presence of 2 μM wildtype Bcl-X_L; C) Incubation sample containing fragments SZ4 and TA2 in presence of 2 μM double mutant ^F131A,D133ABcl-X_L; D) Synthetic SZ4TA2 as the reference compound.