A new role of low barrier hydrogen bond in mediating protein stability by small molecules

Abstract

Low barrier hydrogen bond (LBHB) is a special type of hydrogen bond which occurs where two heteroatoms with similar pKa values share a single proton resulting in an unusually strong and short hydrogen bond. LBHBs in protein play important roles in enzyme catalysis and maintaining protein structural integrity but its other biochemical roles are unknown. Here we report a novel function of LBHB in selectively inducing tubulin protein degradation. A tubulin inhibitor, 3-(3-Phenoxybenzyl) amino-β-carboline (PAC), promotes selective degradation of αβ-tubulin heterodimers by binding to the colchicine site of β-tubulin. Biochemical studies have revealed that PAC specifically destabilizes tubulin, making it prone to aggregation that then predisposes it to ubiquitinylation and then degradation. Structural activity analyses have indicated that the destabilization is mediated by a single hydrogen bond formed between the pyridine nitrogen of PAC and βGlu198, which is identified as a LBHB. In contrast, another two tubulin inhibitors only forming normal hydrogen bonds with βGlu198 exhibit no degradation effect. Thus, the LBHB accounts for the degradation. Most importantly, we screened for compounds capable of forming LBHB with βGlu198 and demonstrated that BML284, a Wnt signaling activator, also promotes tubulin heterodimers degradation in a PAC-like manner as expected. Our study has identified a novel approach for designing tubulin degraders, providing a unique example of LBHB function and suggests that designing small molecules to form LBHBs with protein residues resulting in the highly specific degradation of a target protein could be a new strategy for drug development.

Structure-Based Design and Synthesis of N-Substituted 3-Amino-β-Carboline Derivatives as Potent αβ-Tubulin Degradation Agents

“… a IC 50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SEM from the dose–response curves of at least three independent experiments. b 10a has been reported in ref . c 10b has been reported in refs and . d 10c has been reported in ref . e 10f has been reported in ref . …”

“… a Reagents and conditions: (a) K 2 CO 3 , DMF, iodoethane, 70 °C; (b) LiAlH 4 , THF, 0 °C; (c) MnO 2 , CH 2 Cl 2 , reflux; (d) (1) 3-phenoxyaniline, titanium tetraisopropanolate, THF, rt; (2) NaBH 4 , MeOH, rt; (e) SOCl 2 , reflux; (f) 3-phenoxyphenol, K 2 CO 3 , cat. KI, DMF, 70 °C. b Compound 13 has been reported in ref . …”

“…Up to now, only two tubulin-binding sites, the pironetin site and the seventh site, which were reported by our group, are completely located on α-tubulin. , Which of the abovementioned sites would compound 2 bind to? After extensive studies, we found that compound 2 was a colchicine-targeting inhibitor (interacting with β-tubulin rather than α-tubulin) that could promote tubulin degradation . As far as we know, some colchicine-targeted inhibitors could promote tubulin degradation by covalently binding to Cys239 of β-tubulin. − Interestingly, the cocrystal structure of 2 -tubulin indicated that no covalent bond was formed between 2 and tubulin (Figure ; PDB code: 7CDA).…”

“…Previously, we have verified that the hydrogen bond formed by N-2 of the A ring with E198 was responsible for tubulin degradation. The detailed mechanism of tubulin degradation has been illustrated in our abovementioned work . In order to analyze the structure–activity relationships and improve the antiproliferative activity of compound 2 , four modification strategies (modification of the A, B, and C rings as well as the linker) based on the 2 -tubulin complex have emerged to find new 3-amino-β-carboline derivatives as potent tubulin degradation agents (TDAs) for the treatment of cancer.…”

“…After extensive studies, we found that compound 2 was a colchicine-targeting inhibitor (interacting with β-tubulin rather than α-tubulin) that could promote tubulin degradation. 29 As far as we know, some colchicinetargeted inhibitors could promote tubulin degradation by covalently binding to Cys239 of β-tubulin. 30−35 Interestingly, the cocrystal structure of 2-tubulin indicated that no covalent bond was formed between 2 and tubulin (Figure 1; PDB code: 7CDA).…”