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Fructose 1,6-bisphosphatase (FBPase) has attracted substantial interest as a target associated with cancer and type 2 diabetes. Herein, we found that disulfiram and its derivatives can potently inhibit FBPase by covalently binding to a new C128 allosteric site distinct from the original C128 site in APO FBPase. Further identification of the allosteric inhibition mechanism reveals that the covalent binding of a fragment of 214 will result in the movement of C128 and the dissociation of helix H4 (123− 128), which in turn allows S123 to more easily form new hydrogen bonds with K71 and D74 in helix H3 (69−72), thereby inhibiting FBPase activity. Notably, both disulfiram and 212 might moderately reduce blood glucose output in vivo. Therefore, our current findings not only identify a new covalent allosteric site of FBPase but also establish a structural foundation and provide a promising way for the design of covalent allosteric drugs for glucose reduction.

De novo drug design actively seeks to use sets of chemical rules for the fast and efficient identification of structurally new chemotypes with the desired set of biological properties. Fragment-based de novo design tools have been successfully applied in the discovery of noncovalent inhibitors. Nevertheless, these tools are rarely applied in the field of covalent inhibitor design. Herein, we present a new protocol, called Cov_FB3D, which involves the in silico assembly of potential novel covalent inhibitors by identifying the active fragments in the covalently binding site of the target protein. In this protocol, we propose a BA-SAMP strategy, which combines the noncovalent moiety score with the X-Score as the molecular mechanism (MM) level, and the covalent candidate score with the PM7 as the QM level. The synthetic accessibility of each suggested compound could be further evaluated with machine-learning-based synthetic complexity evaluation (SCScore). An in-depth test of this protocol against the crystal structures of 15 covalent complexes consisting of BTK inhibitors, KRAS inhibitors, EGFR inhibitors, EphB1 inhibitors, MAGL inhibitors, and MAPK inhibitors revealed that most of these inhibitors could be de novo reproduced from the fragments by Cov_FB3D. The binding modes of most generated reference poses could accurately reproduce the known binding mode of most of the reference covalent adduct in the binding site (RMSD ≤ 2 Å). In particular, most of these inhibitors were ranked in the top 2%, using the BA-SAMP strategy. Notably, the novel human ALDOA inhibitor (T1) with potent inhibitory activity (0.34 ± 0.03 μM) and greater synthetic accessibility was successfully de novo designed by this protocol. The positive results confirm the abilities of Cov_FB3D protocol.

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Combination drugs, characterized by high efficacy and few side effects, have received extensive attention from pharmaceutical companies and researchers for the treatment of complex diseases such as heart failure (HF). Traditional combination drug discovery depends on large-scale high-throughput experimental approaches that are time-consuming and costly. Herein we developed a novel, rapid, and potentially universal computer-guided combination drug-network-screening approach based on a set of databases and web services that are easy for individuals to obtain and operate, and we discovered for the first time that the menthol–allethrin combination screened by this approach exhibited a significant synergistic cardioprotective effect in vitro. Further mechanistic studies indicated that allethrin and menthol could synergistically block calcium channels. Allethrin bound to the central cavity of the voltage-dependent L-type calcium channel subunit alpha-1S (CACNA1S) lead to a conformational change in an allosteric site of CACNA1S, thereby enhancing the binding of menthol to this allosteric site. In summary, we reported a potentially universal computational approach to combination drug screening that has been used to discover a new combination of menthol–allethrin against HF in vitro, providing a new synergistic mechanism and prospective agent for HF treatment.