Identification of putative allosteric inhibitors of BCKDK via virtual screening and biological evaluation

Abstract

Abnormal accumulation of branched-chain amino acids (BCAAs) can lead to metabolic diseases and cancers. Branched-chain α-keto acid dehydrogenase kinase (BCKDK) is a key negative regulator of BCAA catabolism, and targeting BCKDK provides a promising therapeutic approach for diseases caused by BCAA accumulation. Here, we screened PPHN and POAB as novel putative allosteric inhibitors by integrating allosteric binding site prediction, large-scale ligand database virtual screening, and bioactivity evaluation assays. Both of them showed a high binding affinity to BCKDK, with K_d values of 3.9 μM and 1.86 μM, respectively. In vivo experiments, the inhibitors demonstrated superior kinase inhibitory activity and notable antiproliferative and proapoptotic effects on diverse cancer cells. Finally, bulk RNA-seq analysis revealed that PPHN and POAB suppressed cell growth through a range of signalling pathways. Taken together, our findings highlight two novel BCKDK inhibitors as potent therapeutic candidates for metabolic diseases and cancers associated with BCAA dysfunctional metabolism.

Keywords:

• BCKDK
• allosteric inhibitors
• virtual screening
• BCAA metabolism
• metabolic diseases and cancer treatment

Acknowledgements

We would like to thank Mrs Xinshuang Zhang for Bulk RNA-seq library construction and sequencing, Mr Xiangyu Zhou for Bulk RNA-seq data analyses, Prof. Yulong Li (Peking University) for distributing the donor plasmids containing the BCKDK sequence, Prof. Zhe Zhang and Dr. Yuefeng Jiang (Peking University) for giving instruction in the expression and purification of BCKDK, Wenjuan Zhang (National Center for Protein Sciences-Beijing, PHOENIX Center) for providing technical support during the SPR experiments and Mr Hao Meng in molecular dynamics simulations associated with Figure 6 during revision.

Authors contributions

Li Zhang and Quanjun Yang conceived, designed and supervised the project. Li Zhang, Quanjun Yang and Chunqiong Li wrote the manuscript. Chunqiong Li led all the experiments and data analyses with the help of Hao Meng on molecular dynamics simulations.

Disclosure statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Additional information

Funding

This study was supported by the institutional funding to Li Zhang and the National Natural Science Foundation of China [No.: 81872494 to Quanjun Yang] and GHfund B [No. 20210702 to Chunqiong Li].