The tumor suppressor p53 is inactivated by mutation in about half of all tumors, making mutant p53 a prime target for cancer therapy. Missense mutations R248W, R273H and R248Q are included in the five most frequent p53 mutations and in total lead to more than 630 000 new diagnosed cases of tumor diseases in the world every year. These mutations, located at or near the protein-DNA interface, lead to p53 inactivation by loss of direct p53-DNA interactions, and by causing conformational changes in the protein, resulting in lowering its stability. Using small molecule drugs to reactivate mutant p53 is a promising therapeutic approach for treating a wide variety of human malignancies. The aim of this research was to study the biological properties of derivatives of aminobenzothiazole considered as selective small-molecule stabilizers of the p53(R248W), p53(R273H) and p53(R248Q) mutants.
Mutant recombinant proteins were expressed in E. coli BL21 (DE3) pLysS and purified using affinity and size exclusion chromatography. Determination of the affinity of small molecule compounds for mutant proteins was performed by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). The cytotoxicity of compounds was assessed on cell lines containing mutant p53 using the MTS test. The biological activity of the compounds was studied using cytofluorimetric analysis of the cell cycle and programmed cell death, quantitative real-time PCR analysis of the expression of p53-dependent genes, and immunoblotting of intracellular protein level changes.
Derivatives of aminobenzothiazole were found to stabilize p53 mutants in human cell lines, reactivating p53 transcriptional activity and the production of its target proteins. The compounds were more selective for p53-mutated than p53-wild type cells.
These studies will provide a more detailed understanding of the molecular mechanisms of reactivation of various forms of mutant p53, which, in turn, is of decisive importance in the development of new personalized anticancer drugs. The study was funded by RSF grant 22-24-20034 and strategically supported by Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030).
Kazan Federal University.
Russian Science Foundation.
All authors have declared no conflicts of interest.