Cancer has always afflicted humans with increasing incidence and mortality, with an estimated 9.6 million deaths in 2018 and the number expected to rise to more than 14 million annually over the next decade[1, 2]. In 2020, there are 4.569 million new cancer cases and 3.003 million cancer deaths in China for the Cancer Today website (https://gco.iarc.fr/today). Cancer has become the leading cause of death in China and is a major public health problem.
The tumor suppressor protein p53 is known as the “guardian of the genome” and plays a central role in preventing tumor development through biological functions such as cell cycle arrest, DNA repair, cell senescence, and cell apoptosis. In about half of cancer cases, p53 is inactivated by mutations or deletions. In many other cancer cases, it is inactivated by overexpression of HDM2.
HDM2 (also known as MDM2) is the main negative regulator for p53. In normal cells, HDM2 and p53 form a negative feedback loop that maintains both p53 and HDM2 at very low levels. In this process, HDM2 is transcriptionally activated by p53, and HDM2 regulates p53 activity through three main mechanisms. (i) HDM2 inhibits p53 transcriptional activity by binding to the transactivation domain of p53 and thereby inhibits p53-mediated transactivation. (ii) HDM2 exports p53 out of the cell nucleus, leaving p53 inaccessible to targeted DNA and reducing its transcriptional ability. (iii) HDM2 functions as an E3 ubiquitin ligase that promotes the degradation of p53 proteasome[3, 6, 7]. Consequently, HDM2 is considered as an effective inhibitor of p53. HDM2 is abnormally up-regulated in human tumors through gene amplification, increased transcription level and enhanced translation, and the overexpression of HDM2 can impair the stability and activities of p53 and have carcinogenic effects. An analysis of nearly 4000 samples from tumors or xenografts from 28 tumor types shows that the frequency of HDM2 amplification in these human tumors was 7%. Therefore, the HDM2 as a drug target that the inhibition of HDM2 can restore the activity of p53.
The HDM2-p53 interaction has been confirmed to be the interaction between the first 120 N-terminal residues of HDM2 and 30 N-terminal residues of p53. Unlike many other protein-protein interfaces, the p53-HDM2 interface forms a narrow and deep cleft. In 1996, the first cocrystal structure of HDM2(residues 17 − 125) with p53(residues 15 − 29) was reported (PDB code 1YCR) . The interaction between HDM2 and p53 is mainly that the three hydrophobic residues of p53 (Phe19, Trp23 and Leu26) interact with the surface of HDM2 to form three hydrophobic cavities. The key residues of these three mimic ligands of p53 occupy the hydrophobic cleft of HDM2 in a “thumb − index − middle finger” conformation.. And some of the other ligands were designed for ubiquitin ligase. The binding modes of all ligands are highly conservative and occupying three hydrophobic groups with a strict arrangement.
In recent years, some highly effective and selective HDM2 inhibitors have been successfully discovered, such as RG7112, RG7338, SAR405838, HDM201, AMG-232 and CGM097 from Novartis . Other compounds such as MI-77301, MI-219 also have strong inhibitory effects on HDM2, as well. However, there are still no relevant medicines used in clinic.
Therefore, designing small molecule inhibitors to block HDM2-p53 interaction has become a possible cancer treatment strategy. In order to develop new HDM2 inhibitors, we have adopted the computer-aided drug design method in this work. Compared with the experimental method, it has become a reliable, cost-effective, and time-saving strategy that can be used to discover new lead compounds. In this study, considering the structural diversity and availability of compounds, the specs database (http://www.specs.net) was first optimized according to the Lipinski and Veber rules, then the specs database was used to virtual screening. Moreover, in order to further understand the binding mode of the HDM2 and the potential modulators, molecular dynamics simulations and MM-PBSA was carried out to calculate the binding free energy.