Analysis of the potential of dehydroabietic acid as an inhibitor of the PI3K/AKT/mTOR signaling pathway based on computer simulation technology
The PI3K/AKT/mTOR signaling pathway plays an important role in the processes of cell proliferation, differentiation, and apoptosis, and is overactivated in many tumor cells. It has been found to promote the development of drug resistance in tumor cells. Inhibiting the activation of this signaling pathway can promote tumor cell apoptosis and restore tumor cell sensitivity to drugs. Therefore, the development of small molecule PI3K/AKT/mTOR signaling pathway inhibitors has become one of the research hotspots for anti-tumor drugs. Dehydroabietic acid (DHA) is an important tricyclic diterpenoid natural resin acid and one of the components of traditional Chinese medicine rosin. It is mainly obtained by separating rosin through disproportionation reaction. It has stable properties and a structure similar to steroid molecules, and has been widely used in the synthesis and development of fluorescent reagents and drug intermediates. Dehydroabietic acid and many of its derivatives exhibit excellent anti-tumor activity. Recent reports have shown that 1H benzo [d] imidazole derivatives of dehydroabietic acid have inhibitory effects on PI3K α and can downregulate the expression of phosphorylated AKT. Our research also found that some B-ring modified dehydroabietic acid derivatives can reduce the expression levels of phosphorylated PI3K, AKT, and mTOR, as well as the phosphorylation levels of their downstream effectors S6 and 4EBP1. Although different derivatives of dehydroabietic acid have shown certain inhibitory effects on the PI3K/AKT/mTOR signaling pathway proteins, it is currently unclear whether dehydroabietic acid itself has the corresponding inhibitory ability and further research is needed.

Most compounds exert their effects by binding to protein molecules, and their binding modes and abilities can be verified through methods such as protein crystal analysis and protein fluorescence quenching. However, the experimental period is long and the cost is high. Molecular docking technology is a means of using computers to perform theoretical calculations and predict the binding between compounds and receptor proteins. Based on the docking binding energy, the binding ability between compounds and proteins can be determined, and potential protein modulators can be screened; Preliminary mechanism research can also be conducted through the obtained docking configuration and other docking scores. In addition, understanding the drug properties and pharmacokinetic characteristics of compounds such as absorption, distribution, metabolism, excretion, and toxicity in the human body can determine whether they can be used as candidate compounds for clinical treatment. In order to reduce research costs, computer-aided programs have gradually been used for preliminary studies of drug likeness and pharmacokinetics.

Therefore, this study used molecular docking technology to predict the binding ability and binding mode of dehydroabietic acid to key proteins in this pathway, and verified the actual inhibition of dehydroabietic acid using protein immunoblotting. Furthermore, a network server was used to simulate drug like properties and pharmacokinetics, in order to preliminarily understand the potential of dehydroabietic acid as an inhibitor of the PI3K/AKT/mTOR signaling pathway and provide theoretical basis for further development of dehydroabietic acid.

The PI3K/AKT/mTOR signaling pathway plays an important role in the processes of cell proliferation, differentiation, and apoptosis, and is also one of the targets for cancer treatment. PI3K is mainly composed of catalytic subunit p110 and regulatory subunit p85. After phosphorylation activation, it can convert its substrate 3,4-phosphophosphatidylinositol (PIP2) into 3,4,5-triphosphate phosphatidylinositol (PIP3). PIP3 can bind to AKT and promote AKT phosphorylation through phosphoinositol dependent protein kinase 1 (PDK1). AKT activated by phosphorylation can directly or indirectly phosphorylate mTOR, thereby further promoting the phosphorylation of downstream effector proteins 4EBP1 and S6 to regulate intracellular biochemical activity.

This study is based on the continuous development of anticancer derivatives of dehydroabietic acid in recent years, some of which have been reported to have inhibitory effects on key proteins in the PI3K/AKT/mTOR signaling pathway. In order to investigate the effect of dehydroabietic acid as a raw material on the PI3K/AKT/mTOR signaling pathway and provide a basis for the development of dehydroabietic acid anticancer derivatives. The binding ability and binding mode of dehydroabietic acid to the ATP binding sites of key proteins PI3K, AKT, and mTOR in the pathway were predicted using molecular docking technology. The inhibitory effect of dehydroabietic acid on the pathway was examined using protein immunoblotting, and the potential of dehydroabietic acid as an oral drug was predicted through preliminary drug like and pharmacokinetic simulations.

The molecular docking results showed that dehydroabietic acid has a certain binding ability to ATP binding sites of various key proteins, with the weakest binding to AKT3 at a minimum binding energy of -6.16 kcal/mol and the strongest binding to AKT1 at a minimum binding energy of -8.04 kcal/mol. The binding ability of dehydroabietic acid to pathway proteins is weaker than that of the original ligand as a highly efficient inhibitor, which is similar to the dehydroabietic acid based PI3K/AKT/mTOR signaling pathway inhibitor molecule DBDA that we have synthesized. In ATP binding sites, the interaction residues between dehydroabietic acid and proteins are mostly hydrophobic residues. Except for Lys890 of PI3K δ, other key residues that play the most important role in ligand protein binding are also hydrophobic residues. Indicating that dehydroabietic acid may exert inhibitory effects by binding to the glandular gyrus of ATP binding sites. The interaction residues between various proteins and dehydroabietic acid overlap significantly with those of the original ligand, indicating that dehydroabietic acid may compete with ATP in a similar manner to the original ligand to inhibit protein action. In addition, structures without hydrogen bonds, such as dehydroabietic acid with PI3K α, AKT2, and AKT3, have higher binding energies than other structures with hydrogen bonds, indicating that hydrogen bonds play an important role in the stable binding of dehydroabietic acid to proteins. In the configuration of dehydroabietic acid binding, all hydrogen bonds are generated at the carboxyl position. However, there are multiple overlapping residues near the isopropyl and benzene rings that interact through hydrogen bonds in the original ligand. This feature suggests that when designing new inhibitors of the dehydroabietic acid based PI3K/AKT/mTOR signaling pathway, the hydrophilic ability of the carboxyl position can be enhanced, and hydrophilic groups can be introduced at the isopropyl and benzene ring positions, which may improve the binding ability of the new compound to proteins.

Protein immunoblotting experiments showed that after treatment with dehydroabietic acid, the expression of PI3K regulated subunit p85 in SCC9 cells was significantly reduced. The decrease in p85 content may hinder the generation of PI3K and become one of the factors that reduce the phosphorylation expression of downstream protein AKT in PI3K. The total protein of AKT and mTOR showed no significant changes compared to the blank group under different concentrations of dehydroabietic acid, but the expression of phosphorylated proteins was significantly reduced, indicating that the phosphorylation process of AKT and mTOR was inhibited. The decreased phosphorylation expression of 4EBP1 downstream of mTOR may be due to the fact that 4EBP1 can also be activated through the MEK/Erk signaling pathway. The downstream phosphorylation expression of another effector protein S6K1 regulated by mTOR was significantly reduced, which may be due to the inhibition of mTOR phosphorylation, leading to a decrease in S6K1 activation and thus reducing the phosphorylation reaction of S6. Overall, in SCC9 cells, the expression of the PI3K/AKT/mTOR signaling pathway was inhibited by dehydroabietic acid, which is consistent with the predicted results in molecular docking.

In the prediction of drug properties and pharmacokinetics, the Lipinski rule of five analysis of dehydroabietic acid shows that its molecular weight, LogP, rotational bond, hydrogen bond acceptor, and hydrogen bond donor values are all within the range required by this empirical rule. Pharmacokinetic prediction simulates the absorption, distribution, metabolism, excretion, and toxicity of dehydroabietic acid in vivo, and dehydroabietic acid has passed most of the tests, indicating that it may be able to exert drug effects well in vivo.

In summary, this study found that dehydroabietic acid itself may become a candidate therapeutic agent for inhibiting the PI3K/AKT/mTOR signaling pathway, in order to alleviate tumor cell resistance and anti-tumor effects, through molecular docking prediction, protein immunoblotting experiments, drug likeness testing, and pharmacokinetic prediction. Further modifications can be made to the structure of dehydroabietic acid to develop more effective inhibitors of the dehydroabietic acid based PI3K/AKT/mTOR signaling pathway.