Virtual Screening,Synthesis And Preliminary Evaluation Of Activity Of Aldosterone Synthase(CYP11B2) Inhibitors

BackgroundAldosterone synthase(CYP11B2),encoded by the CYP11B2 gene,is the rate-limiting enzyme in the last three steps of aldosterone synthesis in the human body.The continuous increase of aldosterone content in plasma is the direct cause of primary hyperaldosteronism,and one of the typical clinical manifestations is hypertension.Primary aldosteronism,referred to as primary aldosteronism,is not only a common factor in secondary hypertension,but also plays a very important role in the induction of various diseases,such as kidney damage,stroke,heart failure and atherosclerosis hardening,etc.Relevant studies have shown that aldosterone and its m RNA expression are found in human tissues such as cardiomyopathy and interstitial tissues,vascular smooth muscle cells,heart,brain and blood vessels,and it is also found that angiotensin II can promote its gene transcription.This finding confirms that aldosterone can be secreted autonomously by local tissues in addition to being governed by the renin-angiotensin-aldosterone system(RAAS).In the clinical use of antihypertensive drugs in combination,the phenomenon of "Aldosterone escape" occurs after long-term use of RAAS inhibitors.Excessive levels of aldosterone can cause damage to various target cells,target tissues and target organs.However,there is no aldosterone synthase inhibitor on the market.Therefore,computer-aided design methods are used to develop small molecule inhibitors targeting aldosterone synthase.It is very necessary to treat aldosterone-related diseases in order to discover small molecule compound inhibitors with clinical potential,and provide new ideas and methods for the development of aldosterone synthase(CYP11B2)inhibitor drugs.Methods and Results1.Virtual Screening and Molecular Dynamics Simulation of Small Molecule InhibitorsMolecular docking of the compound library was carried out using three docking methods,and the obtained results were then used to predict its pharmacokinetics properties through the ADMET module,and finally the molecular dynamics simulation(MD)of the protein-ligand complex was carried out.After molecular docking,it was found that all the obtained compounds had affinity with proteins mainly through hydrophobic interaction,and amino acid residues such as Trp116,Phe130,Trp260,Ala313,Phe487 and Ile488 were the main participants.After MD simulation,some compounds can reach an equilibrium state,indicating that the binding between receptors and ligands is very stable.After MD simulation,it was found that important amino acids such as Phe130,Phe487 and Ile488 can form a strong hydrophobic interaction with small molecules to promote the stable binding between the two,and the amino acid residue Arg110 can form hydrogen bonds with some compounds.The MM-PBSA method was used to calculate the contribution of key amino acids in the binding free energy.The contribution of key amino acids to binding free energy was calculated by the MM-PBSA method,and it was found that the key amino acid residues mainly contributed to the electrostatic interaction energy and van der Waals interaction energy,while the solvation energy was not conducive to the binding of small molecules to proteins.2.Synthesis of aldosterone synthase(CYP11B2)inhibitorsDesign the synthetic scheme for the screened compounds and synthesize candidate compounds under laboratory conditions according to the synthetic methods of the published literature.3.Preliminary biological activity evaluation of aldosterone synthase(CYP11B2)inhibitorsIn human normal bronchial cells HBE and mouse fibroblasts L929,the toxic effects of the compounds on the two cells were tested by MTT method at less than 200 μM.The experimental results showed that the compounds did not reach the half-inhibitory effective concentration on L929 cells,while some compounds High toxicity to HBE cells.Compounds were tested for their toxic effects in target cells,human adrenocortical carcinoma cells NCI-H295 R,and it was found that compounds 5,8,and 9 had much higher cell viability than other compounds at concentrations below 100 μM.Finally,the effects of some compounds on the secretion of aldosterone by target cells were detected by ELISA.The results showed that some compounds had better inhibitory effect,and the inhibitory effect of compound 5 was more prominent,which was consistent with the prediction results of MD simulation.Therefore,compound 5 is more expected to be the lead compound for follow-up research.ConclusionIn this project,9 compounds with high affinity for the target protein were screened by molecular docking and molecular dynamics simulation,and the compounds were synthesized on this basis.Then,the effect of the compound on the secretion of aldosterone by target cells was quantitatively detected by cytotoxicity test and ELISA method,and it was concluded that compound 5 could be used as a small molecule lead compound with the potential to inhibit aldosterone synthase in subsequent studies.It can lay the foundation for the discovery of new aldosterone synthase inhibitors.