| Necroptosis is a different form of programmed cell death from apoptosis.It has been shown that programmed necrosis is strongly associated with many diseases such as neurodegenerative diseases,cardiovascular diseases,acute kidney injury and cancer,and receptor-interacting protein kinase 1(RIPK1)is a key kinase that mediates the signaling pathway of programmed necrosis.Therefore,the development of inhibitors targeting RIPK1 has become a hot topic in current drug design.In recent years,it has been shown that 4,5-dihydropyrazole derivatives can effectively inhibit the activity of RIPK1,but the exact mechanism of inhibition is not clear.The experimental approach allows to obtain the crystal structures of proteins and inhibitors,but it is hard to provide the conformational dynamics of proteins at the molecular level.Molecular dynamics simulation as a complementary method can overcome this limitation and is a powerful tool to study the dynamic behavior of biological macromolecules such as proteins and DNA.Therefore,in this paper,we investigated the binding modes of five 4,5-dihydropyrazole derivatives(1-4 and 2R)to RIPK1 by means of molecular docking,molecular dynamics simulations and binding free energy calculations,and explored the possible reasons for the differences in the inhibitory effects of several derivatives.The findings indicated that all five derivatives were stably present in the hydrophobic pocket formed by residues Leu70,Val75,Val76,Leu78,Leu129,Ala155,Leu159,Asp156,and Phe162.Binding free energy calculations revealed that hydrophobic interactions play an important role in the binding process of derivatives to proteins.Molecular dynamics simulations showed that both Asp156 and the carbonyl oxygen on the chirally identical S-enantiomer derivatives(1-4)formed stable hydrogen bonds,and this strong electrostatic interaction both solidified the position of the derivatives in the hydrophobic pocket of the protein and kept Asp156 in an inactive conformation.The R-enantiomer derivative(2R)with opposite chirality exhibits a larger flip in the binding posture.This flip causes the carbonyl oxygen on the derivative to point differently from the other derivatives and leads to the breakage of the hydrogen bond between the carbonyl oxygen and Asp156.At the same time,the different pointing of the carbonyl oxygen may also affect the position of Ser161,making it unable to form hydrogen bonds with Asp156.The result of both effects together leads to Asp156 not easily maintaining the inactive conformation,thus affecting the inhibitory ability of the derivatives.In addition,derivatives of the same S-enantiomer but with different 1-substituents(1-4)may exert non-competitive inhibitory effects through three pathways.The first pathway is for the derivatives to interact directly with ATP and influence its γ-phosphate position.Since the1-substituents of the derivatives are hydrophobic,they may push the hydrophilic phosphate end of ATP away from the ATP by surface tension,which would cause theγ-phosphate to move away from a suitable position for transfer and thus achieve an inhibitory effect.The second pathway is to affect the conformation of the residue Lys45,one of the catalytic triplets that stabilizes the α-and β-phosphate of ATP.changes in the position of Lys45 may affect the binding stability of ATP.the hydrophobicity,size,and charge of the 1-substituent can affect Lys45,thus The hydrophobicity,size,and charge of the 1-substituent can affect Lys45,thereby affecting the position of ATP to achieve inhibition.The third pathway is to affect the structure of the P-loop and β1 folded sheet of the protein,thus causing differences in the recognition of the substrate peptide.Sequence analysis of other serine kinases with similar structures to RIPK1,as well as its substrate polypeptides,revealed that the residues important for substrate polypeptide recognition are on the P-loop and β1folded sheet.The secondary structure analysis showed that the presence of the derivatives affected the secondary structure of the protein,with more significant effects on the P-loop and the β1 folded sheet.In summary,our results can provide clues for the design and development of such inhibitors in the future. |
