Theoretical Study On The Structure And Function Of LOXL2 And Its Inhibitors

Background and purpose From the beginning of new century,the incidence and mortality of cancer have risen rapidly worldwide and become a major killer that threatening human health.How to effectively prevent and cure cancer is a critical issue for all human being.There is no doubt that the future of cancer prevention and treatment task is a long way to go,and the situation is challenging.In recent years,there have been a number of successful cases of molecular targeted therapy,which is one of the hopes for human cancer treatment,therefore finding effective molecular targets is the critical step.In the last decade,more and more studies have focus on lysyl oxidase(LOX)family as molecular targets.LOXL2 is a member of lysyl oxidase family,in organisms,LOXL2 catalyzes the substrate collagen and elastin in the extracellular matrix,thereby facilitating the formation of the extracellular matrix cross-linked network.However,many studies show that LOXL2 has some functions in cells,which are not clear at present.According to the data,LOXL2 has been proposed by many laboratories around the world as an effective molecular target to intervene the progression of LOXL2 related tumors,or as an effective molecular marker to predict the progression of related cancers.Overall,LOXL2 is not only catalyze the oxidative deamination of the side chain of lysine residues of its target protein in extracellular,but also have the function of promoting tumor invasion and metastasis in intracellular.However,up to now,the function and molecular mechanism of LOXL2 in intracellular have not been clearly defined.Based on the full-length structure of LOXL2,this paper study about the structure and function of LOXL2 through computer simulation,and screened covalent inhibitors for functional disulfide bonds in LOXL2.These results can provide important theory for the treatment of LOXL2 related tumors.Research methods1)The missing domains of LOXL2 were predicted by Alpha Fold2,then the full-length structure of LOXL2 was established by combining with crystal structure.The stability of residues in LOXL2 were predicted by Fold X,Deep DDG,m CSM and Prem PS,and the key residues were screened.2)The redox potentials of all disulfide bonds in LOXL2 were calculated by non-equilibrium simulation method.3)The structure of glycosylated LOXL2 was established by CHARMM-GUI,then four systems(wide type LOXL2 and three glycosylated LOXL2)were simulated by enhanced sampling method(Gaussian accelerated molecular dynamics simulation)to analyze the potential mean force and hydrogen bond analysis of the systems.4)The small molecule database Life Chemicals and Enamine were used to screen for inhibitor of LOXL2,AMDET evaluations were carried out,then bio-assay verifications were performed on the potential inhibitors.Results1)74 key amino acid residues in LOXL2 play an important role in the stability of LOXL2,and the number of key amino acid residues in SRCR1,SRCR2,SRCR3,SRCR4 and the catalytic domain are 14,13,8,11 and 26,respectively.There were 24 Cys,11 Leu,9 Val,9 Trp,8 Phe and 13 other amino acid residues.More than 60% of the key residues are hydrophobic in LOXL2,which is consistent with the fact that hydrophobic interactions are the main force of protein folding.The amino acid residues that mainly affect the stability of LOXL2 are Cys,because in LOXL2,they are all crosslinked to form disulfide bonds,which play the stability role in protein.In addition,the structural distribution of these residues was analyzed: 16% on the alpha helix,38% on the beta fold,and the rest on the loop;2)LOXL2 contains a total of 17 pairs of disulfide bonds,of which 7 pairs of disulfide bonds have redox potentials range from-185 to-296 m V that have role in regulating protein function.They are disulfide bonds Cys573-Cys625,Cys579-Cys695,Cys657-Cys673 and Cys663-Cys685 in the catalytic domain.And Cys351-Cys414,Cys464-Cys530 and Cys477-Cys543 in scavenger receptor cysteine-rich domains.Study results shown that disulfide Cys351-Cys414 has allosteric function that can affect the metastasis and invasion of cancer cells.Disulfide bonds Cys464-Cys530,Cys573-Cys625 and Cys657-Cys673 have function related to enzyme catalytic activity.The remaining disulfide bonds serve to stabilize the structure of LOXL2;3)Results showed that the N288 glycosylation site contributed to LOXL2 degradation.And N455 glycosylation site is proposed to activate the proteolysis process of LOXL2,N644 glycosylation site tends to assist LOXL2 to promote the activity of amine oxidase.We also analysis the hydrogen bond between LOXL2 and glycans structure during the simulation,and screened out the key residues involved in glycosylation process of LOXL2;4)7908 small molecules were obtained by combining the small molecule database Life Chemicals and Enamine.Through covalent docking,empirical screening and ADMET evaluation,8 potential small molecule inhibitors were screened and verified experimentally.Experimental data show that small molecule F50972176 can effectively inhibit the mobility of esophageal squamous cell carcinoma cells,which is expected to become a therapy drug that target LOXL2 related cancers.ConclusionIn this study,the structure and function of LOXL2 were systematically studied by using computational simulation method,the key residues affecting the stability of LOXL2 and the functional disulfide bonds were determined,the effects of glycosylation on its structure and function were studied.Meanwhile,small molecule inhibitors were screened for the functional role of cysteine.Our studies expand the understanding of the relationship between structure and function of LOXL2,and provide therapy drug targeting LOXL2 related cancers.This study is of great significance to the mechanism research and inhibitor development of LOXL2.