Phaleria macrocarpa Flavonoid as a Potent MMP-1 Inhibitor for Endometriosis Therapy: In silico Study

Introduction: Endometriosis is a gynaecological disorder in women and causes infertility. Several therapeutic were developed to reduce endometriosis cases, one of them was matrix metalloproteinase (MMP) inhibitor. This study investigated the potential activity of the flavonoids in Phaleria macrocarpa fruit as MMP1 inhibitors for endometriosis. Methods: In silico modelling was used in this study. Six flavonoid structures were retrieved from PubChem NCBI database. A targeted protein, MMP1 was taken out from Protein Data Bank with ID 1CGE and predicted the active sites. Six flavonoids and MMP1 was interacted by molecular docking using Molegro virtual Docker version 5 and analyzed using PyMol 2.2 and Discovery Studio version 21.1.1. Three-dimensional complex structure of flavonoids – MMP1 showed interaction in the same active sites and performed an amino acid residue Glu219 as catalytic site. Results: Six flavonoids of Phaleria macrocarpa were divided into two patterns and generated varied binding energy. Glycitin and Catechin 7-O-beta-D-xyloside showed low score of binding energy and depicted similar structure with four aromatic rings. 8-Prenylnaringenin performed lower binding energy than naringenin, eriodictyol, and 5-O-methylgenistein. Conclusion: In silico analysis suggested that six flavonoids compounds is potent as MMP1 inhibitor and might be interfered endometriosis pathophysiology. In vivo and in vivo investigations are required for further analysis.


INTRODUCTION
Endometriosis defines as endometrium at the outside of the uterine cavity. Endometriosis cases occur in around 5-20% of reproductive women [1][2][3][4][5][6]. Some therapeutic drugs, herbs, and treatments for endometriosis were reported in recent studies. Those treatments include surgery, laparoscopy, ablation, draining, and drug therapy [3,7,8]. Drug therapy was modeled due to the endometriosis mechanism and targeted protein. A matrix metalloproteinase is an essentials enzyme that regulates the pathophysiology of endometriosis. Matrix metalloproteinase has several types, involving collagenases, gelatinases, stromelysins, and matrilysins. Matrix metalloproteinase also played roles in tissue remodeling, angiogenesis, inflammation, cell proliferation and movement, embryogenesis, and ovulation. MMP activities were regulated by several factors: cytokines, hormones, and growth factors [9-11]. MMP levels increased in cell cancer and were used as a therapeutic target biomarker. Some MMPs was reported that overexpressed in ectopic tissue, peritoneal fluid, and sera of endometrium patients. compound was reported inhibiting proMMP-9 and interfering HT1080 cells migration. A synthetic compound, [3,4-dichloro-N-(1-methylbutyl)benzamide] inhibited MT1-MMP activity by binding the HPX domain in MCF7-β3/MT tumor xenograft. Hydroxylamine, hydroxamic acid, and carboxylic acid formed a complex with Zn2+ to reduce non-specific inhibition of MMPs [11][12][13]. Phaleria macrocarpa fruit is a traditional herb found in some Asia regions, especially Indonesia. Phaleria macrocarpa was traditionally used for reducing cholesterol, atherosclerosis, and diabetes mellitus [14][15][16][17]. Our previous study revealed that Phaleria macrocarpa contained six flavonoids, including eriodictyol, glycitin, 5-O-methylgenistein, catechin 7-O-beta-D-xyloside, 8prenylnaringenin, and naringenin and inhibited peritoneal damage in the endometriosis mice model [18]. A flavonoids compound has been reported high activity to reduce cancer proliferation.
However, flavonoids from Phaleria macrocarpa compounds as MMP1 inhibitors was limited information. Therefore, this study identified six flavonoid compounds identified in the Phaleria macrocarpa fruit as endometriosis therapy.

Data Analysis
Docking models were superimposed between interacted ligand with prepared MMP1 using PyMol 2.2. data was analyzed using Discovery Studio version 21.1.1. binding energy was generated from the summary of MolDock Score, MolDock Grid score, and Rerank score.

Ethics
This study used bioinformatic approach, which were not require any ethic clearance or informed consent.

RESULTS
According to the three -dimensional complex structure, six flavonoids bound to MMP1 in the same active sites (Fig. 1). Same active sites of six flavonoids in MMP1 protein were LEU181. GLU219 and VAL215 were identified on five complex, involved Naringenin -  complexes. Besides that, van der Waals force also performed in all complexes based on the two dimensional structure of complex (Fig. 1).
The binding energy might be correlated with the number of active sites of complex, type of interactions, and ligand structure. Glycitin and Catechin 7-O-beta-Dxyloside have similar structure with four aromatic rings, produced lower score of binding energy. 8-Prenylnaringenin showed lower binding energy than naringenin, Eriodictyol, and 5-O-methylgenistein. Eventhough, all of four flavonoids have similar pattern. Low energy indicated tight interactions of ligandprotein complex. The binding energy score was affected by type of interactions, number of hydrogen and hydrophobic interactions [21,22,[24][25][26].

CONCLUSION
Six flavonoids inhibited matrix metalloproteinase in several binding sites by two alternative inhibition mechanism. Naringenin, Catechin