{"paper_id":"779e28f7-74e3-4b35-8690-2eb6da40a4a2","body_text":"Endometriosis is a prevalent gynaecological disorder characterised by active lesions of endometrial\ntissue, including glandular cells and stroma, located\noutside the uterus that may affect fertility. The overall prevalence of this chronic inflammatory disease may be around 10-15% in reproductive age females\n( 1 ). The exact pathogenesis of endometriosis has not\nbeen completely determined; however, it is believed\nto be influenced by factors such as retrograde menstruation, benign metastasis, hormonal imbalance,\nimmune dysregulation, and genetic and epigenetic\nchanges ( 2 ,  3 ).\nGenes play key roles in female reproductive biology, and the role of the homeobox\n( HOX ) family genes has been shown from the formation of the uterus to\nendometrial receptivity ( 4 ).  HOX  genes express in the Müllerian duct during\nthe development of the female reproductive tract and are regulated by cyclic hormonal\nchanges during each reproductive cycle. This regulation is essential for the growth,\ndifferentiation, and implantation of the endometrium to facilitate successful embryo\nimplantation ( 4 ,  5 ). One of the cofactors for  HOX  genes is ISL LIM homeobox\n2 (ISL2). Our previous study results revealed significantly higher expression of the\n ISL2  gene in endometriotic tissues compared to normal endometrium\n( 6 ).\nOn the other hand, it has been reported that  ISL2  is overexpressed in\nglioma tumours and promotes the angiogenesis, proliferation, and invasion of human brain\nmicrovessel endothelial cells through the vascular endothelial growth factor A\n(VEGFA)-mediated ERK signalling pathway ( 7 ).\nAngiogenesis is a principal factor in the aberrant growth of endometrial tissue outside the\nuterus. VEGFA plays an essential role in promoting angiogenesis due to its higher expression\nin endometriosis patients compared to healthy women ( 8 ). Based on the results of our\nprevious study ( 9 ) and considering the association of  ISL2  with\n HOX  genes and  VEGF , as well as its role in promoting\nangiogenesis, it appears that inhibiting ISL2 could serve as an effective treatment for\nendometriosis.\nIn the current study, we use drug repurposing to target ISL2 in an attempt to identify the most suitable\nfood and drug administration (FDA)-approved drug for\nendometriosis treatment. To substantiate the selection\nof ISL2 as a viable target for this research, we evaluated its expression in the endometrial tissues of patients\nwith endometriosis compared to a control group using\nreverse transcription-polymerase chain reaction (RTPCR) and real-time PCR. Subsequently, computational\nmethods that included molecular dynamics (MD) simulations and molecular docking were utilised to identify\nthe optimal inhibitor for ISL2 from among the FDAapproved drugs.\n\nThe medical Ethics Committee at Royan Institute,\nTehran, Iran approved this study (IR.ACECR.ROYAN.\nREC.1400.157, March 2, 2022). Written informed consent was obtained from all participants, in accordance\nwith the guidelines of the Declaration of Helsinki 2000.\nParticipants’ consent was received before collecting tissue samples.\nThe participants were 20-40 years of age and exhibited\nno signs of endometrial hyperplasia, endometriosis, visible endometrial hyperplasia or neoplasia, inflammatory\ndiseases, myoma, polyps, or reproductive inflammatory\ndiseases. All women were in the secretory and proliferative phases of their menstrual cycles and had not\nreceived hormone therapy for a period of three months\n( Table S1 , See Supplementary Online Information at\n  www.ijfs.ir ). The women with endometriosis were diagnosed with stages III or IV disease, as classified by the\nrevised American Society for Reproductive Medicine\nclassification.\nThis computational study assessed ectopic, eutopic,\nand normal endometrial tissue samples. Ectopic (n=6)\nand eutopic (n=12) tissue samples were collected from\nwomen diagnosed with endometriosis who were undergoing laparoscopy at Royan Institute (Tehran, Iran). Ectopic endometrial lesions were obtained through laparoscopic procedures, while eutopic endometrium biopsy\nspecimens were collected from the uterine cavity in the\nsame procedure using Pipelle sampling. Normal endometrial tissues (control group) were obtained from eight\nhealthy women during diagnostic laparoscopy, and tissue samples were collected from the uterine cavity using\nPipelle sampling. All tissues were gently stirred in Dulbecco’s phosphate-buffered saline (Cat. No. 21600, Life\nTechnologies, USA) for 20 minutes to remove excess\nblood and debris.\nTotal RNA was extracted from the samples with TRIzol reagent (Cat. No. 15596, Life Technologies, USA)\naccording to the manufacturer’s protocol, followed\nby DNase I treatment (DNase I, RNase-free, Cat. No.\nEN0521, Thermo Fisher Scientific, USA) to eliminate\nany genomic DNA contamination. Subsequently, cDNA\nsynthesis was performed using a cDNA synthesis kit\n(ExcelRT™ Reverse Transcription Kit, SMOBIO Technology, Cat. No. RP1300, Taiwan) following the manufacturer’s protocol.\nThe expression of the target gene in all samples was assessed by quantitative real-time\nPCR (qRT-PCR). The SYBR Green q-PCR master mix (RealQ Plus 2x Master Mix Green with high\nROX™, Ampliqon, Cat. No: A323402, Denmark) was utilised for the qRTPCR reactions.  Table S2 \n(See Supplementary Online Information at  www.ijfs.ir ) lists the primer sets, lengths and\nproduct sizes. The primer was designed using PerlPrimer software (version 1.1.21) and\nverified by Gene Runner software (version 3.05, Informer Computer Terminals, USA). The\nqRT-PCR reaction consisted of three stages: a holding stage (10 minutes at 95°C for one\ncycle), a cycling stage (15 seconds at 95°C, followed by one minute at 60°C for 40\ncycles), and the melting curve stage (15 seconds at 95°C, one minute at 60°C, and 15\nseconds at 95°C). The relative expression of  ISL2  in the sample was\ncalculated using the 2 -ΔΔCt  technique with  GAPDH  as the\ninternal control.\nThe LIM domain is a conserved region within the\nISL2 protein, and its malfunction can lead to pathological effects such as tissue detachment, embryonic lethality, and cancer development ( 10 ). The LIM domain\nplays a significant role in forming protein complexes\nand binding ISL2 to promoter genes ( 11 ). Therefore,\ntargeting the LIM domain could be a strategy to inhibit\nISL2. In this study, the LIM domain of the ISL2 protein was selected as the target for molecular docking\nand MD simulations.\nThe crystal structure of the human LIM domain was\nnot available in the protein data bank; therefore, human and mouse LIM sequences were compared using the Basic Local Alignment Search Tool program\n(National Centre for Biotechnology, USA). We confirmed that the mouse LIM domain sequence has\n100% identity with the human LIM domain amino\nacid sequence. Consequently, the crystal structure\nof the LIM domain with the PDB code 3MMK was\ndownloaded from the PDB database ( 12 ). Next, water\nmolecules were eliminated from the coordinate data,\nand hydrogen atoms were added to optimise hydrogen\nbonding interactions. The GROMACS 5.1.4 package\nwas employed to perform energy minimisation of the\nprotein structure ( 13 ). The DrugBank database was\nsearched to obtain 2471 FDA-approved drugs ( 14 ). In\nthis stage, the pocket identification was performed using the CB-Dock web server ( 15 ). The cavity detection\nis an effective method to enhance molecular docking\nand a representation of this pocket is shown in Figure\n1. The cavity volume was 444 Å3, with a cavity centre located at coordinates 34, 38, 21. The cavity size\nwas 9, 13, 9 with a grid spacing of 0.375Å. In terms\nof ligand preparation, the PyRx tool was employed to\nmodify energy minimisation, and ligands were converted into the PDBQT format. The AutoDock Vina\ntool integrated in PyRx was utilised to conduct virtual\nscreening ( 16 ,  17 ) and the docking results were saved\nto evaluate binding affinities. Eventually, ligand-target bonding interactions were examined using Discovery Studio software.\nCavity detection using the CB-DOCK web server. The cavity volume\nis 444 Å3, with a cavity centre located at coordinates 34, 38, 21. The cavity\nsize is 9, 13, 9, with a grid spacing of 0.375Å.\nOnce all the FDA-approved drugs were docked with\nISL2, the medications that had a desirable delta G (∆G\n≤-8 kcal/mol) with ISL2 were chosen for further analyses. We aimed to identify drugs that form a more stable\ncomplex and have the strongest binding to the protein.\nTherefore, root mean square fluctuation (RMSF), root\nmean square deviation (RMSD), the number of hydrogen\nbonds, and the number of contacts between drugs and the\nprotein were calculated using MD simulations.\nThe GROMACS 5.1.4 software (Netherlands) was employed to accomplish all MD simulations in this study\n( 18 ) within the GROMOS 54a7 force field. The ATB\nserver was used to prepare ligand coordinates and topology. The system was neutralised by adding the necessary\nquantities of chloride ions and sodium using modelling\npackages. The periodic boundary conditions were implemented along the axial directions of the simulation box\nin each modelling framework. The SPC water simulation\nwas applied for systemic solubilisation ( 19 ).\nAll covalent bonds were restricted and Van der waals\ninteractions were cut off at 1.2 nm ( 20 ). Energy minimisation of the structures was performed with 50000 steps.\nMD simulations were conducted at a temperature of 310\nK for a duration of 100 nanoseconds ( 21 ,  22 ).\nThe Prism Graphpad was used for all statistical analyses (Insight Partners, version 9.5.1, USA). The data are\npresented as mean ± standard error of the mean (SEM)\nand 99% confidence (**; P<0.01). Gene expression in\nthe three tissue groups was compared using the KruskalWallis test.\n\nqRT-PCR analysis was conducted to evaluate the expression levels of the\n ISL2  gene in both eutopic and ectopic endometrial tissues. Our data\nrevealed a significant increase in  ISL2  expression levels (P=0.0036) in\nthe eutopic tissues of patients with endometriosis compared to the control and ectopic\ngroups. ISL2 expression in ectopic endometrial tissues also increased (P=0.0019) compared\nto the control group ( Fig .2 ).\nComparative expressions of  ISL2  in eutopic and ectopic endometrial tissues\nversus normal endometrial tissues.  ISL2  expression levels increased\nin eutopic (n=13) and ectopic (n=13) endometrial tissues in endometriosis patients\ncompared to normal endometrium from the control group (n=8, **; P<0.01). There\nare no significant changes in  ISL2  levels (ns, P>0.05) in eutopic\nversus ectopic tissues. Each of these tests were repeated twice.\n ISL2 ; ISL LIM homeobox 2.\nAs depicted in Figure 2, the  ISL2  expression showed a significant and\nseveral hundredfold increase in endometriotic tissues of patients compared to the control\ngroup. Therefore, we chose  ISL2  as the target for drug repurposing by\ncomputational methods.\nThe results obtained from computational methods were\nutilised to identify the most suitable FDA-approved drug\nto inhibit ISL2. Initially, we assessed the ability of 2471\nFDA-approved drugs to interact with the identified binding pocket of ISL2.\nThe preliminary docking results indicated that, out of the pool of 2471 FDA-approved\ndrugs, six drugs (Dactinomycin, Paritaprevir, Ivermectin, Ergotamine, Alectinib, and\nSimeprevir) exhibited the most favourable binding energy (∆G ≤-8 kcal.mol -1 )\nwith the identified binding pocket on ISL2. Two-dimensional and three-dimensional\nrepresentations of the complexes formed between these drugs and ISL2 are shown in the\n( Figes.S1-S6 , See Supplementary Online Information at  www.ijfs.ir ). As depicted, Ivermectin\nestablished the greatest number of hydrogen bonds with ISL2.\nNext, we used MD simulations to assess the stability\nof these complexes by calculating RMSD, RMSF, the\nnumber of hydrogen bonds, and the number of contacts\nbetween the drugs and the protein.\nOverall, the RMSD and RMSF analyses provide insights into the stability of the complexes (Figes .3 ,  4 ). As\nshown, the RMSD value converges at 80 ns. The RMSD\nvalue for Ergotamine was higher than the other complexes, which indicated that other complexes were more\nstable than this complex. RMSF analysis examines the\nfluctuation of various parts of the structure from their\nmean positions. Among the complexes, the Paritaprevir\ncomplex had the highest RMSF value, which indicated a\ngreater flexibility of the protein in this complex compared\nto the other complexes.\nRMSD graphs for protein in complexes of  ISL2 /drugs during 100 ns of the MD\nsimulation period. The RMSD value converges at 80 ns. The RMSD value for Ergotamine\nwas higher than the other complexes. ISL2; ISL LIM homeobox 2, RMSD; Root mean square\ndeviation, and MD: Molecular dynamics.\nRMSF graphs for protein in complexes of ISL2/drugs during 100 ns of the MD simulation period. The RMSF analysis examines the fluctuation of\nvarious parts of the structure from their mean positions. ISL2; ISL LIM homeobox 2, RMSF; Root mean square fluctuation, and MD: Molecular dynamics.\nThe most appropriate bonding between ISL2 and the\ndrugs selected in the simulation analyses can be evaluated\nby considering the number of hydrogen bonds and contacts. Our findings indicate that Ivermectin, Paritaprevir,\nand Dactinomycin formed the highest number of hydrogen bonds with ISL2 ( Fig .5 ). The Ivermectin and Paritaprevir complexes had the highest number of contacts\nwith ISL2 ( Fig .6 ).\nThe number of H-bonds between the drugs and ISL2. Ivermectin,\nParitaprevir, and Dactinomycin formed the highest number of hydrogen\nbonds with ISL2. ISL2; ISL LIM homeobox 2.\nThe number of contacts between the drugs and ISL2. Ivermectin\nand Paritaprevir formed the highest number of contacts with ISL2. ISL2;\nISL LIM homeobox 2.\n\nEndometriosis is a female health disorder for which the\nexact cause and definitive treatment remain uncertain.\nConsequently, extensive studies have been conducted to\nselect key genes implicated in the development of this disease and to identify the most effective drugs for its treatment ( 23 ,  24 ). Computational methods allow for identifying structures that have a high likelihood of binding to a\ndrug target, with the intent to reduce cost and time, and\nincrease accuracy. One of the strategies in drug discovery\nfor disease treatment is drug repurposing ( 25 ,  26 ). This approach, which has previously yielded numerous promising\ncandidates, may rationally repurpose drugs that have already undergone the necessary stages for FDA approval for\ntreatment of diseases that have uncertain treatments ( 27 ).\nRecent studies have been conducted to determine the role of ISL2 in cancer development. In\ntumours like oligodendrogliomas,  ISL2  expression showed a significant\nincrease. One potential pathway for disease development due to ISL2 involves the elevated\nexpression of angiopoietin 2 ( ANGPT2 ), which occurs through ISL2 binding to\nthe promoter region of the  ANGPT2  gene. Increased  ANGPT2 \nexpression results in cancer cell proliferation, invasion, and metastasis ( 28 ).\nAdditionally, angiopoietins are integral to angiogenesis, and play a crucial function in the\npathogenesis of endometriosis ( 29 - 31 ). These findings allowed researchers to consider ISL2\nas a critical target for drug design to treat these conditions.\nISL2 induces VEGF A expression, which is one of the pivotal angiogenesis factors that\npromotes tumour growth and development ( 32 ). VEGF A has a substantial impact on the\ndevelopment of endometriosis ( 33 ). Consequently, the increased expression of\n ISL2  could lead to heightened levels of  ANGPT2  and\n VEGF  expressions in endometriosis patients.\nWe observed a significant and several hundredfold increase in  ISL2  gene\nexpression in endometriotic tissues of endometriosis patients compared to the control group.\nBased on the outcomes derived from the initial step of this study, ISL2 was selected as the\ntarget for drug repurposing by computational methods.\nFirst, we observed the interactions of 2471 FDA-approved drugs with the identified binding\npocket within the LIM domain of ISL2. The preliminary docking results revealed that, among\nthese 2471 FDA-approved drugs, six (Dactinomycin, Paritaprevir, Ivermectin, Ergotamine,\nAlectinib, and Simeprevir) exhibited the most favourable binding energies (∆G ≤-8\nkcal.mol -1 ) with the identified pocket within the LIM domain of the ISL2\nprotein. Twodimensional representations of the docking results for these six drugs show that\nIvermectin formed the highest number of hydrogen bonds with the LIM domain\nMD simulations were then employed to assess stability of the six drug complexes. The results indicated that,\nfrom these six complexes, Ivermectin exhibited the lowest RMSD and RMSF, and highest number of hydrogen\nbonds and contacts, which indicated a higher potential for\nforming a stable complex with ISL2.\nOur findings indicate that Ivermectin is more likely to effectively inhibit ISL2.\nCurrently, numerous studies have used  in vivo  and  in vitro \nmethods to investigate the role of Ivermectin as a cancer treatment ( 34 - 36 ). Li and Zhan\n( 37 ) observed that this drug significantly suppressed the growth of various cancer cell\ntypes, including ovarian cancer. Ivermectin also appears to be an effective glioma treatment\n( 38 ). Although the precise molecular mechanism of its anti-cancer action has not been fully\nelucidated, the study outcomes suggest that its effectiveness might be linked to inhibition\nof ISL2.\nThe outcome of our research could improve diagnostic\nmethods and enable development of new therapies for endometriosis. Limitations of this study included challenges\nin sample collection, as the inclusion and exclusion criteria narrowed the pool of eligible participants.\n\nISL2 plays a role in enhancing angiogenesis and it has elevated expression in the endometrial tissues of patients diagnosed with endometriosis. Therefore, inhibiting this protein\ncould be efficacious in modulating endometriosis. Based on\nthe results obtained from the docking and molecular simulation conducted in this study, Dactinomycin, Paritaprevir,\nIvermectin, Ergotamine, Alectinib, and Simeprevir all have\nthe potential to inhibit ISL2. It is recommended that the efficacy of these drugs be assessed in laboratory environments\nand animal models to evaluate their impact.","source_license":"CC0","license_restricted":false}