{"paper_id":"12ed752e-c875-4b82-8edc-e8071def32f9","body_text":"Indoleamine-2,3-Dioxygenase 1 Enzyme Inhibition: A Useful Target to Screen Chemicals for their Therapeutic Potential | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Indoleamine-2,3-Dioxygenase 1 Enzyme Inhibition: A Useful Target to Screen Chemicals for their Therapeutic Potential Gayatri Sawale, Santosh Ghuge, Sadhana Sathaye This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7074793/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Journal of Fluorescence → Version 1 posted 14 You are reading this latest preprint version Abstract Indoleamine 2,3-dioxygenase 1 (IDO1) is a key immunoregulatory enzyme that catalyzes the oxidative cleavage of L-tryptophan to N-formyl kynurenine, playing a critical role in immune tolerance and various pathological conditions, including cancer, autoimmune diseases, cataractogenesis, and neurodegenerative disorders. In this study, molecular docking was performed using a phytochemical library to identify compounds with strong binding affinity and favorable interactions within the IDO1 active site. Based on these in silico findings, selected compounds were further evaluated using a newly developed and cost-effective optimized fluorescence-based assay employing lens homogenate enzyme preparations to quantitatively assess IDO1 activity. Dose–response experiments revealed that several phytochemicals exhibited significant concentration-dependent inhibition of IDO1, with promising IC₅₀ values. The consistency between docking results and experimental inhibition supports the potential of these compounds as IDO1 inhibitors. This integrated in silico–in vitro approach provides a reliable platform for screening IDO1 modulators and identifies promising natural inhibitors for further development as therapeutics for IDO1-associated diseases. Enzyme activity Fluorescence Indoleamine-2 3-dioxygenase In-vitro model Biochemical Investigation Antioxidants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION Antioxidants are essential molecules that protect cells, including DNA and proteins, from oxidative damage caused by free radicals. Antioxidants may be used therapeutically to prevent diseases related to oxidative stress. Superoxide dismutase (SOD) and reduced glutathione (GSH) are two of the body’s inherent antioxidant defense mechanisms. Dietary antioxidants are also essential for maintaining overall health. Such antioxidant compounds act through several mechanisms involving hydrogen atom transfer, single electron transfer and chelation of transition metals [ 1 ]. One among these is the Kynurenine (KYN) pathway wherein the involvement of Indoleamine-2,3-dioxygenase 1 (IDO1) enzyme has been observed, thereby impacting health outcomes. Metabolites of the KYN pathway are linked to numerous health conditions and can cause oxidative damage [ 2 ]. The free-radical scavenging ability or antioxidant activity of phytochemicals may therefore impact cellular signaling pathways and play a crucial role in reducing the risk of diseases. The KYN pathway is a crucial metabolic pathway closely related to many diseases through degrading tryptophan (TRP) and generating toxic KYN metabolites [ 3 ]. IDO activity involved in the KYN pathway is essential for the diagnosis of multiple types of disease. For diseases involving abnormal IDO expression, studying IDO inhibitors can be valuable in reversing or stopping the development and treatment of related diseases. The activity of IDO1 directly depends on the content of its ferrous heme, which binds to dioxygen to enable its insertion into the indole ring of TRP [ 4 ]. Antioxidants have the ability to inactivate IDO through the reduction of either heme levels in the protein or protein expression [ 5 ]. Modern medicine is exploring the mechanisms of phytochemicals to optimize its pharmacological activity. The intricate workings of the processes and the unpredictable interactions between them make this type of research challenging. However, it is helpful to begin by demonstrating the modes of action of individual compounds. Tryptophan is an essential amino acid whose metabolites play key role in various physiological processes. TRP also has antioxidant properties that contribute to host cells’ defense against oxidative stress, participating in the synthesis of molecules with antioxidant activity. TRP is metabolized through two pathways that includes serotonin and KYN pathway capable of performing signaling functions [ 6 ]. IDO1 oxidizes L-tryptophan to N-formyl KYN (NFK) [ 7 ]. Mammals possess two forms of this enzyme, TDO (tryptophan-2,3-dioxygenase) and IDO (indoleamine-2,3-dioxygenase); the IDO gene family includes IDO1 and IDO2. However, compared to IDO1; TDO and IDO2 are poor producers of KYN [ 8 ]. The activation of IDO1, the resultant TRP depletion, and the formation of KYN and its metabolites have important biological effects, which, depending on the circumstance, can be beneficial or deleterious to the host. IDO is an integrated part of the immune system, acting as natural defense against various pathogens and inflammatory stimuli. Interferon-gamma is a potent inducer of IDO synthesis, leading to enhanced tryptophan metabolism. This process can limit viral replication by depleting the essential amino acid tryptophan, which is necessary for viral protein synthesis. This serves as protective strategy against infection and inflammation, but the accumulation of KYN can also have unintended damaging effects, as these metabolites can chemically modify proteins and be cytotoxic. The interplay between tryptophan depletion pathways and the effects of KYN metabolites is complex, with each cell type responding differently to these changes [ 9 ]. In the human lens, IDO activity is present mainly in the anterior epithelium. Several KYNs, such as KYN, 3-hydroxy KYN (3-OH KYN), and 3-hydroxy KYN glucoside, are present in the lens. They are thought to protect the retina by absorbing UV light and are referred to as UV filters. However, several studies show that KYNs are prone to deamination and oxidation. The deamination results in the formation of α,β-unsaturated ketones that chemically react and modify lens proteins. In the normal human lens, KYN modifications increase after 50 years of age, they may also reduce the chaperone function of α-crystallin, which is necessary for maintaining lens transparency [ 10 ]. Another mechanism for cataractogenesis is tryptophan, an amino acid in eye lenses in higher concentration, which forms N- Formyl KYN when it absorbs UV radiation. It may combine with the 3-OH KYN and riboflavin, which are lens photosensitizers; they absorb photons from the light and emit electrons. These electrons react with the molecular O 2 to form a superoxide radical, which reacts with the Na + K + ATPase pump in the eye and may cause swelling of the eye and lens opacification [ 11 , 12 ]. KYN also play a role in reactive oxygen species (ROS)-mediated crystallin modification and KYN-modified crystallin can generate ROS through photochemical reactions, weakened defenses against oxidative stress due to age or cataract formation could exacerbate lens protein modifications [ 13 ]. Although the antioxidant GSH can protect against the Kynurenilation of the lens by forming GSH adducts in in-vitro lens protein and in human lenses, the protection conferred by GSH decreases with age due to the reduced diffusion of GSH to the center of human lenses. Recent studies have demonstrated that expression of IDO1 is upregulated in cataractous lenses compared to healthy lenses, suggesting a potential role of this enzyme in cataract development. Inhibition of IDO1 activity has been found to reduce oxidative stress and inflammation in animal models of cataract, leading to improved lens clarity and visual function [ 14 ]. KYN pathway metabolites have been closely linked to the pathogenesis of several neurodegenerative diseases, including AD (Alzheimer’s Disease) and PD (Parkinson’s Disease) [ 15 ]. In AD and PD, the amyloid peptide upregulates IDO1 expression and increases the production of quinolinic acid in human macrophages and microglia [ 16 ]. These findings suggest that IDO1 inhibition could have therapeutic potential in neurodegenerative diseases by modulating immune response and protecting neural cells. Cancer cells utilize the IDO pathway to suppress the host’s immune response, facilitating the survival, invasion, and metastasis of malignant cells. IDO1 pathways significantly affect T cell response to antigenic stimulation. In tumors, activating L-TRP catabolism by L-TRP‐degrading enzyme IDO1 leads to a local generation of immunosuppressive KYN and L‐TRP depletion, resulting in a poor immune response. Inhibition of L‐TRP‐degrading enzymes might be beneficial and complementary for efficacy improvement in immunotherapy [ 16 , 17 ]. Depletion of TRP and increased KYN in the tumor microenvironment produces an inhibitory signal in T cells, preventing them from attacking the cancer cells. Inhibition of TRP degrading enzymes will eventually increase TRP levels and cause immune stimulation, complementing immunotherapy in cancer treatment. Hence, IDO inhibition relieves the immunoinhibitory microenvironment. Indoximod, a competitive inhibitor of the IDO1 enzyme has been widely used as a standard to optimize the assay methods for IDO1 inhibition [ 18 , 19 ]. In this study, we sought to optimize the assay method using Indoximod as selective standard inhibitor and evaluate a library of antioxidants compounds for their potential as IDO1 inhibitors. The antioxidants compounds selected for this study - gallic acid, quercetin, hydroxychavicol, phloretin, luteolin, ursolic acid and ethyl ferulate – were chosen based on ongoing research in our lab. This compounds have demonstrated potential in targeting the IDO1 pathway and possess known antioxidant properties. 2. MATERIALS AND METHODS Materials Tryptophan, Kynurenine, Indoximod, Gallic acid hydrate, Quercetin, Phloretin, Hydroxychavicol, Luteolin, Ursolic acid and Ethyl ferulate were purchased from Zeta Scientific, India. Reagents required for the assay were purchased from Molychem and Sisco Chem, India. Fresh wild goat eyeballs were procured from the Abattoir (Deonar, Govandi, Mumbai). UV-light bulb (Osram, Make Germany) was used to UV-induced IDO1 overexpression or cataract formation in the lens. Microcentrifuge equipment from Remi Electronics, India and a multimode plate reader from SYNERGY H1 BioTek Instruments, Inc. USA with Gen5 software were used. Black 96 well plate were purchased from Genaxy Scientific Pvt. Ltd. (Cat. No. GEN-0224). Schrodinger Maestro 2023 and GraphPad Prism 10 were used to dock and analyze the data, respectively. Methods Molecular Docking with IDO1 Detailed Network Pharmacology Analysis of gallic acid was studied earlier by M.R. Beg [ 20 ] from our lab, with one of the identified key target IDO1, in continuation we proceed with IDO1 in-silico and in-vitro analysis of gallic acid along with other potential antioxidants compounds. IDO1 protein was selected as the target macromolecule to dock with the ligands. 5EK4 protein in ‘.pdb’ format was downloaded from the website rcsb.org. The ligand or structure of compounds to be evaluated was downloaded from PubChem in 3D ‘.sdf’ format [ 21 ]. Schrodinger Maestro 2023-1 software was used for docking [ 22 ]. Protein and ligands were prepared using Protein Preparation and LigPrep task options. Docking was done with the receptor grid generated on the co-crystallized NLG919 ligand site on the protein including heme with all tautomer of ligands generated. Standard precision, flexible ligand sampling and other default settings were applied for ligand docking. A low (negative) energy glide score (gscore) in Kcal/mol indicates a stable system and thus a likely binding interaction. IDO1 Crude Protein Working Solution Preparation : The generation of ROS is thought to be the mechanism through which UV-B radiation-induced damage to the eye lens is mediated. IDO1 is well-documented as the principal indoleamine 2,3-dioxygenase in ocular tissues, including the lens and cornea, and is upregulated by UV exposure. Studies show that IDO1 is consistently expressed at low levels in corneal and lens tissues normally, but its expression and activity significantly increase after UV irradiation. This upregulation is linked to a protective mechanism against UV-induced oxidative stress and apoptosis in ocular cells. IDO2 is expressed at much lower levels than IDO1 in most tissues and is considered to have a minor physiological role. TDO (tryptophan 2,3-dioxygenase) is primarily a hepatic enzyme and is not typically expressed at significant levels in extrahepatic tissues like the eye lens [ 3 , 23 , 24 ]. Hence, we aimed to extract specific enzyme IDO1 from the lenses. Crude protein extraction was carried out from the lens homogenate [ 25 ]. Fresh goat lens were transported to the Pharmacology Research Lab II, DPST, ICT Mumbai, under refrigeration conditions (2–8°C). Lens were carefully removed by extracapsular extraction from eyeballs and then washed with normal saline solution. Lens were then transferred to an artificial aqueous humor solution to mimic the eye environment and exposed to an artificial UVR source (Osram Ultra Vitalux 300 W 230 V E27) at 13 cm distance for 20 min at room temperature. The resulting opaque lenses were expected to demonstrate overexpression of IDO1. The IDO1 overexpressed lenses were homogenized in lysis buffer of 0.2M Tris-base (pH 7.8) containing 25mM EDTA with 10% w/v homogenate utilizing hand homogenizer and centrifuged at 10000 rpm, 4 o C for 20 min. Supernatants were pulled with 1:1 100% glycerol, and various 1ml aliquots were prepared and stored at -20 o C for experimental use, we called this as enzyme working solution (as shown in Fig. 1 ). Total protein content was estimated according to ‘Bradford’s Method’ [ 26 ], with some modifications and bovine serum albumin (BSA) was used as a standard. Methods for Assessment of IDO Activity Various techniques for assessing the activity of IDO1 used to screen IDO1 inhibitors were reviewed by literature. Mainly, HPLC, fluorescence detection, cell-based assay, NFK GreenScreen™, and absorbance assay techniques have been reported for assessing the activity of IDO1. Each method possesses particular advantages, disadvantages, and cost-differences [ 27 ]. Among all of these, the fluorescence detection method is a novel and sensitive assay for the determination of IDO1 enzymatic activity, which can achieve high throughput screening (HTS) for IDO1 inhibitors. The IDO1 fluorescence assay introduced in 2006 measures the fluorescence of KYN produced from hydrolysis of NFK in sodium hydroxide [ 28 ]. Tomek et al. [ 29 ] described a fluorescence assay used for measuring IDO1 activity. In place of measuring KYN, it detects the in-situ formation of NFK-derived fluorophores (PIP-THQ) with an excitation wavelength of 400 nm and emission wavelength of 500 nm. For the new fluorescence assay, the former steps are performed as described by Takikawa et al. [ 30 ] for an absorbance assay. We have adopted this method for our study, and the optimization is familiar with the available method of fluorescence detection. The fluorescence intensity of the PIP-THQ formed is directly related to the amount of enzyme activity. Piperidine Tetrahydro quinolone (PIP-THQ) is not formed directly from PIP-NFK. While the optimal reaction temperature and time for maximum formation of PIP-THQ are 65°C for 20 min [ 31 ]. Fluorescence-based IDO1 Assay Assay was performed in a black polystyrene 96-well plate. The 50 µl of reaction assay medium containing an in-well concentration of 50 mM potassium phosphate buffer (PBS) (pH 6.5), 10 mM ascorbic acid neutralized with equimolar NaOH, 100 µg/mL catalase, 5 mM methylene blue and L-Tryptophan (or only assay mixture for blank) were added in the well. 10 µl standard or test substance (distilled water for blank) and 60 µl Enzyme working solution was added to the plate. Reaction was carried out at incubation of 37 0 C for 20 hours. After incubation, 30 µl Piperidine (PIP) was added with 200 mM in-well concentration and the plate was covered and heated at 65°C for 20 min. Plate was left at room temperature for 60 min, post-incubation the fluorescence intensity of PIP-THQ was measured at the 400 nm excitation wavelength and 500 nm emission wavelengths (25°C, 7 mm measurement height, 100 gain), using Spectro-fluorimeter microplate reader (BioTek, Synergy H1). N-formyl Kynurenine (Product) standard curve N-formyl KYN (NFK) was synthesized by formylation of 1 mg of L-KYN (5mM) in 2.5 µl of formic acid, make up to 1ml using assay mixture. After 2 hrs, the NFK was formed, which was further used in the standard reaction for the IDO1 assay [ 32 ]. The standard graph of absolute absorbance vs. NFK concentration was plotted to calculate the unknown concentration of NFK formed due to the reaction of lens homogenate enzyme solution. Validation of IDO1 Inhibition by Selective Inhibitor One unit of IDO1 activity is the amount of enzyme that generates 1 µmole of detected N-formyl Kynurenine per minute by oxidative metabolism of 1 µmole L-tryptophan at 37 °C. IDO1 activity is expressed as µmole of detected NFK formed within 1 minute by oxidative metabolism of 1 µmole of L-TRP at 37°C. ∆V/min was calculated by (V1-V0)/time, where V0 is the initial velocity, and V1 is the velocity after starting of the reaction. The amount of NFK produced in each reaction was calculated from the NFK standard curve. Indoximod is the known selective IDO1 inhibitor, if N-formyl Kynurenine production drops significantly in the presence of the Indoximod, this confirms that IDO1 is a major enzyme responsible for the conversion of L-TRP to NFK. IDO1 Inhibition Assay (IC 50 ) For the inhibition assay, 50 µL of 2 mM of tryptophan was used in the assay mixture, and 10 µL of different concentrations of standard & test compounds and 60 µL of enzyme solution were added to each well. Further reaction was carried out for 20 hrs, and post-incubation PIP was added. The plate was heated at 65°C for 20 min. Plate was left at room temperature for 60 min, post-incubation the fluorescence intensity of PIP-THQ was measured. Inhibition expressed as a percentage of inhibited PIP-NFK complex (PIP-THQ) was calculated as (A/B X 100), where A and B are absorbances in the presence and absence of inhibitor, respectively. 3. RESULTS AND DISCUSSIONS In-silico Molecular Docking Study In-silico molecular docking study has shown the interaction of the ligand compounds with the protein IDO1 [ 33 ]. NLG919 (co-crystallized ligand with protein 5EK4), Indoximod (selective inhibitor of IDO1), antioxidants test compounds as Quercetin, Gallic acid, Hydroxychavicol, Phloretin, Luteolin, Ursolic acid and Ethyl Ferulate were taken as library of ligands. Table 1 Maestro Glide Docking Score and Interactions Ligands Docking score (Kcal/mol) Ligands Interaction with receptor amino acids π-π aromatic ring hydrogen bond π-cation NLG919 -7.30168 TYR126, HEM501 N/A HEM501 Indoximod -7.06598 TYR126, HEM501 N/A HEM501 Luteolin -7.30319 TYR126, HEM501 SER167, HEM501 N/A Quercetin -6.61408 HEM501 SER167, HEM501 N/A Phloretin -6.39428 HEM501 SER167, HEM501 N/A Gallic acid -5.83717 HEM501 SER167 N/A Hydroxychavicol -5.53807 TYR126, HEM501 SER167 N/A Ethyl Ferulate -5.06422 HEM501 SER167 N/A Ursolic acid -4.24518 N/A GLY236 N/A The obtained molecular docking data, represented in Table 1 , indicated that IDO1 enzyme has an affinity towards all the tested compounds [ 34 ]. The negative docking score value is directly proportional to the affinity of the protein to compound. As known from the literature, NLG919 a co-crystallized ligand of protein and Indoximod a selective competitive inhibitor of IDO1, its low docking score and common receptor amino acid interactions validated these results. Out of tested antioxidants, Luteolin has shown highest docking score of -7.303 comparable with the ligand NLG919. Other compounds have also shown good docking score, with Ursolic acid with least affinity of -4.245 docking score. Receptor amino acids HEM501, TYR126 and SER167 are mainly involved in the all the ligand interactions, with GLY236 interaction with only Ursolic acid. IDO1 Enzyme Activity As per BSA standard curve, the Goat lens homogenate solution contained total protein content of 74.8989 µg/ml. An assay for the standard N-formyl Kynurenine curve was performed (Fig. 3 ) and enzyme activity was calculated for lens enzyme solutions. The goat lens showed enzyme activity of 0.0860 U and 22.05 U/mg specific activity. The kinetics study for enzyme solutions was performed, by Michaelis-Menton K M and V Max value is found to be 33.01 µM and 16516, respectively and by Lineweaver-Burk K M and V Max value is found to be 31.13 µM and 16316, respectively (Fig. 4 ). IDO1 Enzyme Inhibition IDO1 enzyme inhibition for all the standard and test compounds was performed to calculate IC 50 values and the final results are shown in Fig. 5 . Graph analysis for IC 50 was performed using second-order quadratic non-linear regression method in GraphPad Prism. Standard drug Indoximod and other test compounds Quercetin, Gallic acid, Luteolin, Phloretin, and Hydroxychavicol exhibited IDO1 Inhibition activity. From Fig. 5 , the standard IDO1 inhibitor has shown an excellent IC 50 value of 0.329 mM, which is the most potent among other test compounds. Quercetin and Luteolin demonstrated IC 50 comparable to the standard. Gallic acid and Phloretin exhibited good IC 50 value. A significant IDO1 inhibition was exhibited by the Hydroxychavicol. However, it was less potent among other compounds. The values showed that Quercetin, Luteolin, Gallic acid, Phloretin, and Hydroxychavicol influenced IDO1 inhibition in the KYN pathway. Compounds Ethyl Ferulate and Ursolic acid, which were involved in docking studies, did not show any promising IDO1 enzyme inhibition via our optimized in-vitro method. 4. CONCLUSION The IDO1 overexpression has been linked to several disorders, which lead to the need for easy quantification of IDO1 inhibitors. The optimized assay used crude lens homogenate enzyme solution, that can be used for screening the IDO1 inhibitory activity of many natural and synthetic compounds. It has shown good potential to be incorporated into assays instead of pure enzyme, enabling sensitive and cost-effective enzyme assays. Inhibition by the selective inhibitor Indoximod, shows that this method for evaluating IDO1 is validated. The in-silico docking score, in-vitro IC 50 value of the compounds enable us to interpret the therapeutic effects of these compounds as seen in in-house experiments from our lab. The IDO1 inhibition was demonstrated by the well-researched antioxidants phloretin, luteolin, gallic acid and quercetin. Phloretin and luteolin is being studied as potential adjuvants’ in treating neurodegenerative disorders using in-vivo models in our lab [ 35 ]. IDO1 inhibition activity of gallic acid and quercetin was done to explore it as a target for its probable activity to treat cataract [ 20 ]. Ocular formulation containing these antioxidants has also shown good ex-vivo and in-vivo anti-cataract activity as per granted patent from our lab [ 36 ]. Hydroxychavicol is being investigated as a potential anticancer agent, exhibiting IDO1 inhibition [ 37 ]. The overexpression of this enzyme is implicated in cataractogenesis, carcinogenesis, inflammation, and neuronal degeneration [ 38 ]. In conclusion, the therapeutic potential of IDO1 inhibitors represents a promising avenue for treating various disorders, offering significant prospects for more effective and targeted therapies in the future. Developing combination therapies that include IDO1 inhibitor alongside other pharmacological agents could maximize therapeutic efficacy and minimize resistance mechanism, leading to more comprehensive and effective treatment approach. Abbreviations IDO: Indoleamine-2,3-dioxygenase SOD: Superoxide dismutase GSH: Glutathione TRP: Tryptophan KYN: Kynurenine NFK: N-formyl Kynurenine TDO: Tryptophan-2, 3-dioxygenase ROS: Reactive Oxygen Species AD: Alzheimer's Disease PD: Parkinson's Disease UV: Ultraviolet EDTA: Ethylenediaminetetraacetic Acid BSA: Bovine Serum Albumin HPLC: High pressure liquid chromatography HTS: High throughput screening PIP-THQ: Piperidine Tetrahydro Quinolone PIP: Piperidine AM: Assay Medium PBS: Phosphate buffer saline TYR: Tyrosine HEM: Heme SER: Serine GLY: Glycine Declarations Conflict of Interest The authors have no relevant financial or non-financial interest to disclose. Ethics Approval The CCSEA in 110th meeting has decided that the IAECs and CCSEA has no objection for studies conducted on slaughter house samples and no ethical approval is required. Fresh wild goat eyeballs were procured from the Abattoir (Deonar, Govandi, Mumbai), with due consideration given to obtaining approval letters from both the institute and the abattoir. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by G.S. and S.G. The first draft of the manuscript was written by G.S. All authors commented on previous versions of the manuscript and reviewed by S.S. All authors read and approved the final manuscript. Acknowledgement The authors would like to acknowledge Dr. Shamlan Reshamwala (Centre of Energy Biosciences, Institute of Chemical Technology, Mumbai) his intellectual contribution to analyze the enzyme assay results. The authors gratefully acknowledge the use of facilities of Institute of Chemical Technology, Mumbai. 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Anal Biochem 349:96–102. https://doi.org/10.1016/j.ab.2005.10.039 Tomek P, Palmer BD, Flanagan JU, Fung S-PS, Bridewell DJA, Jamie JF, Ching L-M (2013) Formation of an N-formylkynurenine-derived fluorophore and its use for measuring indoleamine 2,3-dioxygenase 1 activity. Anal Bioanal Chem 405:2515–2524. https://doi.org/10.1007/s00216-012-6650-y Takikawa O, Kuroiwa T, Yamazaki F, Kido R (1988) Mechanism of interferon-gamma action. Characterization of indoleamine 2,3-dioxygenase in cultured human cells induced by interferon-gamma and evaluation of the enzyme-mediated tryptophan degradation in its anticellular activity. J Biol Chem 263:2041–2048 Tomek P, Palmer BD, Kendall JD, Flanagan JU, Ching L-M (2015) Formation of fluorophores from the kynurenine pathway metabolite N-formylkynurenine and cyclic amines involves transamidation and carbon-carbon bond formation at the 2-position of the amine, Biochim. Biophys Acta 1850:1772–1780. https://doi.org/10.1016/j.bbagen.2015.04.007 Simat T, Meyer K, Steinhart H (1994) Synthesis and analysis of oxidation and carbonyl condensation compounds of tryptophan. J Chromatogr A 661:93–99. https://doi.org/10.1016/0021-9673(94)85180-8 Bank RPD, RCSB PDB – 5EK4 (2023) accessed December 6, : Crystal structure of the indoleamine 2,3-dioxygenagse 1 (IDO1) complexed with NLG919 analogue, (n.d.). https://www.rcsb.org/structure/5EK4 Rashid M, Rafique H, Roshan S, Shamas S, Iqbal Z, Ashraf Z, Abbas Q, Hassan M, Qureshi ZUR, Asad MHHB (2020) Enzyme Inhibitory Kinetics and Molecular Docking Studies of Halo-Substituted Mixed Ester/Amide-Based Derivatives as Jack Bean Urease Inhibitors, BioMed Res. Int. (2020) 8867407. https://doi.org/10.1155/2020/8867407 Deshpande RD, Shah DS, Gurram S, Jha DK, Batabyal P, Amin PD, Sathaye S (2023) Formulation, characterization, pharmacokinetics and antioxidant activity of phloretin oral granules. Int J Pharm 645:123386. https://doi.org/10.1016/j.ijpharm.2023.123386 Sathaye S, OCULAR FORMULATION FOR DRY EYES (2025) AND CATARACT AND PREPARATION METHOD THEREOF, 558968 Mohamad NA, Rahman AA (2023) Sheikh Abdul Kadir, Hydroxychavicol as a potential anticancer agent (Review). Oncol Lett 25:34. https://doi.org/10.3892/ol.2022.13620 Zádori D, Veres G, Szalárdy L, Klivényi P, Fülöp F, Toldi J, Vécsei L (2016) Inhibitors of the kynurenine pathway as neurotherapeutics: a patent review (2012–2015), Expert Opin. Ther Pat 26:815–832. https://doi.org/10.1080/13543776.2016.1189531 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 07 Oct, 2025 Read the published version in Journal of Fluorescence → Version 1 posted Editorial decision: Revision requested 08 Aug, 2025 Reviews received at journal 04 Aug, 2025 Reviews received at journal 02 Aug, 2025 Reviews received at journal 29 Jul, 2025 Reviewers agreed at journal 28 Jul, 2025 Reviews received at journal 26 Jul, 2025 Reviewers agreed at journal 24 Jul, 2025 Reviewers agreed at journal 23 Jul, 2025 Reviewers agreed at journal 23 Jul, 2025 Reviewers agreed at journal 23 Jul, 2025 Reviewers invited by journal 23 Jul, 2025 Editor assigned by journal 11 Jul, 2025 Submission checks completed at journal 11 Jul, 2025 First submitted to journal 08 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7074793\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":490206128,\"identity\":\"ad64a2a7-deef-44e2-9bda-4e9abbf3c09e\",\"order_by\":0,\"name\":\"Gayatri Sawale\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Institute of Chemical Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Gayatri\",\"middleName\":\"\",\"lastName\":\"Sawale\",\"suffix\":\"\"},{\"id\":490206129,\"identity\":\"dcaf812d-1140-4009-8552-0983bc394ab9\",\"order_by\":1,\"name\":\"Santosh 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Preparation\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7074793/v1/d67d916d3210cd4db616cee0.png\"},{\"id\":87588250,\"identity\":\"bb0b8b31-a702-421c-82e6-cddb2acab113\",\"added_by\":\"auto\",\"created_at\":\"2025-07-25 14:16:08\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":95100,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eIDO1 Fluorescence Assay Method\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7074793/v1/c34615d1dab4497311918e38.png\"},{\"id\":87588256,\"identity\":\"6c01bf88-defb-4c2f-a616-ba9278306d41\",\"added_by\":\"auto\",\"created_at\":\"2025-07-25 14:16:08\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":91052,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eN-formyl Kynurenine Standard Curve\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7074793/v1/cce7ef51f65ef5b1a3984da0.png\"},{\"id\":87588252,\"identity\":\"1ea33909-a5d2-4cf6-8162-950c883af3d1\",\"added_by\":\"auto\",\"created_at\":\"2025-07-25 14:16:08\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":23164,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eLens IDO1 Enzyme Kinetics Graphs\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7074793/v1/55cf3c5b0cab7b11aac6ed03.png\"},{\"id\":87588258,\"identity\":\"9ecd4fbc-7ab6-41eb-9a29-0d0d89b9fcfd\",\"added_by\":\"auto\",\"created_at\":\"2025-07-25 14:16:08\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":236135,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eGraph of Compounds for % Inhibition of IDO1\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7074793/v1/0aed263595e901f2d2544761.png\"},{\"id\":93419789,\"identity\":\"b0c480cc-e797-452b-8fc6-c7538435bb3d\",\"added_by\":\"auto\",\"created_at\":\"2025-10-13 16:07:40\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1100254,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7074793/v1/13c9c91a-b4d0-4757-93b8-e4b2ac6ffd55.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Indoleamine-2,3-Dioxygenase 1 Enzyme Inhibition: A Useful Target to Screen Chemicals for their Therapeutic Potential\",\"fulltext\":[{\"header\":\"1. INTRODUCTION\",\"content\":\"\\u003cp\\u003eAntioxidants are essential molecules that protect cells, including DNA and proteins, from oxidative damage caused by free radicals. Antioxidants may be used therapeutically to prevent diseases related to oxidative stress. Superoxide dismutase (SOD) and reduced glutathione (GSH) are two of the body\\u0026rsquo;s inherent antioxidant defense mechanisms. Dietary antioxidants are also essential for maintaining overall health. Such antioxidant compounds act through several mechanisms involving hydrogen atom transfer, single electron transfer and chelation of transition metals [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. One among these is the Kynurenine (KYN) pathway wherein the involvement of Indoleamine-2,3-dioxygenase 1 (IDO1) enzyme has been observed, thereby impacting health outcomes. Metabolites of the KYN pathway are linked to numerous health conditions and can cause oxidative damage [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. The free-radical scavenging ability or antioxidant activity of phytochemicals may therefore impact cellular signaling pathways and play a crucial role in reducing the risk of diseases. The KYN pathway is a crucial metabolic pathway closely related to many diseases through degrading tryptophan (TRP) and generating toxic KYN metabolites [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. IDO activity involved in the KYN pathway is essential for the diagnosis of multiple types of disease. For diseases involving abnormal IDO expression, studying IDO inhibitors can be valuable in reversing or stopping the development and treatment of related diseases. The activity of IDO1 directly depends on the content of its ferrous heme, which binds to dioxygen to enable its insertion into the indole ring of TRP [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Antioxidants have the ability to inactivate IDO through the reduction of either heme levels in the protein or protein expression [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Modern medicine is exploring the mechanisms of phytochemicals to optimize its pharmacological activity. The intricate workings of the processes and the unpredictable interactions between them make this type of research challenging. However, it is helpful to begin by demonstrating the modes of action of individual compounds.\\u003c/p\\u003e\\u003cp\\u003eTryptophan is an essential amino acid whose metabolites play key role in various physiological processes. TRP also has antioxidant properties that contribute to host cells\\u0026rsquo; defense against oxidative stress, participating in the synthesis of molecules with antioxidant activity. TRP is metabolized through two pathways that includes serotonin and KYN pathway capable of performing signaling functions [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e]. IDO1 oxidizes L-tryptophan to N-formyl KYN (NFK) [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. Mammals possess two forms of this enzyme, TDO (tryptophan-2,3-dioxygenase) and IDO (indoleamine-2,3-dioxygenase); the IDO gene family includes IDO1 and IDO2. However, compared to IDO1; TDO and IDO2 are poor producers of KYN [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. The activation of IDO1, the resultant TRP depletion, and the formation of KYN and its metabolites have important biological effects, which, depending on the circumstance, can be beneficial or deleterious to the host. IDO is an integrated part of the immune system, acting as natural defense against various pathogens and inflammatory stimuli. Interferon-gamma is a potent inducer of IDO synthesis, leading to enhanced tryptophan metabolism. This process can limit viral replication by depleting the essential amino acid tryptophan, which is necessary for viral protein synthesis. This serves as protective strategy against infection and inflammation, but the accumulation of KYN can also have unintended damaging effects, as these metabolites can chemically modify proteins and be cytotoxic. The interplay between tryptophan depletion pathways and the effects of KYN metabolites is complex, with each cell type responding differently to these changes [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eIn the human lens, IDO activity is present mainly in the anterior epithelium. Several KYNs, such as KYN, 3-hydroxy KYN (3-OH KYN), and 3-hydroxy KYN glucoside, are present in the lens. They are thought to protect the retina by absorbing UV light and are referred to as UV filters. However, several studies show that KYNs are prone to deamination and oxidation. The deamination results in the formation of α,β-unsaturated ketones that chemically react and modify lens proteins. In the normal human lens, KYN modifications increase after 50 years of age, they may also reduce the chaperone function of α-crystallin, which is necessary for maintaining lens transparency [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. Another mechanism for cataractogenesis is tryptophan, an amino acid in eye lenses in higher concentration, which forms N- Formyl KYN when it absorbs UV radiation. It may combine with the 3-OH KYN and riboflavin, which are lens photosensitizers; they absorb photons from the light and emit electrons. These electrons react with the molecular O\\u003csub\\u003e2\\u003c/sub\\u003e to form a superoxide radical, which reacts with the Na\\u003csup\\u003e+\\u003c/sup\\u003eK\\u003csup\\u003e+\\u003c/sup\\u003eATPase pump in the eye and may cause swelling of the eye and lens opacification [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. KYN also play a role in reactive oxygen species (ROS)-mediated crystallin modification and KYN-modified crystallin can generate ROS through photochemical reactions, weakened defenses against oxidative stress due to age or cataract formation could exacerbate lens protein modifications [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. Although the antioxidant GSH can protect against the Kynurenilation of the lens by forming GSH adducts in \\u003cem\\u003ein-vitro\\u003c/em\\u003e lens protein and in human lenses, the protection conferred by GSH decreases with age due to the reduced diffusion of GSH to the center of human lenses. Recent studies have demonstrated that expression of IDO1 is upregulated in cataractous lenses compared to healthy lenses, suggesting a potential role of this enzyme in cataract development. Inhibition of IDO1 activity has been found to reduce oxidative stress and inflammation in animal models of cataract, leading to improved lens clarity and visual function [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eKYN pathway metabolites have been closely linked to the pathogenesis of several neurodegenerative diseases, including AD (Alzheimer\\u0026rsquo;s Disease) and PD (Parkinson\\u0026rsquo;s Disease) [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. In AD and PD, the amyloid peptide upregulates IDO1 expression and increases the production of quinolinic acid in human macrophages and microglia [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. These findings suggest that IDO1 inhibition could have therapeutic potential in neurodegenerative diseases by modulating immune response and protecting neural cells. Cancer cells utilize the IDO pathway to suppress the host\\u0026rsquo;s immune response, facilitating the survival, invasion, and metastasis of malignant cells. IDO1 pathways significantly affect T cell response to antigenic stimulation. In tumors, activating L-TRP catabolism by L-TRP‐degrading enzyme IDO1 leads to a local generation of immunosuppressive KYN and L‐TRP depletion, resulting in a poor immune response. Inhibition of L‐TRP‐degrading enzymes might be beneficial and complementary for efficacy improvement in immunotherapy [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Depletion of TRP and increased KYN in the tumor microenvironment produces an inhibitory signal in T cells, preventing them from attacking the cancer cells. Inhibition of TRP degrading enzymes will eventually increase TRP levels and cause immune stimulation, complementing immunotherapy in cancer treatment. Hence, IDO inhibition relieves the immunoinhibitory microenvironment.\\u003c/p\\u003e\\u003cp\\u003eIndoximod, a competitive inhibitor of the IDO1 enzyme has been widely used as a standard to optimize the assay methods for IDO1 inhibition [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. In this study, we sought to optimize the assay method using Indoximod as selective standard inhibitor and evaluate a library of antioxidants compounds for their potential as IDO1 inhibitors. The antioxidants compounds selected for this study - gallic acid, quercetin, hydroxychavicol, phloretin, luteolin, ursolic acid and ethyl ferulate \\u0026ndash; were chosen based on ongoing research in our lab. This compounds have demonstrated potential in targeting the IDO1 pathway and possess known antioxidant properties.\\u003c/p\\u003e\"},{\"header\":\"2. MATERIALS AND METHODS\",\"content\":\"\\u003cp\\u003e\\u003cb\\u003eMaterials\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003eTryptophan, Kynurenine, Indoximod, Gallic acid hydrate, Quercetin, Phloretin, Hydroxychavicol, Luteolin, Ursolic acid and Ethyl ferulate were purchased from Zeta Scientific, India. Reagents required for the assay were purchased from Molychem and Sisco Chem, India. Fresh wild goat eyeballs were procured from the Abattoir (Deonar, Govandi, Mumbai). UV-light bulb (Osram, Make Germany) was used to UV-induced IDO1 overexpression or cataract formation in the lens. Microcentrifuge equipment from Remi Electronics, India and a multimode plate reader from SYNERGY H1 BioTek Instruments, Inc. USA with Gen5 software were used. Black 96 well plate were purchased from Genaxy Scientific Pvt. Ltd. (Cat. No. GEN-0224). Schrodinger Maestro 2023 and GraphPad Prism 10 were used to dock and analyze the data, respectively.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eMethods\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eMolecular Docking with IDO1\\u003c/strong\\u003e\\u003cp\\u003eDetailed Network Pharmacology Analysis of gallic acid was studied earlier by M.R. Beg [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e] from our lab, with one of the identified key target IDO1, in continuation we proceed with IDO1 \\u003cem\\u003ein-silico\\u003c/em\\u003e and \\u003cem\\u003ein-vitro\\u003c/em\\u003e analysis of gallic acid along with other potential antioxidants compounds. IDO1 protein was selected as the target macromolecule to dock with the ligands. 5EK4 protein in \\u0026lsquo;.pdb\\u0026rsquo; format was downloaded from the website rcsb.org. The ligand or structure of compounds to be evaluated was downloaded from PubChem in 3D \\u0026lsquo;.sdf\\u0026rsquo; format [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Schrodinger Maestro 2023-1 software was used for docking [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. Protein and ligands were prepared using Protein Preparation and LigPrep task options. Docking was done with the receptor grid generated on the co-crystallized NLG919 ligand site on the protein including heme with all tautomer of ligands generated. Standard precision, flexible ligand sampling and other default settings were applied for ligand docking. A low (negative) energy glide score (gscore) in Kcal/mol indicates a stable system and thus a likely binding interaction.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eIDO1 Crude Protein Working Solution Preparation\\u003c/b\\u003e: The generation of ROS is thought to be the mechanism through which UV-B radiation-induced damage to the eye lens is mediated. IDO1 is well-documented as the principal indoleamine 2,3-dioxygenase in ocular tissues, including the lens and cornea, and is upregulated by UV exposure. Studies show that IDO1 is consistently expressed at low levels in corneal and lens tissues normally, but its expression and activity significantly increase after UV irradiation. This upregulation is linked to a protective mechanism against UV-induced oxidative stress and apoptosis in ocular cells. IDO2 is expressed at much lower levels than IDO1 in most tissues and is considered to have a minor physiological role. TDO (tryptophan 2,3-dioxygenase) is primarily a hepatic enzyme and is not typically expressed at significant levels in extrahepatic tissues like the eye lens [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. Hence, we aimed to extract specific enzyme IDO1 from the lenses. Crude protein extraction was carried out from the lens homogenate [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. Fresh goat lens were transported to the Pharmacology Research Lab II, DPST, ICT Mumbai, under refrigeration conditions (2\\u0026ndash;8\\u0026deg;C). Lens were carefully removed by extracapsular extraction from eyeballs and then washed with normal saline solution. Lens were then transferred to an artificial aqueous humor solution to mimic the eye environment and exposed to an artificial UVR source (Osram Ultra Vitalux 300 W 230 V E27) at 13 cm distance for 20 min at room temperature. The resulting opaque lenses were expected to demonstrate overexpression of IDO1. The IDO1 overexpressed lenses were homogenized in lysis buffer of 0.2M Tris-base (pH 7.8) containing 25mM EDTA with 10% w/v homogenate utilizing hand homogenizer and centrifuged at 10000 rpm, 4\\u003csup\\u003eo\\u003c/sup\\u003eC for 20 min. Supernatants were pulled with 1:1 100% glycerol, and various 1ml aliquots were prepared and stored at -20\\u003csup\\u003eo\\u003c/sup\\u003eC for experimental use, we called this as enzyme working solution (as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Total protein content was estimated according to \\u0026lsquo;Bradford\\u0026rsquo;s Method\\u0026rsquo; [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e], with some modifications and bovine serum albumin (BSA) was used as a standard.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eMethods for Assessment of IDO Activity\\u003c/strong\\u003e\\u003cp\\u003eVarious techniques for assessing the activity of IDO1 used to screen IDO1 inhibitors were reviewed by literature. Mainly, HPLC, fluorescence detection, cell-based assay, NFK GreenScreen\\u0026trade;, and absorbance assay techniques have been reported for assessing the activity of IDO1. Each method possesses particular advantages, disadvantages, and cost-differences [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Among all of these, the fluorescence detection method is a novel and sensitive assay for the determination of IDO1 enzymatic activity, which can achieve high throughput screening (HTS) for IDO1 inhibitors. The IDO1 fluorescence assay introduced in 2006 measures the fluorescence of KYN produced from hydrolysis of NFK in sodium hydroxide [\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. Tomek et al. [\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e] described a fluorescence assay used for measuring IDO1 activity. In place of measuring KYN, it detects the in-situ formation of NFK-derived fluorophores (PIP-THQ) with an excitation wavelength of 400 nm and emission wavelength of 500 nm. For the new fluorescence assay, the former steps are performed as described by Takikawa et al. [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e] for an absorbance assay. We have adopted this method for our study, and the optimization is familiar with the available method of fluorescence detection. The fluorescence intensity of the PIP-THQ formed is directly related to the amount of enzyme activity. Piperidine Tetrahydro quinolone (PIP-THQ) is not formed directly from PIP-NFK. While the optimal reaction temperature and time for maximum formation of PIP-THQ are 65\\u0026deg;C for 20 min [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e].\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eFluorescence-based IDO1 Assay\\u003c/strong\\u003e\\u003cp\\u003eAssay was performed in a black polystyrene 96-well plate. The 50 \\u0026micro;l of reaction assay medium containing an in-well concentration of 50 mM potassium phosphate buffer (PBS) (pH 6.5), 10 mM ascorbic acid neutralized with equimolar NaOH, 100 \\u0026micro;g/mL catalase, 5 mM methylene blue and L-Tryptophan (or only assay mixture for blank) were added in the well. 10 \\u0026micro;l standard or test substance (distilled water for blank) and 60 \\u0026micro;l Enzyme working solution was added to the plate. Reaction was carried out at incubation of 37\\u003csup\\u003e0\\u003c/sup\\u003eC for 20 hours. After incubation, 30 \\u0026micro;l Piperidine (PIP) was added with 200 mM in-well concentration and the plate was covered and heated at 65\\u0026deg;C for 20 min. Plate was left at room temperature for 60 min, post-incubation the fluorescence intensity of PIP-THQ was measured at the 400 nm excitation wavelength and 500 nm emission wavelengths (25\\u0026deg;C, 7 mm measurement height, 100 gain), using Spectro-fluorimeter microplate reader (BioTek, Synergy H1).\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eN-formyl Kynurenine (Product) standard curve\\u003c/strong\\u003e\\u003cp\\u003eN-formyl KYN (NFK) was synthesized by formylation of 1 mg of L-KYN (5mM) in 2.5 \\u0026micro;l of formic acid, make up to 1ml using assay mixture. After 2 hrs, the NFK was formed, which was further used in the standard reaction for the IDO1 assay [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. The standard graph of absolute absorbance vs. NFK concentration was plotted to calculate the unknown concentration of NFK formed due to the reaction of lens homogenate enzyme solution.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eValidation of IDO1 Inhibition by Selective Inhibitor\\u003c/strong\\u003e\\u003cp\\u003eOne unit of IDO1 activity is the amount of enzyme that generates 1 \\u0026micro;mole of detected N-formyl Kynurenine per minute by oxidative metabolism of 1 \\u0026micro;mole L-tryptophan at 37 \\u0026deg;C. IDO1 activity is expressed as \\u0026micro;mole of detected NFK formed within 1 minute by oxidative metabolism of 1 \\u0026micro;mole of L-TRP at 37\\u0026deg;C. ∆V/min was calculated by (V1-V0)/time, where V0 is the initial velocity, and V1 is the velocity after starting of the reaction. The amount of NFK produced in each reaction was calculated from the NFK standard curve. Indoximod is the known selective IDO1 inhibitor, if N-formyl Kynurenine production drops significantly in the presence of the Indoximod, this confirms that IDO1 is a major enzyme responsible for the conversion of L-TRP to NFK.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eIDO1 Inhibition Assay (IC\\u003csub\\u003e50\\u003c/sub\\u003e)\\u003c/strong\\u003e\\u003cp\\u003eFor the inhibition assay, 50 \\u0026micro;L of 2 mM of tryptophan was used in the assay mixture, and 10 \\u0026micro;L of different concentrations of standard \\u0026amp; test compounds and 60 \\u0026micro;L of enzyme solution were added to each well. Further reaction was carried out for 20 hrs, and post-incubation PIP was added. The plate was heated at 65\\u0026deg;C for 20 min. Plate was left at room temperature for 60 min, post-incubation the fluorescence intensity of PIP-THQ was measured. Inhibition expressed as a percentage of inhibited PIP-NFK complex (PIP-THQ) was calculated as (A/B X 100), where A and B are absorbances in the presence and absence of inhibitor, respectively.\\u003c/p\\u003e\\u003c/p\\u003e\"},{\"header\":\"3. RESULTS AND DISCUSSIONS\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eIn-silico\\u003c/em\\u003e Molecular Docking Study\\u003c/strong\\u003e\\u003cp\\u003e\\u003cem\\u003eIn-silico\\u003c/em\\u003e molecular docking study has shown the interaction of the ligand compounds with the protein IDO1 [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. NLG919 (co-crystallized ligand with protein 5EK4), Indoximod (selective inhibitor of IDO1), antioxidants test compounds as Quercetin, Gallic acid, Hydroxychavicol, Phloretin, Luteolin, Ursolic acid and Ethyl Ferulate were taken as library of ligands.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eMaestro Glide Docking Score and Interactions\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"5\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"char\\\" char=\\\".\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eLigands\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003eDocking score (Kcal/mol)\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c5\\\" namest=\\\"c3\\\"\\u003e\\u003cp\\u003eLigands Interaction with receptor amino acids\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eπ-π aromatic ring\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003ehydrogen bond\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eπ-cation\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eNLG919\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e-7.30168\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eTYR126, HEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eN/A\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eHEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eIndoximod\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e-7.06598\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eTYR126, HEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eN/A\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eHEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eLuteolin\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e-7.30319\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eTYR126, HEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eSER167, HEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eN/A\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eQuercetin\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e-6.61408\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eHEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eSER167, HEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eN/A\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003ePhloretin\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e-6.39428\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eHEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eSER167, HEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eN/A\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eGallic acid\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e-5.83717\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eHEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eSER167\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eN/A\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eHydroxychavicol\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e-5.53807\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eTYR126, HEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eSER167\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eN/A\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eEthyl Ferulate\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e-5.06422\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eHEM501\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eSER167\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eN/A\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eUrsolic acid\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"char\\\" char=\\\".\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003e-4.24518\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eN/A\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eGLY236\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eN/A\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe obtained molecular docking data, represented in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, indicated that IDO1 enzyme has an affinity towards all the tested compounds [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. The negative docking score value is directly proportional to the affinity of the protein to compound. As known from the literature, NLG919 a co-crystallized ligand of protein and Indoximod a selective competitive inhibitor of IDO1, its low docking score and common receptor amino acid interactions validated these results. Out of tested antioxidants, Luteolin has shown highest docking score of -7.303 comparable with the ligand NLG919. Other compounds have also shown good docking score, with Ursolic acid with least affinity of -4.245 docking score. Receptor amino acids HEM501, TYR126 and SER167 are mainly involved in the all the ligand interactions, with GLY236 interaction with only Ursolic acid.\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eIDO1 Enzyme Activity\\u003c/strong\\u003e\\u003cp\\u003eAs per BSA standard curve, the Goat lens homogenate solution contained total protein content of 74.8989 \\u0026micro;g/ml. An assay for the standard N-formyl Kynurenine curve was performed (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e) and enzyme activity was calculated for lens enzyme solutions. The goat lens showed enzyme activity of 0.0860 U and 22.05 U/mg specific activity. The kinetics study for enzyme solutions was performed, by Michaelis-Menton K\\u003csub\\u003eM\\u003c/sub\\u003e and V\\u003csub\\u003eMax\\u003c/sub\\u003e value is found to be 33.01 \\u0026micro;M and 16516, respectively and by Lineweaver-Burk K\\u003csub\\u003eM\\u003c/sub\\u003e and V\\u003csub\\u003eMax\\u003c/sub\\u003e value is found to be 31.13 \\u0026micro;M and 16316, respectively (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cstrong\\u003eIDO1 Enzyme Inhibition\\u003c/strong\\u003e\\u003cp\\u003eIDO1 enzyme inhibition for all the standard and test compounds was performed to calculate IC\\u003csub\\u003e50\\u003c/sub\\u003e values and the final results are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e.\\u003c/p\\u003e\\u003c/p\\u003e\\u003cp\\u003eGraph analysis for IC\\u003csub\\u003e50\\u003c/sub\\u003e was performed using second-order quadratic non-linear regression method in GraphPad Prism. Standard drug Indoximod and other test compounds Quercetin, Gallic acid, Luteolin, Phloretin, and Hydroxychavicol exhibited IDO1 Inhibition activity. From Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e, the standard IDO1 inhibitor has shown an excellent IC\\u003csub\\u003e50\\u003c/sub\\u003e value of 0.329 mM, which is the most potent among other test compounds. Quercetin and Luteolin demonstrated IC\\u003csub\\u003e50\\u003c/sub\\u003e comparable to the standard. Gallic acid and Phloretin exhibited good IC\\u003csub\\u003e50\\u003c/sub\\u003e value. A significant IDO1 inhibition was exhibited by the Hydroxychavicol. However, it was less potent among other compounds. The values showed that Quercetin, Luteolin, Gallic acid, Phloretin, and Hydroxychavicol influenced IDO1 inhibition in the KYN pathway. Compounds Ethyl Ferulate and Ursolic acid, which were involved in docking studies, did not show any promising IDO1 enzyme inhibition via our optimized \\u003cem\\u003ein-vitro\\u003c/em\\u003e method.\\u003c/p\\u003e\"},{\"header\":\"4. CONCLUSION\",\"content\":\"\\u003cp\\u003eThe IDO1 overexpression has been linked to several disorders, which lead to the need for easy quantification of IDO1 inhibitors. The optimized assay used crude lens homogenate enzyme solution, that can be used for screening the IDO1 inhibitory activity of many natural and synthetic compounds. It has shown good potential to be incorporated into assays instead of pure enzyme, enabling sensitive and cost-effective enzyme assays. Inhibition by the selective inhibitor Indoximod, shows that this method for evaluating IDO1 is validated. The \\u003cem\\u003ein-silico\\u003c/em\\u003e docking score, \\u003cem\\u003ein-vitro\\u003c/em\\u003e IC\\u003csub\\u003e50\\u003c/sub\\u003e value of the compounds enable us to interpret the therapeutic effects of these compounds as seen in in-house experiments from our lab. The IDO1 inhibition was demonstrated by the well-researched antioxidants phloretin, luteolin, gallic acid and quercetin. Phloretin and luteolin is being studied as potential adjuvants\\u0026rsquo; in treating neurodegenerative disorders using \\u003cem\\u003ein-vivo\\u003c/em\\u003e models in our lab [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. IDO1 inhibition activity of gallic acid and quercetin was done to explore it as a target for its probable activity to treat cataract [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. Ocular formulation containing these antioxidants has also shown good \\u003cem\\u003eex-vivo\\u003c/em\\u003e and \\u003cem\\u003ein-vivo\\u003c/em\\u003e anti-cataract activity as per granted patent from our lab [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. Hydroxychavicol is being investigated as a potential anticancer agent, exhibiting IDO1 inhibition [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. The overexpression of this enzyme is implicated in cataractogenesis, carcinogenesis, inflammation, and neuronal degeneration [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eIn conclusion, the therapeutic potential of IDO1 inhibitors represents a promising avenue for treating various disorders, offering significant prospects for more effective and targeted therapies in the future. Developing combination therapies that include IDO1 inhibitor alongside other pharmacological agents could maximize therapeutic efficacy and minimize resistance mechanism, leading to more comprehensive and effective treatment approach.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003eIDO: Indoleamine-2,3-dioxygenase\\u003c/p\\u003e\\n\\u003cp\\u003eSOD: Superoxide dismutase\\u003c/p\\u003e\\n\\u003cp\\u003eGSH: Glutathione\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTRP: Tryptophan\\u003c/p\\u003e\\n\\u003cp\\u003eKYN: Kynurenine\\u003c/p\\u003e\\n\\u003cp\\u003eNFK: N-formyl Kynurenine\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTDO: Tryptophan-2, 3-dioxygenase\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eROS: Reactive Oxygen Species\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAD: Alzheimer\\u0026apos;s Disease\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003ePD: Parkinson\\u0026apos;s Disease\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eUV: Ultraviolet\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eEDTA: Ethylenediaminetetraacetic Acid\\u003c/p\\u003e\\n\\u003cp\\u003eBSA: Bovine Serum Albumin\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eHPLC: High pressure liquid chromatography\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eHTS: High throughput screening\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003ePIP-THQ: Piperidine Tetrahydro Quinolone\\u003c/p\\u003e\\n\\u003cp\\u003ePIP: Piperidine\\u003c/p\\u003e\\n\\u003cp\\u003eAM: Assay Medium\\u003c/p\\u003e\\n\\u003cp\\u003ePBS: Phosphate buffer saline\\u003c/p\\u003e\\n\\u003cp\\u003eTYR: Tyrosine\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eHEM: Heme\\u003c/p\\u003e\\n\\u003cp\\u003eSER: Serine\\u003c/p\\u003e\\n\\u003cp\\u003eGLY: Glycine\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003ch2\\u003eConflict of Interest\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors have no relevant financial or non-financial interest to disclose.\\u003c/p\\u003e\\n\\u003ch2\\u003eEthics Approval\\u003c/h2\\u003e\\n\\u003cp\\u003eThe CCSEA in 110th meeting has decided that the IAECs and CCSEA has no objection for studies conducted on slaughter house samples and no ethical approval is required. Fresh wild goat eyeballs were procured from the Abattoir (Deonar, Govandi, Mumbai), with due consideration given to obtaining approval letters from both the institute and the abattoir.\\u003c/p\\u003e\\n\\u003ch2\\u003eFunding\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\\u003c/p\\u003e\\n\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\n\\u003cp\\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by G.S. and S.G. The first draft of the manuscript was written by G.S. All authors commented on previous versions of the manuscript and reviewed by S.S. All authors read and approved the final manuscript.\\u003c/p\\u003e\\n\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\n\\u003cp\\u003eThe authors would like to acknowledge Dr. Shamlan Reshamwala (Centre of Energy Biosciences, Institute of Chemical Technology, Mumbai) his intellectual contribution to analyze the enzyme assay results. The authors gratefully acknowledge the use of facilities of Institute of Chemical Technology, Mumbai.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eNimse SB, Pal D (2015) Free radicals, natural antioxidants, and their reaction mechanisms. 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Ther Pat 26:815\\u0026ndash;832. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1080/13543776.2016.1189531\\u003c/span\\u003e\\u003cspan address=\\\"10.1080/13543776.2016.1189531\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-fluorescence\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jofl\",\"sideBox\":\"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)\",\"snPcode\":\"10895\",\"submissionUrl\":\"https://submission.nature.com/new-submission/10895/3\",\"title\":\"Journal of Fluorescence\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false},\"keywords\":\"Enzyme activity, Fluorescence, Indoleamine-2,3-dioxygenase, In-vitro model, Biochemical Investigation, Antioxidants\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7074793/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7074793/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eIndoleamine 2,3-dioxygenase 1 (IDO1) is a key immunoregulatory enzyme that catalyzes the oxidative cleavage of L-tryptophan to N-formyl kynurenine, playing a critical role in immune tolerance and various pathological conditions, including cancer, autoimmune diseases, cataractogenesis, and neurodegenerative disorders. In this study, molecular docking was performed using a phytochemical library to identify compounds with strong binding affinity and favorable interactions within the IDO1 active site. Based on these in silico findings, selected compounds were further evaluated using a newly developed and cost-effective optimized fluorescence-based assay employing lens homogenate enzyme preparations to quantitatively assess IDO1 activity. Dose\\u0026ndash;response experiments revealed that several phytochemicals exhibited significant concentration-dependent inhibition of IDO1, with promising IC₅₀ values. The consistency between docking results and experimental inhibition supports the potential of these compounds as IDO1 inhibitors. This integrated in silico\\u0026ndash;in vitro approach provides a reliable platform for screening IDO1 modulators and identifies promising natural inhibitors for further development as therapeutics for IDO1-associated diseases.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Indoleamine-2,3-Dioxygenase 1 Enzyme Inhibition: A Useful Target to Screen Chemicals for their Therapeutic Potential\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-07-25 14:16:03\",\"doi\":\"10.21203/rs.3.rs-7074793/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-08-08T10:18:44+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-08-04T17:38:44+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-08-02T11:32:22+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-07-29T12:03:35+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"60181262337161487728658928993235625075\",\"date\":\"2025-07-28T12:54:52+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-07-26T12:53:25+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"81176319392210108251003079288861096649\",\"date\":\"2025-07-24T08:54:06+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"3873780614573169071442268312245805912\",\"date\":\"2025-07-23T18:43:34+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"101780774000615717298463302025012271050\",\"date\":\"2025-07-23T11:23:27+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"208578962925850595568536441452089372619\",\"date\":\"2025-07-23T11:14:07+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-07-23T11:04:44+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-07-11T18:38:04+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-07-11T18:37:19+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Journal of Fluorescence\",\"date\":\"2025-07-08T12:11:04+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-fluorescence\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jofl\",\"sideBox\":\"Learn more about [Journal of Fluorescence](https://www.springer.com/journal/10895)\",\"snPcode\":\"10895\",\"submissionUrl\":\"https://submission.nature.com/new-submission/10895/3\",\"title\":\"Journal of Fluorescence\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"Springer Hybrid\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":false}}],\"origin\":\"\",\"ownerIdentity\":\"89701c5d-5c9a-4259-ae2e-e09425343851\",\"owner\":[],\"postedDate\":\"July 25th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-10-13T16:03:26+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-7074793\",\"link\":\"https://doi.org/10.1007/s10895-025-04564-9\",\"journal\":{\"identity\":\"journal-of-fluorescence\",\"isVorOnly\":false,\"title\":\"Journal of Fluorescence\"},\"publishedOn\":\"2025-10-07 15:58:26\",\"publishedOnDateReadable\":\"October 7th, 2025\"},\"versionCreatedAt\":\"2025-07-25 14:16:03\",\"video\":\"\",\"vorDoi\":\"10.1007/s10895-025-04564-9\",\"vorDoiUrl\":\"https://doi.org/10.1007/s10895-025-04564-9\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7074793\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7074793\",\"identity\":\"rs-7074793\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}