Protein click chemistry and its potential for medical applications.

A revolution in chemical biology occurred with the introduction of click chemistry. Click chemistry plays an important role in protein chemistry modifications, providing specific, sensitive, rapid, and easy-to-handle methods. Under physiological conditions, click chemistry often overlaps with bioorthogonal chemistry, defined as reactions that occur rapidly and selectively without interfering with biological processes. Click chemistry is used for the posttranslational modification of proteins based on covalent bond formations. With the contribution of click reactions, selective modification of proteins would be developed, representing an alternative to other technologies in preparing new proteins or enzymes for studying specific protein functions in different biological processes. Click-modified proteins have potential in diverse applications such as imaging, labeling, sensing, drug design, and enzyme technology. Due to the promising role of proteins in disease diagnosis and therapy, this review aims to highlight the growing applications of click strategies in protein chemistry over the last two decades, with a special emphasis on medicinal applications.

Structural and functional alterations of polydopamine-coated hemoglobin: New insights for the development of successful oxygen carriers.

One of the major factors that is currently hindering the development of hemoglobin (Hb)-based oxygen carriers (HBOCs) is the autoxidation of Hb into nonfunctional methemoglobin. Modification with polydopamine (PDA), which is a biocompatible free radical scavenger has shown the ability to protect Hb against oxidation. Due to its tremendous potential in the development of successful HBOCs, herein, we conduct a thorough evaluation of the effect of PDA on the stability, aggregation, structure and function of the underlying Hb. By UV-vis spectrometry we show that PDA can prevent Hb's aggregation while thermal denaturation studies indicate that, although PDA coating resulted in a lower midpoint transition temperature, it was also able to protect the protein from full denaturation. These results are further corroborated by differential scanning calorimetry. Circular dichroism reveals that PDA can promote changes in Hb's secondary structure while, by UV-vis spectroscopy, we show that PDA also interacts with the porphyrin complex located in Hb's hydrophobic pocket. Last but not least, affinity studies show that PDA-coated Hb has a higher capability for oxygen release. Such an effect is further enhanced at lower pH. Importantly, through molecular docking simulations we provide a plausible explanation for the observed experimental results.

The structural and functional consequences of melatonin and serotonin on human αB-crystallin and their dual role in the eye lens transparency.

Crystallins are the major soluble lens proteins, and α-crystallin, the most important protective protein of the eye lens, has two subunits (αA and αB) with chaperone activity. αB-crystallin (αB-Cry) with a relatively wide tissue distribution has an innate ability to interact effectively with the misfolded proteins, preventing their aggregation. Melatonin and serotonin have also been identified in relatively high concentrations in the lenticular tissues. This study investigated the effect of these naturally occurring compounds and medications on the structure, oligomerization, aggregation, and chaperone-like activity of human αB-Cry. Various spectroscopic methods, dynamic light scattering (DLS), differential scanning calorimetry (DSC), and molecular docking have been used for this purpose. Based on our results, melatonin indicates an inhibitory effect on the aggregation of human αB-Cry without altering its chaperone-like activity. However, serotonin decreases αB-Cry oligomeric size distribution by creating hydrogen bonds, decreases its chaperone-like activity, and at high concentrations increases protein aggregation.

Cellulose membrane coated Mo-doped TiO2nanotube sheets for sustained oxidation of biomolecules.

Titanium dioxide nanotubes (TNT) are widely researched materials for the photocatalytic generation of free radicals, which are useful in wastewater treatment. We aimed to prepare Mo-doped TNT sheets, covered with a cellulose membrane to avoid TNT surface inactivation by protein adsorption. We studied the susceptibility of serum albumin (SA) bound to different molar ratios of palmitic acid (PA) to denaturation and fibrillation by this system, which is meant to mimic oxidative stress conditions such as non-alcoholic fatty liver disease. The results demonstrated that cellulose membrane-covered TNT successfully oxidized the SA, identified by structural changes to the protein. Increasing the molar ratio of PA to protein-enhanced thiol group oxidation while protecting the protein against structural changes. Finally, we propose that in this photocatalyzed oxidation system, the protein is oxidized by a non-adsorptive mechanism mediated by H2O2. Therefore, we suggest that this system could be used as a sustained oxidation system to oxidize biomolecules as well as potentially in wastewater treatment.

Modulation of Nrf2/HO-1 by Natural Compounds in Lung Cancer.

Oxidative stresses (OSs) are considered a pivotal factor in creating various pathophysiological conditions. Cells have been able to move forward by modulating numerous signaling pathways to moderate the defects of these stresses during their evolution. The company of Kelch-like ECH-associated protein 1 (Keap1) as a molecular sensing element of the oxidative and electrophilic stress and nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) as a master transcriptional regulator of the antioxidant response makes a master cytoprotective antioxidant pathway known as the Keap1/Nrf2 pathway. This pathway is considered a dual-edged sword with beneficial features for both normal and cancer cells by regulating the gene expression of the array of endogenous antioxidant enzymes. Heme oxygenase-1 (HO-1), a critical enzyme in toxic heme removal, is one of the clear state indicators for the duality of this pathway. Therefore, Nrf2/HO-1 axis targeting is known as a novel strategy for cancer treatment. In this review, the molecular mechanism of action of natural antioxidants on lung cancer cells has been investigated by relying on the Nrf2/HO-1 axis.

A New Mediterranean Flour Moth-Derived Chitosan: Characterization and Co-electrospun Hybrid Fabrication.

In this study, the chitin of adult Mediterranean flour moth (Ephestia kuheniella) (Cht) was extracted and then converted to chitosan by deacetylation process to achieve the chitosan derived from E. kuheniella (Chsfm). The new chitosan-based scaffold was produced using the polyvinyl alcohol (PVA) co-electrospinning technique. The degree of deacetylation was obtained using the distillation-titration and Fourier transform infrared spectroscopy. The surface morphology and crystallinity index of Chsfm were observed using scanning electron microscopy and X-ray diffraction analysis, respectively, and compared with the commercial chitosan (Chsc). Thermogravimetric analysis was used to estimate two chitosans' water content and thermal stability. The average molecular mass analysis was performed using viscometry. Moreover, the minimum inhibitory concentration and DPPH assay were used to study the antimicrobial activity and antioxidant potential of the Chsfm, respectively. Accordingly, Chsfm was smoother with fewer pores and flakes than Chsc, and its crystallinity index was higher than Chsc. The water content and thermal stability were lower and similar for Chsfm compared to Chsc. The average molecular mass of Chsfm was ~ 5.8 kDa, making it classified as low molecular weight chitosan. The antimicrobial activity of Chsfm against a representative Gram-negative bacteria; E. coli resulted to be the same as Chsc. However, less effective than Chsc against a representative Gram-positive bacteria is S. aureus. The Chsfm/PVA ratio scaffold was optimized at 30:70 to fabricate a uniform nanofiber scaffold.

Insulin fibrillation: Strategies for inhibition.

Insulin and its homologous are the most utilized protein drugs due to their role in diabetic patients' treatment. Insulin forms amyloid-like fibrils in vivo at the injection site. Therefore, the study of its fibrillation mechanism and designing efficient inhibitors have high importance in the pharmaceutical industry. Insulin fibrils are formed at both acidic and neutral pH in vitro. Overall, this process involves the dissociation of hexameric form to monomeric, partially dissociating the native monomeric form, nuclei formation, and finally converting oligomers to large ordered aggregates. Intermediate and terminal species are different pathologically. This review is focused on the research works dedicated to the inhibition of insulin fibril formation. The inhibitors include various polyphenols, natural compounds, nanoparticles, and synthetic chemicals/peptides, as well as the classification of inhibitors targets concerning protein fibrillation. Although most inhibitors stabilize the native structure of the protein and prevent the formation of partially folded species, there are other inhibitors that hinder other steps in the course of fibrillation. Also, several inhibitors were able to dissociate the pre-existing fibrils. Finding inhibition strategies could be beneficial for developing new inhibitors that are more efficient and can block the amyloid pathway in a specific desired stage.

Enzymatically triggered delignification through a novel stable laccase: A mixed in-silico /in-vitro exploration of a complex environmental microbiota.

Laccases have been broadly applied as a multitasking biocatalyst in various industries, but their applications tend to be limited by easy deactivation, lack of adequate stability, and susceptibility under complex conditions. Identifying stable laccase as a green-biocatalyst is crucial for developing cost-effective biorefining processes. In this direction, we attempted in-silico screening a stable metagenome-derived laccase (PersiLac1) from tannery wastewater in a complex environment. The laccase exhibited high thermostability, retaining 53.19% activity after 180 min at 70 °C, and it was stable in a wide range of pH (4.0-9.0). After 33 days of storage at 50°C, pH 6.0, the enzyme retained 71.65% of its activity. Various metal ions, inhibitors, and organic solvents showed that PersiLac1 has a stable structure. The stable PersiLac1 could successfully remove lignin and phenolic from quinoa husk and rice straw. In the separate hydrolysis and fermentation process (SHF) after 72 h, hydrolysis was obtained 100% and 73.4% for quinoa husk and rice straw, and fermentation by the S. cerevisiae was be produced 41.46 g/L and 27.75g/L ethanol, respectively. Results signified that the novel lignin-degrading enzyme was confirmed to have great potential for industrial application as a green-biocatalyst based on enzymatically triggered to delignification and detoxify lignocellulosic biomass.

The oxidative and neurotoxic potentials of the ambient PM2.5 extracts: The efficient multi-solvent extraction method.

The health effects of ambient air particulate matter with a diameter of ≤2.5 µm (PM2.5) on the central nervous system are well known and the induced oxidative stress has been shown as their main neuropathologic outcome. Ambient air PM2.5 sampling methods mostly use air sampler systems that collect PM2.5 on filters, which is followed by a PM2.5 extraction approach. Inefficient extraction may lead to compositional bias and unreal interpretation of the results. This study aimed to compare our proposed multi-solvent extraction (MSE) approach for PM2.5 extraction with a conventional aqueous extraction (AqE) method using the analysis of oxidative effects and cytotoxicity in the human neuroblastoma SH-SY5Y cell line. Ambient PM2.5 samples were collected from an urban traffic location in Tehran city, the capital of Iran, using a high-volume sampler. The developed MSE method was proved to have superior advantages over the AqE method including an increased extraction efficiency (as much as 96 against 48% for PMms and PMaq, respectively), and decreased artifacts and compositional biases. Ambient PM2.5, besides PMms and PMaq were analyzed for water-soluble ions, metals, and major elements. Dithiothreitol, ascorbic acid, lipid peroxidation, and cell viability assays on SH-SY5Y cells represented the significantly higher oxidative potential for PMms compared to PMaq. The increased cytotoxicity may occur because of the increased oxidative potential of PMms and possibly is associated with higher efficiency of the MSE over the AqE method for removal of total redox-active PM components.

The novel homologue of the human α-glucosidase inhibited by the non-germinated and germinated quinoa protein hydrolysates after in vitro gastrointestinal digestion.

Quinoa (Chenopodium quinoa Willd) is a potential source of protein with ideal amino acid profiles which its bioactive compounds can be improved during germination and gastrointestinal digestion. The present investigation studies the impact of germination for 24 hr and simulated gastrointestinal digestion on α-glucosidase inhibitory activity of the quinoa protein and bioactive peptides against the novel homologue of human α-glucosidase, PersiAlpha-GL1. The sprouted quinoa after gastroduodenal digestion was the most effective α-glucosidase inhibitor showing 81.10% α-glucosidase inhibition at concentration 4 mg/ml with the half inhibition rate (IC50 ) of 0.07 mg/ml. Based on the kinetic analysis, both the germinated and non-germinated samples before and after digestion were competitive-type inhibitors of α-glucosidase. Results of this study showed the improved α-glucosidase inhibitory activity of the quinoa bioactive peptides after germination and gastrointestinal digestion and highlighted the potential of metagenome-derived PersiAlpha-GL1 as a novel homologue of the human α-glucosidase for developing the future anti-diabetic drugs. PRACTICAL APPLICATIONS: This study aimed to evaluate the effect of germination and gastrointestinal digestion of the quinoa protein and bioactive peptides on α-glucosidase inhibitory activity against the novel PersiAlpha-GL1. Metagenomic data were used to identify the novel α-glucosidase structurally and functionally homologue of human intestinal. The results showed the highest inhibition on PersiAlpha-GL1 by a germinated quinoa after gastroduodenal digestion and confirmed the potential of PersiAlpha-GL1 to enhance the effectiveness of the anti-diabetic drugs for industrial application.

Application of free and immobilized novel bifunctional biocatalyst in biotransformation of recalcitrant lignocellulosic biomass.

Herein, an innovative, green, and practical biocatalyst was developed using conjugation of a novel bifunctional mannanase/xylanase biocatalyst (PersiManXyn1) to the modified cellulose nanocrystals (CNCs). Firstly, PersiManXyn1 was multi-stage in-silico screened from rumen macrobiota, and then cloned, expressed, and purified. Next, CNCs were synthesized from sugar beet pulp using enzymatic and acid hydrolysis processes, and then Fe3O4 NPs were anchored on their surface to produce magnetic CNCs (MCNCs). This hybrid was modified by dopamine providing DA/MCNCs nano-carrier. The bifunctional PersiManXyn1 demonstrated the superior hydrolysis activity on corn cob compared with the monofunctional xylanase enzyme (PersiXyn2). Moreover, the immobilization of PersiManXyn1 on the nano-carrier resulted in an improvement of the thermal stability, kinetic parameters (Kcat), and storage stability of the enzyme. Incorporation of the Fe3O4 NPs on the CNCs made magnetic nano-carrier with high magnetization value (25.8 emu/g) which exhibited rapid response toward the external magnetic fields. Hence, the immobilized biocatalyst could be easily separated from the products by a magnet, and reused up to 8 cycles with maintaining more than 50% of its original activity. The immobilized PersiManXyn1 generated 22.2%, 38.7%, and 35.1% more reducing sugars after 168 h hydrolysis of the sugar beet pulp, coffee waste, and rice straw, respectively, compared to the free enzyme. Based on the results, immobilization of the bifunctional PersiManXyn1 exhibited the superb performance of the enzyme to improve the conversion of the lignocellulosic wastes into high value products and develop the cost-competition biomass operations.

The concept of protein folding/unfolding and its impacts on human health.

Proteins have evolved in specific 3D structures and play different functions in cells and determine various reactions and pathways. The newly synthesized amino acid chains once depart ribosome must crumple into three-dimensional structures so can be biologically active. This process of protein that makes a functional molecule is called protein folding. The protein folding is both a biological and a physicochemical process that depends on the sequence of it. In fact, this process occurs more complicated and in some cases and in exposure to some molecules like glucose (glycation), mistaken folding leads to amyloid structures and fatal disorders called conformational diseases. Such conditions are detected by the quality control system of the cell and these abnormal proteins undergo renovation or degradation. This scenario takes place by the chaperones, chaperonins, and Ubiquitin-proteasome complex. Understanding of protein folding mechanisms from different views including experimental and computational approaches has revealed some intermediate ensembles such as molten globule and has been subjected to biophysical and molecular biology attempts to know more about prevalent conformational diseases.

Anti-Viral Potential and Modulation of Nrf2 by Curcumin: Pharmacological Implications.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is an essential transcription factor that maintains the cell's redox balance state and reduces inflammation in different adverse stresses. Under the oxidative stress, Nrf2 is separated from Kelch-like ECH-associated protein 1 (Keap1), which is a key sensor of oxidative stress, translocated to the nucleus, interacts with the antioxidant response element (ARE) in the target gene, and then activates the transcriptional pathway to ameliorate the cellular redox condition. Curcumin is a yellow polyphenolic curcuminoid from Curcuma longa (turmeric) that has revealed a broad spectrum of bioactivities, including antioxidant, anti-inflammatory, anti-tumor, and anti-viral activities. Curcumin significantly increases the nuclear expression levels and promotes the biological effects of Nrf2 via the interaction with Cys151 in Keap1, which makes it a marvelous therapeutic candidate against a broad range of oxidative stress-related diseases, including type 2 diabetes (T2D), neurodegenerative diseases (NDs), cardiovascular diseases (CVDs), cancers, viral infections, and more recently SARS-CoV-2. Currently, the multifactorial property of the diseases and lack of adequate medical treatment, especially in viral diseases, result in developing new strategies to finding potential drugs. Curcumin potentially opens up new views as possible Nrf2 activator. However, its low bioavailability that is due to low solubility and low stability in the physiological conditions is a significant challenge in the field of its efficient and effective utilization in medicinal purposes. In this review, we summarized recent studies on the potential effect of curcumin to activate Nrf2 as the design of potential drugs for a viral infection like SARS-Cov2 and acute and chronic inflammation diseases in order to improve the cells' protection.

The Stabilizing Mechanism of Immobilized Metagenomic Xylanases on Bio-Based Hydrogels to Improve Utilization Performance: Computational and Functional Perspectives.

While polysaccharide-based superabsorbent hydrogels (SHs) have attracted increasing interest as proficient carriers in the enzyme immobilization, the nature of the favored interactions between the SHs and enzymes is still unclear. Herein, a combined experimental and computational study was employed to investigate the dominant parameters affecting on the stabilization of two metagenomic xylanases on the SHs. The thermostable enzymes (PersiXyn3 and PersiXyn4) with similar domains were screened, cloned, expressed, and purified from cattle rumen metagenome. Then, the enzymes were immobilized on the carboxymethyl cellulose-g-poly(acrylic acid-co-acrylamide) hydrogel which resulted in increasing their activity and stability. The carboxymethyl cellulose (CMC)-based characteristic of the hydrogel provided high numbers of H-bondings/ionic bridges, causing an improvement in the stability, hydrolysis performance, and reusability of the immobilized enzymes. More specifically, enzyme immobilization resulted in ∼40% increase in the content of the reducing sugars released after treatment of paper pulp. After 16 reuse cycles, the immobilized PersiXyn4 displayed 35.9% activity, but the immobilized PersiXyn3 retained just 8.2% of its initial activity. The comparative investigations illustrated that a higher number of positively charged amino acids in the binding site of the enzyme provided stronger electrostatic attractions between it and negative functionalities of the hydrogel. This was suggested as the main reason for the higher affinity of PersiXyn4 toward hydrogel and explained the better hydrolysis performance and reusability of the immobilized PersiXyn4 on the SH. These findings are essential for designing novel innovative SH carriers and the successful engineering of optimal enzyme assemblies through the prediction of the immobilized enzyme's stabilities.

Anticancer Activity Assessment and DNA Binding Properties of Two Binuclear Platinum (II) Complexes using Spectroscopic and Molecular Simulation Approaches.

BACKGROUND: Nowadays, the biological properties and anticancer activities of platinum-based drugs and metal coordination complexes have been receiving particular attention. These compounds have revealed clinical potential in cancer chemotherapy. OBJECTIVE: In this research, two binuclear platinum complexes including [Pt2Cl2(bhq)2(µ-dppm)] (1) and [(p- MeC6H4)(bhq) Pt(µ-dppm)Pt(bhq)(CF3CO2)] (2) with bhq: benzo[h] quinolone and dppm: bis(diphenylphosphino) methane have been synthesized and evaluated for their anticancer activity against A2780 and A2780/RCIS cancer cell lines. METHODS: The DNA binding and interaction of AMP/GMP nucleotide with these complexes were explored by several experimental and theoretical methods, including UV-Visible, fluorescence spectroscopic techniques and docking analysis. These complexes have demonstrated significant anticancer properties against cisplatinsensitive (A2780) and cisplatin-resistant (A2780/RCIS) human ovarian cancer cell lines. RESULTS: The obtained results indicated that these complexes interact with DNA. Additionally, the fluorescence emission measurements indicated that the platinum complexes binding with DNA structure occurs through nonintercalative interaction. The molecular docking assessments have also revealed the binding of these platinum complexes through DNA grooves. Moreover, the results have indicated that complex 1 exhibited more anticancer activity than complex 2. CONCLUSION: The results of the DNA binding with these platinum complexes confirmed their potential antitumor properties. The substitution of -C6H4CH3 and -CO2CF3 groups in complex 2 with two chlorine atoms in complex 1 acquired the significant improvement of the anticancer activity against the cancer cell.

Stability of multi-subunit proteins and conformational lock.

One of the important physicochemical features of the proteins specifically multi-subunit types is their stability at high temperatures. The kinetics of the dissociation and denaturation of proteins possessing at least two subunits has certain challenges because the overall mechanism of dissociation can include hidden reversible and/or irreversible steps (conformational lock). There are numerous proteins related to diseases which are in equilibrium with oligomer forms. This equilibrium plays an important role in holding the activity levels of these proteins in vitro and in vivo. The stability of proteins is an essential milestone to study conformational changes of the proteins in the living cell. This concept is of great importance when proteins are used in biomedicine fields. The quaternary structure of multi-subunit proteins includes properties that affect function and kinetics of denaturation. The kinetics of denaturation may include some hidden stages of conformational transitions in the protein and steps of reversible dissociation of the oligomer. The transforming factors affect different areas especially active centers in the clefts of each subunit. The distinctive agent among all, is the temperature which sequentially destructs independent inter-subunit contact sites. Recent experimental data regarding dissociative mechanism for irreversible thermal denaturation of multi-subunit proteins have been summarized in the present paper.

Experimental investigation and molecular dynamics simulation of the binding of ellagic acid to bovine liver catalase: Activation study and interaction mechanism.

Ellagic acid (ELA), as a polyphenolic natural compound and food additive, which has reported to possess anti-carcinogenic, antioxidant, antidepressant, ameliorative and anti-mutagenic effects. In the current work, the effects of ELA on the conformation and catalytic activity of catalase were investigated by using spectroscopic techniques including ultraviolet visible (UV-vis), fluorescence, synchronous fluorescence and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy as well as molecular dynamics (MD) simulation. Kinetic studies showed that the enzymatic activity of catalase increases in the presence of ELA (almost 2-fold higher than free enzyme activity). Moreover, analysis of fluorescence data revealed two binding sites for ELA on the catalase and static type of quenching mechanism. The binding constants between ELA and catalase were obtained to be 47.35 × 107 M-1 (at 298 K) and 17.60 × 107 M-1 (at 310 K) and the binding distance was calculated to be 2.83 nm. Thermodynamic data showed that hydrogen bonds have a main role in the ELA-catalase complex formation. The best binding sites for ELA were, in the middle of ß-barrel and wrapping domain and in the middle of ß-barrel and helical domain, according to molecular docking data. MD simulation results were confirmed that ELA can increase catalase activity through increasing the distance between an upper side α-helix structure and a down side random coil structure.

Novel X-ray sequences and crystal structures of Persian and Starry sturgeon methemoglobins: Highlighting the role of heme pocket waters in causing autoxidation.

Although there is a high sequence similarity between mammalian and fish hemoglobin (Hb), the oxidation and heme loss rates can vary greatly between them such that fish Hbs oxidise much more rapidly than mammalian Hbs. There is to date no sequence or structural data for any sturgeon Hb to reveal the level of autoxidation in these fish. In this study, novel high resolution X-ray sequences and crystal structures of methemoglobin (Met-Hb) from two sturgeon fish including Persian sturgeon (Acipenser percisus) and Starry sturgeon (Acipenser stellatus) belonging to the Caspian sea has been determined. A comprehensive sequence and structure comparison between these sturgeon Met-Hbs and a number of non-sturgeon and normal and sickle cell anaemia human Hb in varying heme states has been carried out highlighting (i) the structural variability in the heme propionate groups; (ii) the existence of certain residues or their displacement and shift in the heme pocket allowing entry of water molecules into the heme pocket; (iii) the importance of the number of water molecules in the heme pocket; (iv) the hydrogen bonding between oxygens of A and D propionate groups and that of waters in the heme pocket; and (v) the role of heme binding waters causing oxidative stress and heme autoxidation.

Mining of camel rumen metagenome to identify novel alkali-thermostable xylanase capable of enhancing the recalcitrant lignocellulosic biomass conversion.

The aim of this study was to isolate and characterize novel alkali-thermostable xylanase genes from the mixed genome DNA of camel rumen metagenome. In this study, a five-stage computational screening procedure was utilized to find the primary candidate enzyme with superior properties from the camel rumen metagenome. This enzyme was subjected to cloning, purification, and structural and functional characterization. It showed high thermal stability, high activity in a broad range of pH (6-11) and temperature (30-90 °C) and effectivity in recalcitrant lignocellulosic biomass degradation. Our results demonstrated the power of in silico analysis to discover novel alkali-thermostable xylanases, effective for the bioconversion of lignocellulosic biomass.

The impact of water molecules on binding affinity of the anti-diabetic thiazolidinediones for catalase: Kinetic and mechanistic approaches.

Water molecules play a vital role in efficient drug binding to its target. Thiazolidinediones (TZDs), a class of anti-diabetic drugs, are widely used for treatment of type 2 diabetes mellitus. In the present study, the possible contribution of water molecules to the binding of TZDs to catalase, a potential target in the liver, was investigated by different experimental and theoretical methods. These studies indicated that TZDs could significantly improve the catalase catalytic function with a significant contribution from water molecules. As a probe for the differential number of released water molecules during the catalase transition from E to E* states, the activity of TZDs-catalase complexes was demonstrated to be mainly dependent on water activity. However, free catalase decomposed the substrate more independently. In addition, the spectrofluorimetry studies showed that the binding of TZDs to catalase needed the release of water molecules from the enzyme's binding pocket. The thermodynamic studies indicated that the binding enthalpy and entropy of TZDs for catalase were decreased with lower water activity. The favorable process contributes to release of water molecules from the binding pocket through the formation of hydrophobic interactions between catalase and TZDs in an enthalpic manner. Molecular docking simulations confirmed that the depletion of water molecules from the binding cavity is essential for effective interactions between TZDs and catalase.