An explorative study on Staphylococcus aureus MurE inhibitor: induced fit docking,binding free energy calculation,and molecular dynamics

Staphylococcus aureus MurE enzyme catalyzes the addition of l-lysine as third residue of the peptidoglycan peptide moiety. Due to the high substrate specificity and its ubiquitous nature among bacteria, MurE enzyme is considered as one of the potential target for the development of new therapeutic agents. In the present work, induced fit docking (IFD), binding free energy calculation, and molecular dynamics (MD) simulation were carried out to elucidate the inhibition potential of 2-thioxothiazolidin-4-one based inhibitor 1 against S. aureus MurE enzyme. The inhibitor 1 formed majority of hydrogen bonds with the central domain residues Asn151, Thr152, Ser180, Arg187, and Lys219. Binding free-energy calculation by MM-GBSA approach showed that van der Waals (ΔG_vdW, ?57.30?kcal/mol) and electrostatic solvation (ΔG_solv, ?36.86?kcal/mol) energy terms are major contributors for the inhibitor binding. Further, 30-ns MD simulation was performed to validate the stability of ligand–protein complex and also to get structural insight into mode of binding. Based on the IFD and MD simulation results, we designed four new compounds D1–D4 with promising binding affinity for the S. aureus MurE enzyme. The designed compounds were subjected to the extra-precision docking and binding free energy was calculated for complexes. Further, a 30-ns MD simulation was performed for D1/4C13 complex.     

Characterization of substrate specificity and inhibitory mechanism of bile salt hydrolase from Lactobacillus reuteri CRL 1098 using molecular docking analysis

Objectives

To elucidate the molecular mechanisms involved in the substrate interaction of the bile salt hydrolase of Lactobacillus reuteri CRL 1098 (LrBSH) with bile acids (BAs) and to evaluate potential enzyme inhibitors based on computer and in vitro modeling assays.

Results

Asp19, Asn79, and Asn171 participated in the LrBSH interaction with all BAs tested while Leu56 and Glu 222 played an important role in the interaction with glyco- and tauro-conjugated BAs, respectively. A great percentage of hydrophobic and polar interactions were responsible for the binding of LrBSH with glyco- and tauro-conjugated BAs, respectively. Remarkably, the four binding pocket loops participated in the substrate binding site of LrBSH unlike most of the reported BSHs. Inhibition assays showed that ascorbic acid, citric acid, penicillin G, and ciprofloxacin decreased LrBSH activity by 47.1%, 40.14%, 28.8%, and 9%, respectively. Docking analysis revealed that tetracycline and caffeic acid phenethyl ester had the low binding energy (?7.32 and ?7.19 kcal/mol, respectively) and resembled the interaction pattern of GDCA (?6.88 kcal/mol) while penicillin (?6.25 kcal/mol) and ascorbic acid (?5.98 kcal/mol) interacted at a longer distance.

Conclusion

This study helps to delve into the molecular mechanisms involved in the recognition of substrates and potential inhibitors of LrBSH.

     

A systematic computational analysis of human matrix metalloproteinase 13 (MMP-13) crystal structures and structure-based identification of prospective drug candidates as MMP-13 inhibitors repurposable for osteoarthritis

Abstract

Osteoarthritis (OA) is the most common form of arthritis with no available disease-modifying treatments, and is a major cause of disability. Matrix metalloproteinase 13 (MMP-13) is vital for OA progression and thus, inhibition of MMP-13 is an effective strategy to treat OA. Since the past few decades, drug repurposing has gained substantial popularity worldwide as a time- and cost-effective approach to find new indications for the existing drugs. Therefore, more than 40 X-ray co-crystal structures of the human MMP-13 with bound inhibitors are investigated to gain the structural insights such as conserved direct interactions with binding site residues, namely Ala-238, Thr-245 and Thr-247. Afterwards, enrichment study using active and decoy set of ligands revealed three MMP-13 structures (PDB-IDs: 1XUC, 3WV1 and 5BPA) with optimal enrichment performance. Docking-based screening of existing drugs against the three crystal structures followed by binding free-energy calculation suggested drugs namely eltrombopag, cilostazol and domperidone as potential MMP-13 inhibitors that need further experimental validation. These insights may serve as a potential starting point of further experimental validation and structure-based drug design/repurposing of MMP-13 inhibitors for the treatment of OA. Abbreviations 2D two-dimensional

3D three-dimensional

FDA Food and Drug Administration

MM-GBSA Molecular Mechanics Generalized Born Surface Area

MMPs matrix metalloproteinases

MMP-13 matrix metalloproteinase 13

NMR nuclear magnetic resonance

OA osteoarthritis

PDB Protein Data Bank

PDB-ID Protein Data Bank ID

PLIP protein–ligand interaction profiler

ROC receiver operating characteristic,

RMSD root mean square deviation

Communicated by Ramaswamy H. Sarma     

Drug targeted virtual screening and molecular dynamics of LipU protein of Mycobacterium tuberculosis and Mycobacterium leprae

The lipolytic protein LipU was conserved in mycobacterium sp. including M. tuberculosis (MTB LipU) and M. leprae (MLP LipU). The MTB LipU was identified in extracellular fraction and was reported to be essential for the survival of mycobacterium. Therefore to address the problem of drug resistance in pathogen, LipU was selected as a drug target and the viability of finding out some FDA approved drugs as LipU inhibitors in both the cases was explored. Three-dimensional (3D) model structures of MTB LipU and MLP LipU were generated and stabilized through molecular dynamics (MD). FDA approved drugs were screened against these proteins. The result showed that the top-scoring compounds for MTB LipU were Diosmin, Acarbose and Ouabain with the Glide XP score of ?12.8, ?11.9 and ?11.7 kcal/mol, respectively, whereas for MLP LipU protein, Digoxin (?9.2 kcal/mol), Indinavir (?8.2 kcal/mol) and Travoprost (?8.2 kcal/mol) showed highest affinity. These drugs remained bound in the active site pocket of MTB LipU and MLP LipU structure and interaction grew stronger after dynamics. RMSD, RMSF and Rg were found to be persistent throughout the simulation period. Hydrogen bonds along with large number of hydrophobic interactions stabilized the complex structures. Binding free energies obtained through Prime/MM-GBSA were found in the significant range from ?63.85 kcal/mol to ?34.57 kcal/mol for MTB LipU and ?71.33 kcal/mol to ?23.91 kcal/mol for MLP LipU. The report suggested high probability of these drugs to demolish the LipU activity and could be probable drug candidates to combat TB and leprosy disease.     

Synthesis,cyclooxygenase inhibition and anti-inflammatory evaluation of new 1,3,5-triaryl-4,5-dihydro-1H-pyrazole derivatives possessing methanesulphonyl pharmacophore

A new series of 1,3,5-triaryl-4,5-dihydro-1H-pyrazole derivatives 13a–p were synthesized via aldol condensation of 3/4-nitroacetophenones with appropriately substituted aldehydes followed by cyclization of the formed chalcones with 4-methanesulfonylphenylhydrazine hydrochloride. All the synthesized compounds were evaluated for their cyclooxygenase (COX) inhibition, anti-inflammatory activity and ulcerogenic liability. All compounds were more potent inhibitors for COX-2 than COX-1. While most compounds showed good anti-inflammatory activity, compounds 13d, 13f, 13k and 13o were the most potent derivatives (ED₅₀?=?66.5, 73.4, 79.8 and 70.5?μmol/kg, respectively) in comparison with celecoxib (ED₅₀?=?68.1?μmol/kg). Compounds 13d, 13f, 13k and 13o (ulcer index?=?3.89, 4.86, 4.96 and 3.92, respectively) were 4–6 folds less ulcerogenic than aspirin (ulcer index?=?22.75) and showed approximately ulceration effect similar to celecoxib (ulcer index?=?3.35). In addition, molecular docking studies were performed for compounds 13d, 13f, 13k and 13o inside COX-2 active site which showed acceptable binding interactions (affinity in kcal/mol ?2.1774, ?6.9498) in comparison with celecoxib (affinity in kcal/mol ?6.5330).     

Synthesis and biological activity of pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-4-one derivatives: in silico approach

Xanthine oxidase (XO) is responsible for the pathological condition called gout. Inhibition of XO activity by various pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidine-4-one derivatives was assessed and compared with the standard inhibitor allopurinol. Out of 10 synthesized compounds, two compounds, viz. 3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-4-one (3b) and 3-amino-6-(4-chloro-2-hydroxy-5-methylphenyl)-1H-pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-4-one (3g) were found to have promising XO inhibitory activity of the same order as allopurinol. Both compounds and allopurinol inhibited competitively with comparable Ki (3b: 3.56?µg, 3g: 2.337?µg, allopurinol: 1.816?µg) and IC₅₀ (3b: 4.228?µg, 3g: 3.1?µg, allopurinol: 2.9?µg) values. The enzyme–ligand interaction was studied by molecular docking using Autodock in BioMed Cache V. 6.1 software. The results revealed a significant dock score for 3b (?84.976?kcal/mol) and 3g (?90.921?kcal/mol) compared with allopurinol (?55.01?kcal/mol). The physiochemical properties and toxicity of the compounds were determined in silico using online computational tools. Overall, in vitro and in silico study revealed 3-amino-6-(4-chloro-2-hydroxy-5-methylphenyl)-1H-pyrazolo[3,4-d]thiazolo[3,2–a]pyrimidin-4-one (3g) as a potential lead compound for the design and development of XO inhibitors.     

Structure investigation,enrichment analysis and structure-based repurposing of FDA-approved drugs as inhibitors of BET-BRD4

We report herein detailed structural insights into the ligand recognition modes guiding bromodomain selectivity, enrichment analysis and docking-based database screening for the identification of the FDA-approved drugs that have potential to be the human BRD4 inhibitors. Analysis of multiple X-ray structures prevailed that the lysine-recognition sites are highly conserved, and apparently, the dynamic ZA loop guides the specific ligand-recognition. The protein–ligand interaction profiling revealed that both BRD2 and BRD4 shared hydrophobic interaction of bound ligands with PRO-98/PRO-82, PHE-99/PHE-83, LEU-108/LEU-92 and direct H-bonding with ASN-156/ASN-140 (BRD2/BRD4), while on the other hand the water-mediated H-bonding of bound ligands with PRO-82, GLN-85, PRO-86, VAL-87, ASP-88, LEU-92, TYR-97 and MET-132, and aromatic π–π stacking with TRP-81 prevailed as unique interaction in BRD4, and were not observed in BRD2. Subsequently, through ROC curve analysis, the best enrichment was found with PDB-ID 4QZS of BRD4 structures. Finally, through docking-based database screening study, we found that several drugs have better binding affinity than the control candidate lead (+)-JQ1 (Binding affinity?=?-7.9?kcal/mol), a well-known BRD4 inhibitor. Among the top-ranked drugs, azelastine, a selective histamine H1 receptor antagonist, showed the best binding affinity of –9.3?kcal/mol and showed interactions with several key residues of the acetyl lysine binding pocket. Azelastine may serve as a promising template for further medicinal chemistry. These insights may serve as basis for structure-based drug design, drug repurposing and the discovery of novel BRD4 inhibitors.

Communicated by Ramaswamy H. Sarma     

Chloro and bromo-pyrazole curcumin Knoevenagel condensates augmented anticancer activity against human cervical cancer cells: design,synthesis, in silico docking and in vitro cytotoxicity analysis

Abstract

With an endeavor to develop novel curcumin analogs as potential anti-cancer agents, we designed and synthesized a series of Knoevenagel condensates by clubbing pyrazole carbaldehydes at the active methylene carbon atom of the curcumin backbone. Molecular docking studies were carried out to target the proposed derivatives on human kinase β (IKKβ), a potential anti-cancer target. The chloro derivative displayed five hydrogen bond interactions with a docking score of ?11.874?kcal/mol higher than curcumin (docking score =??7.434?kcal/mol). This was supported by the fact that the propellant shaped derivatives fitted aptly into the binding pocket. Molecular simulations studies were also conducted on the lead molecule and the results figured out that the stable complexes were developed as the minimal deviations per residue of protein within the range of 0.11–0.92 Å. The screened compounds were synthesized, characterized and evaluated in vitro for cytotoxicity against cervical cancer cell line, HeLa using standard cell proliferation assay. Chloro derivative and bromo analog demonstrated IC₅₀ (half maximal inhibitory concentration) value of 14.2 and 18.6 µg/ml, respectively, significantly lower than 42.4 µg/ml of curcumin and higher than 0.008 µg/ml of paclitaxel. Induction of apoptosis was evaluated in the terms of cleavage of caspase-3 enzyme and they also exhibited 69.6 and 65.4% of apoptosis significantly higher than 19.9% induced by curcumin. In conclusion, chloro and bromo derivatives must be evaluated under a set of stringent in vitro and in vivo parameters for translating in to a clinically viable product.

Communicated by Ramaswamy H. Sarma     

Cardiotonic steroids as potential Na+/K+-ATPase inhibitors – a computational study

Abstract

Cardiotonic steroids (CTS) are steroidal drugs, processed from the seeds and dried leaves of the genus Digitalis as well as from the skin and parotid gland of amphibians. The most commonly known CTS are ouabain, digoxin, digoxigenin and bufalin. CTS can be used for safer medication of congestive heart failure and other related conditions due to promising pharmacological and medicinal properties. Ouabain isolated from plants is widely utilized in in vitro studies to specifically block the sodium potassium (Na⁺/K⁺-ATPase) pump. For checking, whether ouabain derivatives are robust inhibitors of Na⁺/K⁺-ATPase pump, molecular docking simulation was performed between ouabain and its derivatives using YASARA software. The docking energy falls within the range of 8.470?kcal/mol to 7.234?kcal/mol, in which digoxigenin was found to be the potential ligand with the best docking energy of 8.470?kcal/mol. Furthermore, pharmacophore modeling was applied to decipher the electronic features of CTS. Molecular dynamics simulation was also employed to determine the conformational properties of Na⁺/K⁺-ATPase-ouabain and Na⁺/K⁺-ATPase-digoxigenin complexes with the plausible structural integrity through conformational ensembles for 100?ns which promoted digoxigenin as the most promising CTS for treating conditions of congestive heart failure patients.     

Theoretical Investigation of the Molecular Structure of the πκ DNA Base Pair

Abstract

The structure of the nonclassical πκ base pair (7–methyl-oxoformycin … 2,4-diaminopyrimidine) was studied at the ab initio Hartree-Fock (HF) and MP2 levels using the 6–31G* and 6–31G** basis sets. The πκ base pair is bound by three parallel hydrogen bonds with the donor-acceptor-donor recognition pattern. Recently, these bases were proposed as an extension of the genetic alphabet from four to six letters (Piccirilli et al. Nature 343, 33(1990)). By the HF/6- 31G* method with full geometry optimization we calculated the 12 degree propeller twist for the minimum energy structure of this complex. The linearity of hydrogen bonds is preserved in the twisted structure by virtue of the pyramidal arrangement of the κ-base amino groups. The rings of both the π and κ molecules remain nearly planar. This nonplanar structure of the πκ base pair is only 0.1 kcal/mol more stable than the planar (Cs) conformation. The HF/6- 31G* level gas-phase interaction energy of πκ (—13.5 kcal/mol) calculated by us turned out to be nearly the same as the interaction energy obtained previously for the adenine-thymine base pair (—13.4 kcal/mol) at the same computational level. The inclusion of p-polarization functions on hydrogens, electron correlation effects (MP2/6–31G** level), and the correction for the basis set superposition error (BSSE) increase this energy to -14.0 kcal/mol.     

Thiol/disulphide homeostasis in pregnant women with Familial Mediterranean fever

Objective: To investigate the presence of oxidative stress (OS) in pregnant women with Familial Mediterranean fever (FMF) in the first trimester by evaluating thiol/disulphide homeostasis.

Study design: A total of 31 pregnant women with a diagnosis of FMF, between 11⁰ and 13⁶ weeks of gestation, were compared with 51 healthy pregnant controls at the same gestational weeks. A recently defined method was used to measure plasma native thiol, total thiol and disulphide levels.

Results: There were no differences between groups in terms of maternal age, body mass index and numbers of gravida and parity. Antenatal complications (45.2% vs. 9.8%, P?=?0.001) and primary caesarean section (22.6% vs. 5.9%, P?=?0.037) were higher in the FMF group. Pregnant women with FMF had significantly lower first trimester serum levels of native thiol (297.5?μmol/l (153.2–441.8) vs. 366.1?μmol/l (288.7–432.4), P?=?0.000), total thiol (327.2?μmol/l (171.0–471.0) vs. 389.9?μmol/l (317.1–449.8), P?=?0.000) and higher levels of disulphide (14.2?±?4.5?μmol/l vs. 12.4?±?3.4?μmol/l, P?=?0.045). No differences were found in these parameters among FMF patients with and without antenatal complications.

Conclusions: The main outcome demonstrates a relation between OS and pregnant women with FMF in the first trimester of gestation. OS in the first trimester may be a major aetiological factor of unfavourable pregancy outcomes in this group of patients.     

In silico identification of mimicking molecule(s) triggering von Willebrand factor in human: a molecular drug target for regulating coagulation pathway

Abstract

Blood coagulation is a complex and dynamic process wherein the body activates its emergency mechanism to stop bleeding and wound healing via the interactions of prothrombotic and antithrombotic agents. von Willebrand factor (VWF) is a complex glycoprotein and initial component of the hemostasis pathway which serves a multipurpose role in blood coagulation process. There are reports of various plants that contain several bioactive compounds possessing properties of inducing blood coagulation directly or indirectly. In the present study, efforts have been made to identify bioactive compounds that may play a significant role in regulation of the coagulation cascade by accelerating VWF and thus enhance the hemostasis process. An antidiuretic peptide drug, Desmopressin, works on VWF and releases them in circulation. Forty-seven compounds from different plant sources were screened through molecular docking, out of which two compounds, Emodin and Peruvianoside II, showed more binding affinity than the reference drug Desmopressin. Emodin and Peruvianoside II showed binding energies ?7.2 and ?7.0?kcal/mol, respectively, when docked with VWF, whereas Desmopressin displayed less binding energy (?6.9?kcal/mol). Emodin belongs to anthraquinone from Rumex hastasus and Peruvianoside II belongs to flavanone glycosides from Thevetia peruviana. The mimicking potential of top identified molecules with respect to the drug was confirmed through simulation analysis. Besides, the molecular dynamics simulation (MDS) study (for 20?ns) showed that the Peruvianoside II protein complex was energetically more stable than Emodin protein complex. Based on the results, Peruvianoside II possesses great potential and thus may be considered for development of drugs for hemostasis.     

Molecular Dynamics/Free Energy Perturbation Studies of the Thermostable V74I Mutant of Ribonuclease HI

Abstract

Recent site-directed mutagenesis and thermodynamic studies have shown that the V74I mutant of Escherichia coli ribonuclease HI (RNase HI) is more stable than the wild type protein [Ishikawa et al., Biochemistry 32, 6171 (1993)]. In order to clarify the stabilization mechanism of this mutant, we calculated the free energy change due to the mutation Val 74→Ile in both the native and denatured states by free energy perturbations based on molecular dynamics (MD) simulations. We carried out inclusive MD simulations for the protein in water; i.e., fully solvated, no artificial constraints applied, and all long-range Coulomb interactions included. We found that the free energy of the mutant increased slightly relative to the wild type, in the native state by 1.60 kcal/mol, and in the denatured state by 2.25 kcal/mol. The unfolding free energy increment of the mutant (0.66 ± 0.19 kcal/mol) was in good agreement with the experimental value (0.6 kcal/mol). The hysteresis error in the free energy calculations, i.e., forward and reverse perturbations, was only ±0.19 kcal/mol. These results show that the V74I mutant is stabilized relative to the wild type by the increased free energy of the denatured state and not by a decrease in the free energy of the native state as had been proposed earlier based on the mutant X-ray structure. It was found that the stabilization was caused by a loss of solvation energy in the mutant denatured state and not by improved packing interactions inside the native protein.     

A multitechnique approach to probe the interaction of a therapeutic tyrosine kinase inhibitor nintedanib and bovine serum albumin

Drug and protein interaction provides a structural guideline in the rational drug designing and in the synthesis of new and improved drugs with greater efficacy. We have examined here the interaction tendency and mechanism of nintedanib (NTB), an anticancer drug (tyrosine kinase inhibitor) with bovine serum albumin (BSA), by spectroscopic techniques. The decline in Stern–Volmer quenching constants and binding constant with the temperature rise suggests that BSA forms a complex with NTB. Binding constant obtained by modified Stern–Volmer equation at 3 temperatures was realized to be of the order of ~10⁴?M^?1. Negative ΔG (~?5.93?kcal?mol^?1), ΔH (?3.74?kcal?mol^?1), and ΔS (?1.50?kcal?mol^?1) values exhibited a spontaneous and exothermic reaction between BSA and NTB. NTB molecule interacts with BSA by forming hydrogen bonds, as elucidated by fluorescence results. Moreover, a minor increment in the helical conformation of BSA upon its binding to NTB was observed by circular dichroism spectroscopy. The modification in protein’s symmetry and a decline in hydrodynamic radii were observed in the presence of NTB (from ~3.6 to ~3?nm) as obtained by the dynamic light scattering measurement results.     

Combined pharmacophore modeling, 3D-QSAR and docking studies to identify novel HDAC inhibitors using drug repurposing

Abstract

Histone deacetylases (HDACs), a critical family of epigenetic enzymes, has emerged as a promising target for antitumor drugs. Here, we describe our protocol of virtual screening in identification of novel potential HDAC inhibitors through pharmacophore modeling, 3D-QSAR, molecular docking and molecular dynamics (MD) simulation. Considering the limitation of current virtual screening works, drug repurposing strategy was applied to discover druggable HDAC inhibitor. The ligand-based pharmacophore and 3D-QSAR models were established, and their reliability was validated by different methods. Then, the DrugBank database was screened, followed by molecular docking. MD simulation (100?ns) was performed to further study the stability of ligand binding modes. Finally, results indicated the hit DB03889 with high in silico inhibitory potency was suitable for further experimental analysis.

Communicated by Ramaswamy H. Sarma     

Identification of ALK5 inhibitor via structure-based virtual screening and ADMET prediction

Abstract

TGF-β plays a critical role in the initiation and progression of fibrosis in various organ systems such as kidney, heart, lung and liver. TGF-β and its receptors (ALK5 and TβR II) are able to control the cellular growth and promote several biological responses. To date, many pharmaceutical companies have employed virtual screening to identify potent inhibitors against ALK5. Nevertheless, none of these studies had involved the in silico ADMET evaluation and Raccoon filtering. In our experiment, all 57423 molecules were downloaded from TCM database and were filtered and converted to PDBQT formats by Raccoon software. Then 24?189 structures were run through AutoDock Vina in PyRx 0.8, 164 molecules were selected and further evaluated by ADMET Predictor 6.5, and 56 structures were selected and docked by Glide 6.2. Finally, the top 10 hits were identified as promising oral ALK5 inhibitors according to their Glide scores. The Glide scores of the best two compounds, 40686 and 33534, were ?10.75 and ?10.30?kcal/mol, respectively. This research provides a set of combined and detailed virtual screening protocol and is helpful for explaining the mechanism of receptor–ligand interactions.     

Inhibition of acetylcholinesterase by two arylderivatives: 3a-Acetoxy-5H-pyrrolo(1,2-a) (3,1)benzoxazin-1,5-(3aH)-dione and cis-N-p-Acetoxy-phenylisomaleimide

Two arylderivatives, 3a-Acetoxy-5H-pyrrolo(1,2-a) (3,1)benzoxazin-1,5-(3aH)-dione 3 and cis-N-p-Acetoxy-phenylisomaleimide 4, were synthesized from anthranilic acid and para-aminophenol, respectively. The inhibitory effects of these compounds on acetylcholinesterase (AChE) activity were evaluated in vitro as well as by docking simulations. Both compounds showed inhibition of AChE activity (K_i = 4.72 ± 2.3 μM for 3 and 3.6 ± 1.8 μM for 4) in in vitro studies. Moreover, they behaved as irreversible inhibitors and made π–π interaction with W84 and hydrogen bonded with S200 and Y337 according to experimental data and docking calculations. The docking calculations showed ΔG bind (kcal/mol) of ? 9.22 for 3 and ? 8.58 for 4. These two compounds that can be use as leads for a new family of anti-Alzheimer disease drugs.     

Probing the structural interactions between methotrexate and dexamethasone with muscle cystatin: a biophysical study

Abstract

Drug protein interactions have gained considerable attention over the past many years. In the current communication the association of muscle cystatin (MC) with anti-rheumatic drugs methotrexate and dexamethasone was studied by thiol proteinase inhibitor assay, ultra violet (UV) absorption, fluorescence spectroscopy, and fluorescence transform infra-red spectroscopy (FTIR). A static pattern of quenching was noticed between muscle cystatin and methotrexate (MTX). Binding constant (K_a) of methotrexate to muscle cystatin was found to be 1?×?10^?7 M^?1 and the stoichiometry of binding was calculated to be one. Fluorescence measurement of the emission quenching reveals that the quenching process of cystatin by dexamethasone (DXN) was also static. The stoichiometry of binding and binding constant was also obtained. Additional evidence regarding MTX–MC and DXN–MC was obtained from UV spectroscopy and FTIR spectroscopic results. Such spectroscopic studies would help in modelling new candidate drugs for rheumatoid arthritis based on their cystatin binding profile.

Communicated by Ramaswamy H. Sarma