Divya Dube

Structural Basis of Recognition of Pathogen-associated Molecular Patterns and Inhibition of Proinflammatory Cytokines by Camel Peptidoglycan Recognition Protein

Peptidoglycan recognition proteins (PGRPs) are involved in the recognition of pathogen-associated molecular patterns. The well known pathogen-associated molecular patterns include LPS from Gram-negative bacteria and lipoteichoic acid (LTA) from Gram-positive bacteria. In this work, the crystal structures of two complexes of the short form of camel PGRP (CPGRP-S) with LPS and LTA determined at 1.7- and 2.1-Å resolutions, respectively, are reported. Both compounds were held firmly inside the complex formed with four CPGRP-S molecules designated A, B, C, and D. The binding cleft is located at the interface of molecules C and D, which is extendable to the interface of molecules A and C. The interface of molecules A and B is tightly packed, whereas that of molecules B and D forms a wide channel. The hydrophilic moieties of these compounds occupy a common region, whereas hydrophobic chains interact with distinct regions in the binding site. The binding studies showed that CPGRP-S binds to LPS and LTA with affinities of 1.6 × 10−9 and 2.4 × 10−8 m, respectively. The flow cytometric studies showed that both LPS- and LTA-induced expression of the proinflammatory cytokines TNF-α and IL-6 was inhibited by CPGRP-S. The results of animal studies using mouse models indicated that both LPS- and LTA-induced mortality rates decreased drastically when CPGRP-S was administered. The recognition of both LPS and LTA, their high binding affinities for CPGRP-S, the significant decrease in the production of LPS- and LTA-induced TNF-α and IL-6, and the drastic reduction in the mortality rates in mice by CPGRP-S indicate its useful properties as an antibiotic agent.

Structural Insights into the Dual Strategy of Recognition by Peptidoglycan Recognition Protein, PGRP-S: Structure of the Ternary Complex of PGRP-S with Lipopolysaccharide and Stearic Acid

Peptidoglycan recognition proteins (PGRPs) are part of the innate immune system. The 19 kDa Short PGRP (PGRP-S) is one of the four mammalian PGRPs. The concentration of PGRP-S in camel (CPGRP-S) has been shown to increase considerably during mastitis. The structure of CPGRP-S consists of four protein molecules designated as A, B, C and D forming stable intermolecular contacts, A–B and C–D. The A–B and C–D interfaces are located on the opposite sides of the same monomer leading to the the formation of a linear chain with alternating A–B and C–D contacts. Two ligand binding sites, one at C–D contact and another at A–B contact have been observed. CPGRP-S binds to the components of bacterial cell wall molecules such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) from both Gram-positive and Gram-negative bacteria. It also binds to fatty acids including mycolic acid of the Mycobacterium tuberculosis (Mtb). Previous structural studies of binary complexes of CPGRP-S with LPS and stearic acid (SA) have shown that LPS binds to CPGRP-S at C–D contact (Site-1) while SA binds to it at the A–B contact (Site-2). The binding studies using surface plasmon resonance showed that LPS and SA bound to CPGRP-S in the presence of each other. The structure determination of the ternary complex showed that LPS and SA bound to CPGRP-S at Site-1 and Site-2 respectively. LPS formed 13 hydrogen bonds and 159 van der Waals contacts (distances ≤4.2 Å) while SA formed 56 van der Waals contacts. The ELISA test showed that increased levels of productions of pro-inflammatory cytokines TNF-α and IFN-γ due to LPS and SA decreased considerably upon the addition of CPGRP-S.

Multiligand Specificity of Pathogen-associated Molecular Pattern-binding Site in Peptidoglycan Recognition Protein

The peptidoglycan recognition protein PGRP-S is an innate immunity molecule that specifically interacts with microbial peptidoglycans and other pathogen-associated molecular patterns. We report here two structures of the unique tetrameric camel PGRP-S (CPGRP-S) complexed with (i) muramyl dipeptide (MDP) at 2.5 Å resolution and (ii) GlcNAc and β-maltose at 1.7Å resolution. The binding studies carried out using surface plasmon resonance indicated that CPGRP-S binds to MDP with a dissociation constant of 10−7 m, whereas the binding affinities for GlcNAc and β-maltose separately are in the range of 10−4 m to 10−5 m, whereas the dissociation constant for the mixture of GlcNAc and maltose was estimated to be 10−6 m. The data from bacterial suspension culture experiments showed a significant inhibition of the growth of Staphylococcus aureus cells when CPGRP-S was added to culture medium. The ELISA experiment showed that the amount of MDP-induced production of TNF-α and IL-6 decreased considerably after the introduction of CPGRP-S. The crystal structure determinations of (i) a binary complex with MDP and (ii) a ternary complex with GlcNAc and β-maltose revealed that MDP, GlcNAc, and β-maltose bound to CPGRP-S in the ligand binding cleft, which is situated at the interface of molecules C and D of the homotetramer formed by four protein molecules A, B, C, and D. In the binary complex, the muramyl moiety of MDP is observed at the C-D interface, whereas the peptide chain protrudes into the center of tetramer. In the ternary complex, GlcNAc and β-maltose occupy distinct non-overlapping positions belonging to different subsites.

3D-QSAR based pharmacophore modeling and virtual screening for identification of novel pteridine reductase inhibitors.

Pteridine reductase is a promising target for development of novel therapeutic agents against Trypanosomatid parasites. A 3D-QSAR pharmacophore hypothesis has been generated for a series of L. major pteridine reductase inhibitors using Catalyst/HypoGen algorithm for identification of the chemical features that are responsible for the inhibitory activity. Four pharmacophore features, namely: two H-bond donors (D), one Hydrophobic aromatic (H) and one Ring aromatic (R) have been identified as key features involved in inhibitor-PTR1 interaction. These features are able to predict the activity of external test set of pteridine reductase inhibitors with a correlation coefficient (r) of 0.80. Based on the analysis of the best hypotheses, some potent Pteridine reductase inhibitors were screened out and predicted with anti-PTR1 activity. It turned out that the newly identified inhibitory molecules are at least 300 fold more potent than the current crop of existing inhibitors. Overall the current SAR study is an effort for elucidating quantitative structure-activity relationship for the PTR1 inhibitors. The results from the combined 3D-QSAR modeling and molecular docking approach have led to the prediction of new potent inhibitory scaffolds.

Structural Studies on Molecular Interactions between Camel Peptidoglycan Recognition Protein, CPGRP-S, and Peptidoglycan Moieties N-Acetylglucosamine and N-Acetylmuramic Acid

Background: PGRP-S is an innate immunity protein that protects hosts from invading microbes. Results: CPGRP-S forms linear polymers with alternating A-B and C-D contacts, and both GlcNAc and MurNAc bind at the same subsite. Conclusion: The mode of binding of camel PGRP-S is different from other PGRPs. Significance: The better antibacterial properties of camel PGRP-S can be exploited for therapeutic applications. Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10−9, 2.6 × 10−7, and 1.8 × 10−7 m, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.

Implication of novel CYP2C9*57 (p.Asn204His) variant in coumarin hypersensitivity

INTRODUCTION Polymorphisms in CYP2C9 can vary the rate of metabolic clearance of oral anticoagulants, risking toxicity in patients. The present study focused on exploring the genetic etiology of idiopathic hyper sensitivity to coumarin anticoagulants in a patient who presented with multiple bleeding episodes and supra-elevated International Normalized Ratios. MATERIALS AND METHODS Bidirectional gene sequencing of CYP2C9 and VKORC1 was carried out. Using allele-specific polymerase chain reaction, the identified novel variant was genotyped in 309 patients on anticoagulation therapy. The pharmacoproteomic significance of the novel genetic variant was elucidated by structural demonstration of binding of coumarin molecules within the mutant CYP2C9 204His protein model and in silico bioinformatic evolutionary analyses. Three-dimensional structure model of the mutant protein was constructed on the basis of the published X-ray crystal structure of human CYP2C9 protein (Protein Data Bank, 1R9O). RESULTS The patient was identified to have a novel heterozygous missense mutation in exon 4 of CYP2C9 gene (g.9172A > C; p.Asn204His; CYP2C9*57). The variant was absent in the 309 genotyped patients. In silico bioinformatic analyses indicated the variant to have a deleterious effect on the protein. Analysis of 3D structure model of the mutant protein revealed that the substituted His204 led to restricted binding of the coumarin drug within the binding site of CYP2C9 enzyme, thereby inhibiting its metabolic clearance and thus explaining the enhanced pharmacologic effect and bleeding in the patient. CONCLUSIONS The study elucidates the structurally deleterious role of the novel CYP2C9*57 missense mutation in coumarin toxicity.

Crystal structure determination and inhibition studies of a novel xylanase and α-amylase inhibitor protein (XAIP) from Scadoxus multiflorus.

A novel plant protein isolated from the underground bulbs of Scadoxus multiflorus, xylanase and α‐amylase inhibitor protein (XAIP), inhibits two structurally and functionally unrelated enzymes: xylanase and α‐amylase. The mature protein contains 272 amino acid residues which show sequence identities of 48% to the plant chitinase hevamine and 36% to xylanase inhibitor protein‐I, a double‐headed inhibitor of GH10 and GH11 xylanases. However, unlike hevamine, it is enzymatically inactive and, unlike xylanase inhibitor protein‐I, it inhibits two functionally different classes of enzyme. The crystal structure of XAIP has been determined at 2.0 Å resolution and refined to Rcryst and Rfree factors of 15.2% and 18.6%, respectively. The polypeptide chain of XAIP adopts a modified triosephosphate isomerase barrel fold with eight β‐strands in the inner circle and nine α‐helices forming the outer ring. The structure contains three cis peptide bonds: Gly33–Phe34, Tyr159–Pro160 and Trp253–Asp254. Although hevamine has a long accessible carbohydrate‐binding channel, in XAIP this channel is almost completely filled with the side‐chains of residues Phe13, Pro77, Lys78 and Trp253. Solution studies indicate that XAIP inhibits GH11 family xylanases and GH13 family α‐amylases through two independent binding sites located on opposite surfaces of the protein. Comparison of the structure of XAIP with that of xylanase inhibitor protein‐I, and docking studies, suggest that loops α3–β4 and α4–β5 may be involved in the binding of GH11 xylanase, and that helix α7 and loop β6–α6 are suitable for the interaction with α‐amylase.

Structural basis of the binding of fatty acids to peptidoglycan recognition protein, PGRP-S through second binding site

Short peptidoglycan recognition protein (PGRP-S) is a member of the mammalian innate immune system. PGRP-S from Camelus dromedarius (CPGRP-S) has been shown to bind to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Its structure consists of four molecules A, B, C and D with ligand binding clefts situated at A-B and C-D contacts. It has been shown that LPS, LTA and PGN bind to CPGRP-S at C-D contact. The cleft at the A-B contact indicated features that suggested a possible binding of fatty acids including mycolic acid of Mycobacterium tuberculosis. Therefore, binding studies of CPGRP-S were carried out with fatty acids, butyric acid, lauric acid, myristic acid, stearic acid and mycolic acid which showed affinities in the range of 10(-5) to 10(-8) M. Structure determinations of the complexes of CPGRP-S with above fatty acids showed that they bound to CPGRP-S in the cleft at the A-B contact. The flow cytometric studies showed that mycolic acid induced the production of pro-inflammatory cytokines, TNF-α and IFN-γ by CD3+ T cells. The concentrations of cytokines increased considerably with increasing concentrations of mycolic acid. However, their levels decreased substantially on adding CPGRP-S.

Immunoglobulin heavy chain variable region gene repertoire and B-cell receptor stereotypes in Indian patients with chronic lymphocytic leukemia

Abstract In chronic lymphocytic leukemia (CLL), the geographical bias in immunoglobulin heavy-chain variable (IGHV) gene usage lead us to analyze IGHV gene usage and B-cell receptor stereotypy in 195 patients from India. IGHV3, IGHV4, and IGHV1 families were the most frequently used. 20.5% sequences had stereotyped BCR and were clustered in 12 pre-defined and 6 novel subsets. Unmutated IGHV was significantly associated with reduced time to first treatment (p < 0.033) and poor overall survival (OS; p = 0.01). We observed a significant difference in OS between IGHV1, IGHV3, and IGHV4 family cases (p = 0.045) in early stage patients. Regarding subfamily usage, only IGHV1-69 expression was found to have statistically significant poor outcome (p = 0.017). Our results from the analysis of various molecular and clinical features suggest that the expression of specific IGHV gene influences the outcome in early stage CLL, and hence its assessment may be added to the clinical leukemia laboratory armamentarium.

Pharmacophore Mapping, In Silico Screening and Molecular Docking to Identify Selective Trypanosoma brucei Pteridine Reductase Inhibitors.

Trypanosoma brucei Pteridine reductase (TbPTR1) is of vital importance and is an established drug target for dreaded Human African trypanosomiasis (HAT). Pharmacophore perception strategy has been employed to identify key chemical features responsible for the biological activity for TbPTR1. The findings suggest that three different pharmacophore features can be associated with T. brucei anti‐PTR1 activity namely: H‐bond donors (D), Hydrophobic aromatic (H) and Ring aromatic (R). The resulting hypothesis is able to predict the activity of other existing TbPTR1 inhibitors with a correlation coefficient (r) of 0.89. An in silico database screening, based on the best hypothesis, has been used to identify some potential nanomolar range TbPTR1 inhibitors. These compounds were then checked by molecular docking and subjected to ADMET analysis. Further, a detailed comparison of the pharmacophore behavior and differential analysis of binding pockets of T. brucei and L. major was made which revealed subtle differences in terms of their shape and charge properties. This investigation can form the basis for tweaking the specificity of compounds for generating new improved species specific inhibitor molecules for Pteridine reductase in these different parasitic protozoans.