Mili Kapoor

Evidence of a trimolecular complex involving LPS, LPS binding protein and soluble CD14 as an effector of LPS response

The kinetics of the interaction of lipopolysaccharide (LPS), lipopolysaccharide binding protein (LBP) and CD14 was studied using surface plasmon resonance. The association and dissociation rate constants for the binding of LPS and rsCD14 were 2.9×104 M−1 s−1 and 0.07 s−1 respectively, yielding a binding constant of 4.2×105 M−1. Significantly, the presence of LBP increased not only the association rate but also the association constant for the interaction between LPS and CD14 by three orders of magnitude. Our experimental results suggest that LBP interacts with LPS and CD14 to form a stable trimolecular complex that has significant functional implications as it allows monocytes to detect the presence of LPS at a concentration as low as 10 pg/ml or 2 pM, and to respond by secreting interleukin‐6. Thus, LBP is not merely transferring LPS to CD14 but it forms an integral part of the LPS–rLBP–rsCD14 complex.

Slow-tight binding inhibition of enoyl-acyl carrier protein reductase from Plasmodium falciparum by triclosan

Triclosan is a potent inhibitor of FabI (enoyl-ACP reductase, where ACP stands for acyl carrier protein), which catalyses the last step in a sequence of four reactions that is repeated many times with each elongation step in the type II fatty acid biosynthesis pathway. The malarial parasite Plasmodium falciparum also harbours the genes and is capable of synthesizing fatty acids by utilizing the enzymes of type II FAS (fatty acid synthase). The basic differences in the enzymes of type I FAS, present in humans, and type II FAS, present in Plasmodium, make the enzymes of this pathway a good target for antimalarials. The steady-state kinetics revealed time-dependent inhibition of FabI by triclosan, demonstrating that triclosan is a slow-tight-binding inhibitor of FabI. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI) that isomerizes to a second enzyme-inhibitor complex (EI*), which dissociates at a very slow rate. The rate constants for the isomerization of EI to EI* and the dissociation of EI* were 5.49x10(-2) and 1x10(-4) s(-1) respectively. The K(i) value for the formation of the EI complex was 53 nM and the overall inhibition constant K(i)* was 96 pM. The results match well with the rate constants derived independently from fluorescence analysis of the interaction of FabI and triclosan, as well as those obtained by surface plasmon resonance studies [Kapoor, Mukhi, N. Surolia, Sugunda and A. Surolia (2004) Biochem. J. 381, 725-733].

Functional characterization of β-ketoacyl-ACP reductase (FabG) from Plasmodium falciparum☆

The malaria parasite, Plasmodium falciparum, unlike its human host, utilizes type II fatty acid synthesis, in which steps of fatty acid biosynthesis are catalyzed by independent enzymes. Due to this difference, the enzymes of this pathway are a potential target of newer antimalarials. Here we report the functional characterization of Plasmodium FabG expressed in Escherichia coli. The purified recombinant FabG from P. falciparum is soluble and active. The K(m) of the enzyme for acetoacetyl-CoA was estimated to be 75 microM with a V(max) of 0.0054 micromol/min/ml and a k(cat) value of 0.014s(-1). NADPH exhibited negative cooperativity for its interaction with FabG. We have also modeled P. falciparum FabG using Brassica napus FabG as the template. This model provides a structural rationale for the specificity of FabG towards its cofactor, NADPH.

Mutational analysis of the triclosan-binding region of enoyl-ACP (acyl-carrier protein) reductase from Plasmodium falciparum.

Triclosan, a known antibacterial, acts by inhibiting enoyl-ACP (acyl-carrier protein) reductase (ENR), a key enzyme of the type II fatty acid synthesis (FAS) system. Plasmodium falciparum, the human malaria-causing parasite, harbours the type II FAS; in contrast, its human host utilizes type I FAS. Due to this striking difference, ENR has emerged as an important target for the development of new antimalarials. Modelling studies, and the crystal structure of P. falciparum ENR, have highlighted the features of ternary complex formation between the enzyme, triclosan and NAD+ [Suguna, A. Surolia and N. Surolia (2001) Biochem. Biophys. Res. Commun. 283, 224-228; Perozzo, Kuo, Sidhu, Valiyaveettil, Bittman, Jacobs, Fidock, and Sacchettini (2002) J. Biol. Chem. 277, 13106-13114; and Swarnamukhi, Kapoor, N. Surolia, A. Surolia and Suguna (2003) PDB1UH5]. To address the issue of the importance of the residues involved in strong specific and stoichiometric binding of triclosan to P. falciparum ENR, we mutated the following residues: Ala-217, Asn-218, Met-281, and Phe-368. The affinity of all the mutants was reduced for triclosan as compared with the wild-type enzyme to different extents. The most significant mutation was A217V, which led to a greater than 7000-fold decrease in the binding affinity for triclosan as compared with wild-type PfENR. A217G showed only 10-fold reduction in the binding affinity. Thus, these studies point out significant differences in the triclosan-binding region of the P. falciparum enzyme from those of its bacterial counterparts.

Kinetic and structural analysis of the increased affinity of enoyl-ACP (acyl-carrier protein) reductase for triclosan in the presence of NAD +

The binding of enoyl-ACP (acyl-carrier protein) reductase from Plasmodium falciparum (PfENR) with its substrates and inhibitors has been analysed by SPR (surface plasmon resonance). The binding of the substrate analogue crotonoyl-CoA and coenzyme NADH to PfENR was monitored in real time by observing changes in response units. The binding constants determined for crotonoyl-CoA and NADH were 1.6x10(4) M(-1) and 1.9x10(4) M(-1) respectively. Triclosan, which has recently been demonstrated as a potent antimalarial agent, bound to the enzyme with a binding constant of 1.08x10(5) M(-1). However, there was a 300-fold increase in the binding constant in the presence of NAD+. The increase in the binding constant was due to a 17 times increase in the association rate constant (k(1)) from 741 M(-1) x s(-1) to 1.3x10(4) M(-1) x s(-1) and a 16 times decrease in the dissociation rate constant (k(-1)) from 6.84x10(-3) s(-1) to 4.2x10(-4) s(-1). These values are in agreement with those determined by steady-state kinetic analysis of the inhibition reaction [Kapoor, Reddy, Krishnasastry, N. Surolia and A. Surolia (2004) Biochem. J. 381, 719-724]. In SPR experiments, the binding of NAD+ to PfENR was not detected. However, a binding constant of 6.5x10(4) M(-1) was obtained in the presence of triclosan. Further support for these observations was provided by the crystal structures of the binary and ternary complexes of PfENR. Thus the dramatic enhancement in the binding affinity of both triclosan and NAD+ in the ternary complex can be explained by increased van der Waals contacts in the ternary complex, facilitated by the movement of residues 318-324 of the substrate-binding loop and the nicotinamide ring of NAD+. Interestingly, the results of the present study also provide a rationale for the increased affinity of NAD+ for the enzyme in the ternary complex.

Synthesis and Evaluation of Substituted Pyrazoles: Potential Antimalarials Targeting the Enoyl-Acp Reductase of Plasmodium Falciparum

A series of 1,5- and 1,3-diarylsubstituted pyrazoles were designed, synthesized, and evaluated for their ability to inhibit enoyl-ACP reductase of Plasmodium falciparum. The inhibitory activity of these synthesized compounds was evaluated in a continuous spectrophotometric assay. Of all the compounds analyzed, NAS-81 and NAS-39 inhibited the enzyme with IC50 values of 30 \mu M and 50 \mu M, respectively. The mode of ligand binding was investigated by docking the synthetic inhibitors at the active site of the crystal structure of the enzyme.

Crystallization and preliminary crystallographic analysis of beta-hydroxyacyl ACP dehydratase (FabZ) from Plasmodium falciparum.

The malarial parasite Plasmodium falciparum synthesizes fatty acids by the type II mechanism. In this cycle, the dehydration of the beta-hydroxyacyl acyl carrier protein is catalyzed by FabZ. Purified FabZ has been crystallized using the hanging-drop vapour-diffusion and microbatch techniques. The crystals are orthorhombic, with space group I222 or I2(1)2(1)2(1) and unit-cell parameters a = 71.78, b = 81.99, c = 97.49 A. A complete data set to a resolution of 2.5 A has been collected under cryoconditions (100 K) using a MAR imaging-plate detector system mounted on a rotating-anode X-ray generator.

One Step Synthesis of Novel Antimicrobial 2‐Hydroxy Diaryl Ethers Through Domestic Microwave Heating

Abstract Herein we report the one step synthesis of novel 2‐hydroxy diaryl ethers that have been synthesized without any protection and deprotection. The reaction was completed within 5 minutes using a domestic microwave. These 2‐hydroxy diaryl ethers (1–6) have also been evaluated for their antimicrobial activity.

Exploring enzyme amplification to characterize specificities of protein-carbohydrate recognition.

Publisher Summary A considerable amount of evidence has accumulated indicating that a large number of glycoconjugates become altered during normal and abnormal cellular development. Understanding these diverse biological processes at the molecular level requires knowledge of the structure and biochemistry of cellular oligosaccharides. Because of their exquisite carbohydrate specificity, ready availability, and diversity lectins mostly from plants have become indispensable tools in biological research for the investigation of sugars of glycoconjugates. This chapter describes the principle and procedure employed for enzyme-linked lectinsorbent assay (ELLA) for measuring the carbohydrate-binding activity of lectins using a microtiter assay. The procedure is broadly divided into direct and indirect methods and is discussed with representative lectin examples optimized in authors laboratory. ELLA provides a sensitive biochemical assay with high specificity and broad flexibility to determine the sugar-binding affinities for different classes of lectins and has been applied to various protein–carbohydrate interaction studies

Exploring kinetics and mechanism of protein-sugar recognition by surface plasmon resonance.

Publisher Summary The study of biomolecular recognition is of basic importance in understanding processes of molecular recognition and biological function. Lectins are a class of non-enzymatic carbohydrate-binding proteins found ubiquitously in nature. These lectin–ligand attachments are critical in several biological processes, such as cell signaling, life cycling of pathogens, fertilization, and inflammatory responses and are put to use in biomedical research as carbohydrate probes based on the binding to surface sugars. The kinds of forces involved in lectin–sugar interactions are generally weak and include non-covalent forces, yet the specificity required for a given cellular adhesive event is great. Determination of kinetic/thermodynamic parameters involved in protein–sugar interactions at the molecular level provides a basic framework for understanding the mechanism of recognition. Biosensor-based techniques, such as surface plasmon resonance, provide an alternative method of studying lectin–carbohydrate interactions. This chapter provides a critical evaluation of this method in studies of protein–carbohydrate recognition by presenting a few representative examples.