Krishanpal Karmodiya

Synthesis and exploration of novel curcumin analogues as anti-malarial agents

Curcumin, a major yellow pigment and active component of turmeric, has been shown to possess anti-inflammatory and anti-cancer activities. Recent studies have indicated that curcumin inhibits chloroquine-sensitive (CQ-S) and chloroquine-resistant (CQ-R) Plasmodium falciparum growth in culture with an IC(50) of approximately 3.25 microM (MIC=13.2 microM) and IC(50) 4.21 microM (MIC=14.4 microM), respectively. In order to expand their potential as anti-malarials a series of novel curcumin derivatives were synthesized and evaluated for their ability to inhibit P. falciparum growth in culture. Several curcumin analogues examined show more effective inhibition of P. falciparum growth than curcumin. The most potent curcumin compounds 3, 6, and 11 were inhibitory for CQ-S P. falciparum at IC(50) of 0.48, 0.87, 0.92 microM and CQ-R P. falciparum at IC(50) of 0.45 microM, 0.89, 0.75 microM, respectively. Pyrazole analogue of curcumin (3) exhibited sevenfold higher anti-malarial potency against CQ-S and ninefold higher anti-malarial potency against CQ-R. Curcumin analogues described here represent a novel class of highly selective P. falciparum inhibitors and promising candidates for the design of novel anti-malarial agents.

Inhibitors of Nonhousekeeping Functions of the Apicoplast Defy Delayed Death in Plasmodium falciparum

ABSTRACT Targeting of apicoplast replication and protein synthesis in the apicomplexan Toxoplasma gondii has conventionally been associated with the typical “delayed death” phenotype, characterized by the death of parasites only in the generation following drug intervention. We demonstrate that antibiotics like clindamycin, chloramphenicol, and tetracycline, inhibitors of prokaryotic protein synthesis, invoke the delayed death phenotype in Plasmodium falciparum, too, as evident from a specific reduction of apicoplast genome copy number. Interestingly, however, molecules like triclosan, cerulenin, fops, and NAS-91, inhibitors of the recently discovered fatty acid synthesis pathway, and succinyl acetone, an inhibitor of heme biosynthesis that operates in the apicoplast of the parasite, display rapid and striking parasiticidal effects. Our results draw a clear distinction between apicoplast functions per se and the apicoplast as the site of metabolic pathways, which are required for parasite survival, and thus subserve the development of novel antimalarial therapy.

15-Deoxyspergualin Primarily Targets the Trafficking of Apicoplast Proteins in Plasmodium falciparum *

15-Deoxyspergualin, an immunosuppressant with tumoricidal and antimalarial properties, has been implicated in the inhibition of a diverse array of cellular processes including polyamine synthesis and protein synthesis. Endeavoring to identify the mechanism of antimalarial action of this molecule, we examined its effect on Plasmodium falciparum protein synthesis, polyamine biosynthesis, and transport. 15-Deoxyspergualin stalled protein synthesis in P. falciparum through Hsp70 sequestration and subsequent phosphorylation of the eukaryotic initiation factor eIF2α. However, protein synthesis inhibition as well as polyamine depletion were invoked only by high micromolar concentrations of 15-deoxyspergualin, in contrast to the submicromolar concentrations sufficient to inhibit parasite growth. Further investigations demonstrated that 15-deoxyspergualin in the malaria parasite primarily targets the hitherto underexplored process of trafficking of nucleus-encoded proteins to the apicoplast. Our finding that 15-deoxyspergualin kills the malaria parasite by interfering with targeting of nucleus-encoded proteins to the apicoplast not only exposes a chink in the armor of the malaria parasite, but also reveals new realms in our endeavors to study this intriguing biological process.

Mass Spectrometry-Based Systems Approach for Identification of Inhibitors of Plasmodium falciparum Fatty Acid Synthase

ABSTRACT The emergence of strains of Plasmodium falciparum resistant to the commonly used antimalarials warrants the development of new antimalarial agents. The discovery of type II fatty acid synthase (FAS) in Plasmodium distinct from the FAS in its human host (type I FAS) opened up new avenues for the development of novel antimalarials. The process of fatty acid synthesis takes place by iterative elongation of butyryl-acyl carrier protein (butyryl-ACP) by two carbon units, with the successive action of four enzymes constituting the elongation module of FAS until the desired acyl length is obtained. The study of the fatty acid synthesis machinery of the parasite inside the red blood cell culture has always been a challenging task. Here, we report the in vitro reconstitution of the elongation module of the FAS of malaria parasite involving all four enzymes, FabB/F (β-ketoacyl-ACP synthase), FabG (β-ketoacyl-ACP reductase), FabZ (β-ketoacyl-ACP dehydratase), and FabI (enoyl-ACP reductase), and its analysis by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS). That this in vitro systems approach completely mimics the in vivo machinery is confirmed by the distribution of acyl products. Using known inhibitors of the enzymes of the elongation module, cerulenin, triclosan, NAS-21/91, and (−)-catechin gallate, we demonstrate that accumulation of intermediates resulting from the inhibition of any of the enzymes can be unambiguously followed by MALDI-TOF MS. Thus, this work not only offers a powerful tool for easier and faster throughput screening of inhibitors but also allows for the study of the biochemical properties of the FAS pathway of the malaria parasite.

Analyses of co-operative transitions in Plasmodium falciparumβ-ketoacyl acyl carrier protein reductase upon co-factor and acyl carrier protein binding

The type II fatty acid synthase pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials because of its intrinsic differences from the type I pathway operating in humans. β‐Ketoacyl‐acyl carrier protein reductase is the only enzyme of this pathway that has no isoforms and thus selective inhibitors can be developed for this player of the pathway. We report here intensive studies on the direct interactions of Plasmodiumβ‐ketoacyl‐acyl carrier protein reductase with its cofactor, NADPH, acyl carrier protein, acetoacetyl‐coenzyme A and other ligands in solution, by monitoring the intrinsic fluorescence (λmax 334 nm) of the protein as a result of its lone tryptophan, as well as the fluorescence of NADPH (λmax 450 nm) upon binding to the enzyme. Binding of the reduced cofactor makes the enzyme catalytically efficient, as it increases the binding affinity of the substrate, acetoacetyl‐coenzyme A, by 16‐fold. The binding affinity of acyl carrier protein to the enzyme also increases by approximately threefold upon NADPH binding. Plasmodiumβ‐ketoacyl‐acyl carrier protein reductase exhibits negative, homotropic co‐operative binding for NADPH, which is enhanced in the presence of acyl carrier protein. Acyl carrier protein increases the accessibility of NADPH to β‐ketoacyl‐acyl carrier protein reductase, as evident from the increase in the accessibility of the tryptophan of β‐ketoacyl‐acyl carrier protein reductase to acrylamide, from 81 to 98%. In the presence of NADP+, the reaction proceeds in the reverse direction (Ka = 23.17 µm−1). These findings provide impetus for exploring the influence of ligands on the structure–activity relationship of Plasmodiumβ‐ketoacyl‐acyl carrier protein reductase.

Design, synthesis, and application of novel triclosan prodrugs as potential antimalarial and antibacterial agents

A number of new triclosan-conjugated analogs bearing biodegradable ester linkage have been synthesized, characterized and evaluated for their antimalarial and antibacterial activities. Many of these compounds exhibit good inhibition against Plasmodium falciparum and Escherichia coli. Among them tertiary amine containing triclosan-conjugated prodrug (5) inhibited both P. falciparum (IC(50); 0.62microM) and E. coli (IC(50); 0.26microM) at lower concentrations as compared to triclosan. Owing to the presence of a cleavable ester moiety, these new prodrugs are hydrolyzed under physiological conditions and parent molecule, triclosan, is released. Further, introduction of tertiary/quaternary functionality increases their cellular uptake. These properties impart them with higher potency to their antimalarial as well as antibacterial activities. The best compound among them 5 shows close to four-fold enhanced activities against P. falciparum and E. coli cultures as compared to triclosan.

Deciphering the key residues in Plasmodium falciparumβ‐ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein

The type II fatty acid synthase (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials, due to its intrinsic differences from the type I pathway operating in humans. β‐Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH‐dependent reduction of β‐ketoacyl‐ACP to β‐hydroxyacyl‐ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. In this article, we report intensive studies on the direct interactions of Plasmodium FabG and Plasmodium ACP in solution, in the presence and absence of its cofactor, NADPH, by monitoring the change in intrinsic fluorescence of P. falciparum FabG (PfFabG) and by surface plasmon resonance. To address the issue of the importance of the residues involved in strong, specific and stoichiometric binding of PfFabG to P. falciparum ACP (PfACP), we mutated Arg187, Arg190 and Arg230 of PfFabG. The activities of the mutants were assessed using both an ACP‐dependent and an ACP‐independent assay. The affinities of all the PfFabG mutants for acetoacetyl‐ACP (the physiological substrate) were reduced to different extents as compared to wild‐type PfFabG, but were equally active in biochemical assays with the substrate analog acetoacetyl‐CoA. Kinetic analysis and studies of direct binding between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical residues for the PfFabG–PfACP interactions. Our studies thus reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of PfFabG for interactions with PfACP.

A unique and differential effect of denaturants on cofactor mediated activation of Plasmodium falciparum β-ketoacyl-ACP reductase

The urea and guanidinium chloride (GdmCl) induced unfolding of FabG, a β‐ketoacyl‐ACP reductase of Plasmodium falciparum, was examined in detail using intrinsic fluorescence of FabG, UV‐circular dichroism (CD), spectrophotometric enzyme activity measurements, glutaraldehyde cross‐linking, and size exclusion chromatography. The equilibrium unfolding of FabG by urea is a multistep process as compared with a two‐state process by GdmCl. FabG is fully unfolded at 6.0M urea and 4.0M GdmCl. Approximately 90% of the enzyme activity could be recovered on dialyzing the denaturants, showing that denaturation by both urea and GdmCl is reversible. We found two states in the reversible unfolding process of FabG in presence of NADPH; one is an activity‐enhanced state and the other, an inactive state in case of equilibrium unfolding with urea. On the contrary, in presence of NADPH, there is no stabilization of FabG in case of equilibrium unfolding with GdmCl. We hypothesize that the hydrogen‐bonding network may be reorganized by the denaturant in the activity‐enhanced state formed in presence of 1.0M urea, by interrupting the association between dimer–dimer interface and help in accommodating the larger substrate in the substrate binding tunnel thus, increasing the activity. Furthermore, binding of the active site organizer, NADPH leads to compaction of the FabG in presence of urea, as evident by acrylamide quenching. We have shown here for the first time, the detailed inactivation kinetics of FabG, which have not been evaluated in the past from any of the FabG family of enzymes from any of the other sources. These findings provide impetus for exploring the influences of ligands on the structure–activity relationship of Plasmodium β‐ketoacyl‐ACP reductase. Proteins 2008.

Conformational stability and thermodynamic characterization of homotetrameric Plasmodium falciparum β‐ketoacyl‐ACP reductase

The conformational stability of the homotetrameric Plasmodium falciparumβ‐ketoacyl‐ACP reductase (FabG) was determined by guanidinium chloride‐induced isothermal and thermal denaturation. The reversible unfolding transitions were monitored by intrinsic fluorescence, circular dichroism (CD) spectroscopy and by measuring the enzyme activity of FabG. The denaturation profiles were analyzed to obtain the thermodynamic parameters associated with unfolding of the protein. The data confirm the simple A4 ↔ 4A model of unfolding, based on the corroboration of CD data by fluorescence transition and similar ΔG estimation for denaturation curves obtained at four different concentration of the FabG. Denaturation is well described by the linear extrapolation model for denaturant‐protein interactions. In addition, the conformational stability (ΔGs) as well as the ΔCp for the protein unfolding is quite high, 22.68 kcal/mole and 5.83 kcal/(mole K), respectively, which may be a reflection of the relatively large size of the tetrameric molecule (Mr 120, 000) and a large buried hydrophobic core in the folded protein. This study provides a prototype for determining conformational stability of other members of the short‐chain alcohol dehydrogenase/reductase superfamily of proteins to which PfFabG belongs. iubmb Life, 59: 441‐449, 2007