Krishna P. Bhabak

A Simple and Efficient Strategy To Enhance the Antioxidant Activities of Amino-Substituted Glutathione Peroxidase Mimics

The glutathione peroxidase (GPx) activities of some diaryl diselenides incorporating tertiary amino groups were studied with H(2)O(2), Cum-OOH, and tBuOOH as substrates and with PhSH as thiol co-substrate. Simple replacement of a hydrogen atom with a methoxy group dramatically enhances the GPx activity. The introduction of methoxy substituents ortho to selenium in N,N-dialkylbenzylamine-based compounds makes the basicity of the amino groups perfect for the catalysis. The presence of 6-OMe groups prevents possible SeN interactions in the selenols, increasing their zwitterionic characters. The methoxy substituents also protect the selenium in the selenenic acid intermediates from overoxidation to seleninic acids or irreversible inactivation to selenonic acid derivatives. The additional substituents also play a crucial role in the selenenyl sulfide intermediates, by preventing thiol exchange reactions-which would normally lead to an inactivation pathway-at the selenium centers. The strengths of SeN interactions in the selenenyl sulfide intermediates are dramatically reduced upon introduction of the methoxy substituents, which not only reduce the thiol exchange reactions at selenium but also enhance the nucleophilic attack of the incoming thiols at sulfur. The facile attack of thiols at sulfur in the selenenyl sulfides also prevents the reactions between the selenenyl sulfides and H(2)O(2) that can regenerate the selenenic acids (reverse-GPx cycle). These studies reveal that the simple 6-OMe groups play multiple roles in each of the catalytically active intermediates by introducing steric and electronic effects that are required for efficient catalysis.

Amide-Based Glutathione Peroxidase Mimics: Effect of Secondary and Tertiary Amide Substituents on Antioxidant Activity

A series of secondary and tertiary amide-substituted diselenides were synthesized and studied for their GPx-like antioxidant activities using H(2)O(2), Cum-OOH, and tBuOOH as substrates and PhSH as thiol co-substrate. The effect of substitution at the free -NH group of the amide moiety in the sec-amide-based diselenides on GPx activity was analyzed by detailed experimental and theoretical methods. It is observed that substitution at the free -NH group significantly enhances the GPx-like activities of the sec-amide-based diselenides, mainly by reducing the Se...O nonbonded interactions. The reduction in strength of the Se...O interaction upon introduction of N,N-dialkyl substituents not only prevents the undesired thiol exchange reactions, but also reduces the stability of selenenyl sulfide intermediates. This leads to a facile disproportionation of the selenenyl sulfide to the corresponding diselenide, which enhances the catalytic activity. The mechanistic investigations indicate that the reactivity of diselenides having sec- or tert-amide moieties with PhSH is extremely slow; indicating that the first step of the catalytic cycle involves the reaction between the diselenides and peroxide to produce the corresponding selenenic and seleninic acids.

Synthesis and structure-activity correlation studies of secondary- and tertiary-amine-based glutathione peroxidase mimics.

In this study, a series of secondary- and tertiary-amino-substituted diaryl diselenides were synthesized and studied for their glutathione peroxidase (GPx) like antioxidant activities with H(2)O(2), cumene hydroperoxide, or tBuOOH as substrates and with PhSH or glutathione (GSH) as thiol cosubstrates. This study reveals that replacement of the tert-amino groups in benzylamine-based diselenides by sec-amino moieties drastically enhances the catalytic activities in both the aromatic thiol (PhSH) and GSH assay systems. Particularly, the N-propyl- and N-isopropylamino-substituted diselenides are 8-18 times more active than the corresponding N,N-dipropyl- and N,N-diisopropylamine-based compounds in all three peroxide systems when GSH is used as the thiol cosubstrate. Although the catalytic mechanism of sec-amino-substituted diselenides is similar to that of the tert-amine-based compounds, differences in the stability and reactivity of some of the key intermediates account for the differences in the GPx-like activities. It is observed that the sec-amino groups are better than the tert-amino moieties for generating the catalytically active selenols. This is due to the absence of any significant thiol-exchange reactions in the selenenyl sulfides derived from sec-amine-based diselenides. Furthermore, the seleninic acids (RSeO(2)H) derived from the sec-amine-based compounds are more stable toward further reactions with peroxides than their tert-amine-based analogues.

A synthetic model for the inhibition of glutathione peroxidase by antiarthritic gold compounds.

In this paper, inhibition of the glutathione peroxidase activity of two synthetic organoselenium compounds, bis[2-(N,N-dimethylamino)benzyl]diselenide (5) and bis[2-(N,N-dimethylamino)benzyl]selenide (9), by gold(I) thioglucose (1), chloro(triethylphosphine)gold(I), chloro(trimethylphosphine)gold(I), and chloro(triphenylphosphine)gold(I) is described. The inhibition is found to be competitive with respect to a peroxide (H(2)O(2)) substrate and noncompetitive with respect to a thiol (PhSH) cosubstrate. The diselenide 5 reacts with PhSH to produce the corresponding selenol (6), which upon treatment with 1 equiv of gold(I) chlorides produces the corresponding gold selenolate complexes 11-13. However, the addition of 1 equiv of selenol 6 to complexes 11-13 leads to the formation of bis-selenolate complex 14 by ligand displacement reactions involving the elimination of phosphine ligands. The phosphine ligands eliminated from these reactions are further converted to the corresponding phosphine oxides (R(3)P=O) and selenides (R(3)P=Se). In addition to the replacement of the phosphine ligand by selenol 6, an interchange between two different phosphine ligands is also observed. For example, the reaction of complex 11 having a trimethylphosphine ligand with triphenylphosphine produces complex 13 by phosphine interchange reactions via the formation of intermediates 15 and 16. The reactivity of selenol 6 toward gold(I) phosphines is found to be similar to that of selenocysteine.

Effective inhibition of acid and neutral ceramidases by novel B-13 and LCL-464 analogues

Induction of apoptosis mediated by the inhibition of ceramidases has been shown to enhance the efficacy of conventional chemotherapy in several cancer models. Among the inhibitors of ceramidases reported in the literature, B-13 is considered as a lead compound having good in vitro potency towards acid ceramidase. Furthermore, owing to the poor activity of B-13 on lysosoamal acid ceramidase in living cells, LCL-464 a modified derivative of B-13 containing a basic ω-amino group at the fatty acid was reported to have higher potency towards lysosomal acid ceramidase in living cells. In a search for more potent inhibitors of ceramidases, we have designed a series of compounds with structural modifications of B-13 and LCL-464. In this study, we show that the efficacy of B-13 in vitro as well as in intact cells can be enhanced by suitable modification of functional groups. Furthermore, a detailed SAR investigation on LCL-464 analogues revealed novel promising inhibitors of aCDase and nCDase. In cell culture studies using the breast cancer cell line MDA-MB-231, some of the newly developed compounds elevated endogenous ceramide levels and in parallel, also induced apoptotic cell death. In summary, this study shows that structural modification of the known ceramidase inhibitors B-13 and LCL-464 generates more potent ceramidase inhibitors that are active in intact cells and not only elevates the cellular ceramide levels, but also enhances cell death.

Novel amide- and sulfonamide-based aromatic ethanolamines: effects of various substituents on the inhibition of acid and neutral ceramidases.

In the present study we describe the design and synthesis of a series of amide- and sulfonamide-based compounds as inhibitor of recombinant acid and neutral ceramidases. Inhibition of ceramidases has been shown to induce apoptosis and to increase the efficacy of conventional chemotherapy in several cancer models. B-13, lead in vitro inhibitor of acid ceramidase has been recently shown to be virtually inactive towards lysosomal acid ceramidase in living cells at lower concentrations and for a shorter time of treatment, suggesting the development of more potent inhibitors. In this study, a detailed SAR investigation has been performed to understand the effect of different substituents on the phenyl ring of amide- and sulfonamide-based compounds that partially resemble the structure of well-known inhibitors such as B-13, D-e-MAPP as well as NOE. Our results suggest that the electronic effects of the substituents on phenyl ring in B-13 and D-e-MAPP analogues have negligible effects either in enhancing the inhibition potencies or for selectivity towards aCDase over nCDase. However, the hydrophobicity and the steric effects of longer alkyl chains (n-Pr, n-Bu or t-Bu groups) at the phenyl ring were found to be important for an enhanced selectivity towards aCDase over nCDase.

Novel fluorescent ceramide derivatives for probing ceramidase substrate specificity.

Ceramidases are key regulators of cell fate. The biochemistry of different ceramidases and of their substrate ceramide appears to be complex, mainly due to specific biophysical characteristics at the water-membrane interface. In the present study, we describe the design and synthesis of a set of fluorescently labeled ceramides as substrates for acid and neutral ceramidases. For the first time we have replaced the commonly used polar NBD-dye with the lipophilic Nile Red (NR) dye. Analysis of kinetic data reveal that although both the dyes do not have any noticeable preference for the substitution at acyl or sphingosine (Sph) part in ceramide towards hydrolysis by acid ceramidase, the ceramides with acyl-substituted NBD and Sph-substituted NR dyes have been found to be a better substrate for neutral ceramidase.

Development of a novel FRET probe for the real-time determination of ceramidase activity.

Fretful novelty: We developed two novel doubly labelled fluorescent ceramide analogues that exhibit significant FRET and undergo hydrolysis by ceramidases. We present a fluorescent sphingolipid FRET probe that allows homogeneous ratiometric determination of enzyme activity in real-time.

Novel Drugs Targeting Sphingolipid Metabolism

While the evidence for an involvement of sphingolipids (SLs) in a variety of diseases is rapidly increasing, the development of sphingolipid-related drugs is still in its infancy. In fact, the recently FDA-approved fingolimod or FTY-720 (see chapter by J. Pfeilschifter for more information) is the first drug on the market to interfere with sphingolipid signaling. The reasons for this lagging are manifold and within this chapter we try to name some of them. Ceramide is in the center of sphingolipid metabolism. We describe the most important and most recent inhibitors for enzymes controlling cellular ceramide levels.

Atom based 3D-QSAR studies on 2,4-dioxopyrimidine-1-carboxamide analogs: Validation of experimental inhibitory potencies towards acid ceramidase

Ceramide (Cer), the central lipid molecule in sphingolipid biosynthesis and degradation, which plays a key role in sphingolipid signaling, induces cell differentiation and apoptosis. Cellular degradation of ceramide to sphingosine is catalyzed by a family of ceramidases (CDases). Pharmacological inhibition of ceramidases and more particularly, acid ceramidase (aCDase) is suggestive of a chemotherapeutic approach as it increases the cellular concentration of ceramide inducing apoptosis. In the present report, we have utilized atom-based 3D-QSAR method to analyze the structural aspects on a series of 2,4-dioxopyrimidine-1-carboxamide (carmofur) derivatives as potent inhibitors of aCDase. In this approach the experimental dataset was divided into training (83%) and test (17%) sets and the best model was chosen based on randomized trial distributions consisting of five compounds in a test set with a wide range of activity profile and superior values of statistical parameters such as Q(2) and R(2) values. The reported experimental results by Piomelli and co-workers on the inhibition of aCDase by the carmofur derivatives were correlated using robust 3D-QSAR as well as docking methods. With careful structure-activity correlation studies the carmofur analogs were classified into four sub-categories (Set 1-4) to understand the effect of each structural features separately. This approach led us to short-list most active carmofur derivatives such as compounds 26, 30 and 32 with the incorporation of more than one structural features in a single molecule. However, the inhibition potency might further be enhanced by designing compound 33 upon the incorporation of all features in a single compound. Compound 33 that was missing in the experimental study by Piomelli and co-workers (J. Med. Chem. 2013, 56, 3518), could be identified using 3D-QSAR studies. Moreover, the importance of structural features in lead inhibitors such as 26, 30 and 32 along with 33 was further justified by their efficient molecular interactions at the active site of homology modeled protein human N-acyl ethanolamine hydrolyzing acid amidase (hNAAA) as evidenced by molecular docking study. Furthermore, efficient molecular interaction of some representative inhibitors with hNAAA led to the understanding that hNAAA could be a possible alternative of aCDase for developing potent inhibitors.