Vikash Kumar Dubey

Glutaraldehyde-Activated Chitosan Matrix for Immobilization of a Novel Cysteine Protease, Procerain B

Proteases have several applications in the food industry. We report the immobilization of procerain B, a novel cysteine protease, on glutaraldehyde-activated chitosan beads through covalent attachment. Glutaraldehyde not only serves as a cross-linking agent but also links the procerain B on the surface of bead through primary amine group (either lysine side chain or N-terminal) by Schiff base linkage. Immobilized procerain B was characterized for optimum functional range and stability with respect to pH and temperature. The chitosan-immobilized procerain B has broad pH and thermal optima. The effects of substrate concentration and reusability of immobilized beads were also studied. It showed nearly 50% activity until the 10th use.

Rational approaches for drug designing against leishmaniasis.

Leishmaniasis has been ignored for many years mainly because it plagues remote and poor areas. However, recently, it has drawn attention of several investigators, and active research is going on for antileishmanial drug discovery. The current available drugs have high failure rates and significant side effects. Recently, liposomal preparations of amphotericin B are available and have proved to be a better drug, but they are very expensive. Miltefosine is one of the few orally administered drugs that are effective against Leishmania. However, it has exhibited teratogenicity, hence, should not be administered to pregnant women. Thus, the search for novel and improved antileishmanial drugs continue. A rational approach to design and develop new antileishmanials can be to identify several metabolic and biochemical differences between host and parasite that can be exploited as drug target. Moreover, many natural products also have significant antileishmanial activity and are yet to be exploited. In the current review, we aim to bring together various drug targets of Leishmania, recent development in the field, future prospects, and hope in the area.

Molecular docking studies of selected tricyclic and quinone derivatives on trypanothione reductase of Leishmania infantum

Visceral leishmaniasis, most lethal form of Leishmaniasis, is caused by Leishmania infantum in the Old world. Current therapeutics for the disease is associated with a risk of high toxicity and development of drug resistant strains. Thiol‐redox metabolism involving trypanothione and trypanothione reductase, key for survival of Leishmania, is a validated target for rational drug design. Recently published structure of trypanothione reductase (TryR) from L. infantum, in oxidized and reduced form along with Sb(III), provides vital clues on active site of the enzyme. In continuation with our attempts to identify potent inhibitors of TryR, we have modeled binding modes of selected tricyclic compounds and quinone derivatives, using AutoDock4. Here, we report a unique binding mode for quinone derivatives and 9‐aminoacridine derivatives, at the FAD binding domain. A conserved hydrogen bonding pattern was observed in all these compounds with residues Thr335, Lys60, His461. With the fact that these residues aid in the orientation of FAD towards the active site forming the core of the FAD binding domain, designing selective and potent compounds that could replace FAD in vivo during the synthesis of Trypanothione reductase can be deployed as an effective strategy in designing new drugs towards Leishmaniasis. We also report the binding of Phenothiazine and 9‐aminoacridine derivatives at the Z site of the protein. The biological significance and possible mode of inhibition by quinone derivatives, which binds to FAD binding domain, along with other compounds are discussed.

Miltefosine-unresponsive Leishmania donovani has a greater ability than miltefosine-responsive L. donovani to resist reactive oxygen species

Resistance of Leishmania parasites to miltefosine, which is only available oral drug, is a great concern. We have analyzed global gene expression profiles of miltefosine‐unresponsive and miltefosine‐responsive Leishmania donovani in order to understand the various metabolic processes involved in miltefosine drug resistance. The microarray data clearly indicated a role of oxidative metabolism in miltefosine resistance. Furthermore, fluorescence microscopy experiments suggested that miltefosine‐unresponsive L. donovani resists the accumulation of reactive oxygen species and subsequent mitochondrial membrane damage leading to apoptotic death. In contrast, in miltefosine‐responsive L. donovani, the accumulation of reactive oxygen species causes apoptotic death. Overall, this study provides fundamental insights into miltefosine resistance in L. donovani.

Molecular Docking and in Vitro Antileishmanial Evaluation of Chromene-2-thione Analogues.

Leishmaniases are an epidemic in various countries, and the parasite is developing resistance against available drugs. Thus, development of new drugs against Leishmania is an open area of investigation for synthetic organic chemists. To meet this challenge, a series of chromene-2-thione derivatives have been synthesized and docked into the active site of trypanothione reductase (TryR) enzyme required for redox balance of the parasite. These were screened on promastigote, axenic amastigote, and intracellular amastigote stages of Leishmania donovani and found to show high levels of antileishmanial activity together with minimal toxicity to human peripheral blood mononuclear cells. Compounds 3b and 3k were found to be the most active among the tested compounds. Although the compounds show moderate antileishmanial activity, they identify a chemical space to design and develop drugs based on these chromene-2-thione derivatives against the Leishmania parasite.

Iridoid glucosides from Nyctanthes arbortristis result in increased reactive oxygen species and cellular redox homeostasis imbalance in Leishmania parasite

We report here the effect of iridoid glucosides, isolated from Nyctanthes arbortristis, on redox homeostasis of Leishmania parasite. These compounds led to an increase in reactive oxygen species by inhibiting a crucial enzyme of redox metabolism of the parasite. Our experiments clearly showed that these compounds are highly active as antileishmanial agents. The in vitro experiments on intra-macrophageal amastigotes showed significant killing of parasite even at very low concentration. Determination of mechanism of action of iridoid glucosides showed that increased ROS level leads to oxidative stress, cell membrane damage and apoptosis of Leishmania sp. Our cellular toxicity assays on Human embryonic kidney (HEK 293) and mouse macrophage (J774A.1) cell lines showed these compounds to be very safe for therapeutics application.

Evaluation of selected antitumor agents as subversive substrate and potential inhibitor of trypanothione reductase: an alternative approach for chemotherapy of Leishmaniasis

Trypanothione reductase (TryR) is a validated drug target against Leishmaniasis. Using integrated computational and experimental approaches, the authors report doxorubicin and mitomycin C, known antitumor agents, as novel inhibitors of TryR of leishmania parasite. Interestingly, these compounds also act as subversive substrates and subvert the physiological function of enzyme by converting it from an anti-oxidant to a pro-oxidant. Possible mechanism of subversive substrate is discussed. Both doxorubicin and mitomycin C show significant effect on redox homeostasis of the parasite and high-leishmanicidal activity. The toxicity studies as well as available toxicity data in literature indicate these compounds to have acceptable toxicity in limited dose.

Molecular mechanism underlying antileishmanial effect of oxabicyclo[3.3.1]nonanones: Inhibition of key redox enzymes of the pathogen

We report oxabicyclo[3.3.1]nonanones as inhibitors of key redox enzymes, trypanothione synthetase (TryS), and trypanothione reductase (TryR) of Leishmania. Further, detailed cellular effects of 4-(4,4,8-Trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2-yl)-benzoic acid methyl ester, a oxabicyclo[3.3.1]nonanones, on the parasite were investigated. As these compounds inhibit key redox enzymes (TryR amd TryS), treatment of these compounds resulted in increased reactive oxygen species (ROS), mitochondrial membrane damage, activation of caspase like proteases, and DNA damage that finally leads to apoptosis. Although the compound has modest IC50 value against parasite (4.9±0.4 μM), they identify a novel chemical space to design and develop drugs based on these compounds against the Leishmania parasite. This is first report of oxabicyclo[3.3.1]nonanones as antileishmanial.

Redesigning symmetry‐related “mini‐core” regions of FGF‐1 to increase primary structure symmetry: Thermodynamic and functional consequences of structural symmetry

Previous reports detailing mutational effects within the hydrophobic core of human acidic fibroblast growth factor (FGF‐1) have shown that a symmetric primary structure constraint is compatible with a stably folded protein. In the present report, we investigate symmetrically related pairs of buried hydrophobic residues in FGF‐1 (termed “mini‐cores”) that are not part of the central core. The effect upon the stability and function of FGF‐1 mutations designed to increase primary structure symmetry within these “mini‐core” regions was evaluated. At symmetry‐related positions 22, 64, and 108, the wild‐type protein contains either Tyr or Phe side chains. The results show that either residue can be readily accommodated at these positions. At symmetry‐related positions 42, 83, and 130, the wild‐type protein contains either Cys or Ile side chains. While positions 42 and 130 can readily accommodate either Cys or Ile side chains, position 83 is substantially destabilized by substitution by Ile. Tertiary structure asymmetry in the vicinity of position 83 appears responsible for the inability to accommodate an Ile side chain at this position, and is known to contribute to functional half‐life. A mutant form of FGF‐1 with enforced primary structure symmetry at positions 22, 64, and 108 (all Tyr) and 42, 83, and 130 (all Cys) is shown to be more stable than the reference FGF‐1 protein. The results support the hypothesis that a symmetric primary structure within a symmetric protein superfold represents a solution to achieving a foldable, stable polypeptide, and highlight the role that function may play in the evolution of asymmetry within symmetric superfolds.

Unraveling the rationale behind organic solvent stability of lipases.

Organic solvent-stable lipases have pronounced impact on industrial economy as they are involved in synthesis by esterification, interesterification, and transesterification. However, very few of such natural lipases have been isolated till date. A study of the recent past provided few pillars to rely on for this work. The three-dimensional structure, inclusive of the surface and active site, of 29 organic solvent-stable lipases was analyzed by subfamily classification and protein solvent molecular docking based on fast Fourier transform correlation approach. The observations revealed that organic solvent stability of lipases is their intrinsic property and unique with respect to each lipase. In this paper, factors like surface distribution of charged, hydrophobic, and neutral residues, interaction of solvents with catalytically immutable residues, and residues interacting with essential water molecules required for lipase activity, synergistically and by mutualism contribute to render a stable lipase organic solvent. The propensity of surface charge in relation to stability in organic solvents by establishing repulsive forces to exclude solvent molecules from interacting with the surface and prohibiting the same from gaining entry to the protein core, thus stabilizing the active conformation, is a new finding. It was also interesting to note that lipases having equivalent surface-exposed positive and negative residues were stable in a wide range of organic solvents, irrespective of their LogP values.