Anil Kumar Shukla

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.

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.

Evaluation of plumbagin and its derivative as potential modulators of redox thiol metabolism of Leishmania parasite

Trypanothione and trypanothione reductase (TryR)-based redox metabolism found in Leishmania and other trypanosomatids exemplify the unique features of this group of organisms. Its absence in mammalian hosts, together with the sensitivity of trypanosomes against oxidative stress, makes this enzyme a unique target for exploitation for potential antileishmanial chemotherapeutics. Plumbagin, a plant-derived naphthoquinone, is reported to possess antileishmanial properties by inhibiting TryR. We here report the kinetics of the inhibitory mechanism of plumbagin and its derivative, 2-methoxy 1, 4-naphthoquinone. Interestingly, apart from acting as inhibitor, these compounds also act as subversive substrates and subvert the physiological function of enzyme by converting it from an antioxidant to a prooxidant. Both naphthoquinones show a significant effect on redox homeostasis and results in increased reactive oxygen species, resulting in morphological changes and parasite death.

Deciphering molecular mechanism underlying antileishmanial activity of Nyctanthes arbortristis, an Indian medicinal plant

ETHNOPHARMACOLOGICAL RELEVANCE Nyctanthes arbortristis L. (Oleaceae) is widely used in the traditional medicine of India. The plant is shown to have antibacterial and antileishmanial activities. AIM OF THE STUDY Evaluation of iridoid glucosides from the plant as inhibitor of trypanothione reductase (TryR), a validated drug target enzyme of the Leishmania parasite. The study contributes towards understanding mechanism of antileishmanial effect of the plant. MATERIALS AND METHODS TryR of Leishmania parasite is expressed and purified. Iridoid glucosides are isolated from the plant and tested as inhibitor of TryR enzyme of the parasite. RESULTS Inhibitory constant (K(i)) of various iridoid glucosides ranges from 3.24±0.05 μM to 6.49±0.05 μM. Thus, the molecular mechanism underlying antileishmanial activity of these compounds is mediated through inhibition of TryR. CONCLUSION The current study also points out towards potential application of iridoid glucosides as novel drugs against the disease.

Screening natural products database for identification of potential antileishmanial chemotherapeutic agents

Leishmaniasis is a parasitic infection caused by unicellular protozoan organism belonging to the family Trypanosomatidae. Among various forms of the disease, visceral leishmaniasis is the most lethal and caused by Leishmania infantum or Leishmania donovani. The redox metabolism of parasite requires a key enzyme, trypanothione reductase which is a validated drug target. In the past decade, it was observed that these protozoan parasites had developed resistance against many of available drugs. Importantly in the case of visceral leishmaniasis drug resistance is very high in the Indian subcontinent, a major endemic region of Leishmania donovani infection. In search for new drugs, we aim to identify potential natural product inhibitors of trypanothione reductase which can be further developed as anti-leishmanial drug. We have performed in silico virtual screening of a natural product data set of 800 diverse chemical entities. Leishmania infantum trypanothione reductase crystal structure (PDB ID: 2JK6) was used in the virtual screening process, docking studies to identify potential lead compounds. The compounds were sorted based upon their binding energy and the top 50 ranked protein-inhibitor complexes were clustered using AuPosSOM to ligand foot print the interactions. We report a few alkaloids and sterols for the first time, which could be potential trypanothione reductase inhibitors. The footprinting of protein-inhibitor interactions into clusters has also provided clues on various possible orientations that inhibitors can attain at the active site of Trypanothione reductase. Moreover, biological significance of the interactions has also been discussed.

Nanospheres encapsulating anti-leishmanial drugs for their specific macrophage targeting, reduced toxicity, and deliberate intracellular release.

The current work focuses on the study of polymeric, biodegradable nanoparticles (NPs) for the encapsulation of doxorubicin and mitomycin C (anti-leishmanial drugs), and their efficient delivery to macrophages, the parasites home. The biodegradable polymer methoxypoly-(ethylene glycol)-b-poly (lactic acid) (MPEG-PLA) was used to prepare polymeric NPs encapsulating doxorubicin and mitomycin C. The morphology, mean diameter, and surface area of spherical NPs were determined by transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), and BET surface area analysis. X-ray diffraction was performed to validate drug encapsulation. An in vitro release profile of the drugs suggested a fairly slow release. These polymeric NPs were efficiently capable of releasing drug inside macrophages at a slower pace than the free drug, which was monitored by epi-fluorescence microscopy. Encapsulation of doxorubicin and mitomycin C into NPs also decreases cellular toxicity in mouse macrophages (J774.1A).

Biophysical and Folding Parameters of Trypanothione Reductase from Leishmania infantum

Out of various tropical diseases caused by trypanosomatids, leishmaniasis is a life-threatening disease caused by the leishmania parasite. We are targeting the thiol metabolic pathway of the parasite for drug development, and trypanothione reductase (TryR) is a key enzyme of this pathway. It is important to gather significant knowledge about biophysical and intrinsic properties of this enzyme which will be helpful in better understanding of this drug-target enzyme. We report here the modulation of activity and stability of TryR from Leishmania infantum in the presence of various denaturants and pHs. The enzyme is quite stable under high concentration of denaturants and showed better stability compared to TryR of Leishmania donovani, whose sequence differs at only on position (Ala363→Gly). Structural basis of the destabilizing effects is discussed.

A simple method based on multiple alignment and phylogeny to derive a correlation between the protein fold and sequence via motif search

Predicting information regarding the structure of the protein from its sequence still remains an uphill task. Though both are intimately linked, it has been found difficult so far to get a direct correlation between the two. In our present approach we use a simple method based on multiple alignment and phylogeny to derive a correlation between the protein structure and sequence via motif search. The protein families which we have considered are SH2 like, Homeodomain, Leucine rich repeat, Alphabeta knot trefoilknot and ferritin like helix bundle. We have been able to successfully predict the protein families with an average prediction of accuracy of 81%, the highest being 89% and the lowest being 73% on our test data set.