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The illness known as leishmaniasis has not become a household name like malaria, although it stands as the second-largest parasitic disease, surpassed only by malaria. As no licensed vaccine is available, treatment for leishmaniasis mostly relies on chemotherapy. Inefficiency and drug resistance are the major impediments in current therapeutics. In this scenario, identification of novel molecular drug candidates is indispensable to develop robust antileishmanials. The exploration of structure-based drugs to target enzymes/molecules of Leishmania which differ structurally/functionally from their equivalents in mammalian hosts not only helps in developing a new class of antileishmanials, but also paves the way to understand Leishmania biology. This review provides a comprehensive overview on possible drug candidates relating to various Leishmania molecular pathways.

The illness known as leishmaniasis has not become a household name like malaria, although it stands as the second-largest parasitic disease, surpassed only by malaria. As no licensed vaccine is available, treatment for leishmaniasis mostly relies on chemotherapy. Inefficiency and drug resistance are the major impediments in current therapeutics. In this scenario, identification of novel molecular drug candidates is indispensable to develop robust antileishmanials. The exploration of structure-based drugs to target enzymes/molecules of

Visceral Leishmaniasis is a deadly parasitic disease caused by Leishmania donovani. Paucity exists in the discovery of novel chemotherapeutics against Leishmaniasis. In this study, we synthesized a natural product inspired Diversity Oriented Synthesis library of L. donovani Trypanothione reductase (LdTR) inhibitor β-carboline-quinazolinone hybrids, which are different in stereochemical architecture and diverse in the bioactive chemical space. It is noteworthy that chirality affects drug-to-protein binding affinity since proteins in any living system are present only in one of the chiral forms. Upon evaluation of the hybrids, one of the chiral forms i.e. Compound 1 showed profound cytotoxic effect in micromolar range as compared to its other chiral form i.e. Compound 2. In-silico docking studies confirmed high binding efficiency of Compound 1 with the catalytic pocket of LdTR. Treatment of L. donovani parasites with Compound 1 inhibits LdTR activity, induces imbalance in redox homeostasis by enhancing ROS, disrupts the mitochondrial membrane potential, modifies actin polymerization and alters the surface topology and architecture. All these cellular modifications eventually led to apoptosis-like death of promastigotes. Furthermore, we synthesized the analogues of Compound 1 and found that these compounds show profound antileishmanial activity in the nanomolar range both in promastigotes and intracellular amastigotes. The enhanced inhibitory potential of these compounds was further supported by in-silico analysis of protein-ligand interactions which revealed high binding efficiency towards the catalytic pocket of LdTR. Taken together, this study reports the serendipitous discovery of β-carboline-quinazolinone hybrids with enhanced antileishmanial activity along with the in-depth structure-activity relationships and mechanism of action of these analogues.

Infectious diseases are menace to the mankind, having a major contribution to the human morbidity and mortality. Trypanosomatids have a pervasive effect in the world, causing devastating but neglected diseases such as leishmaniasis, Chagas disease, and African sleeping sickness affecting 27 million people worldwide with 150,000 deaths annually. Trypanosomatids developing drug resistance is the current bottleneck in providing promising chemotherapeutics for these diseases which forces the continuous quest for new drugs and drug candidates. Balancing redox homeostasis is crucial for cell survival which has various implications in the biology of these parasites. Reactive oxygen species (ROS) act as signaling molecules, involving in various pathways and crucial for survival. Conversely, various chemotherapeutic drugs against trypanosomatids-caused ROS induction result in oxidative stress, eventually leading to apoptotic manifestations. Oxidative stress is one of the host defense mechanisms to control the infection, while detoxification is one of the crucial counteracts at the parasite front for successful host-parasite interaction. Therefore, oxidative stress is a good tool for better understanding of parasite biology, pathogenesis, and host-pathogen interactions. It is noteworthy that trypanosomatids have divergence from all other prokaryotes and eukaryotes at their redox system, majorly trypanothione-trypanothione reductase (TR)-based redox metabolism. The absence of this system in mammalians and structural/functional differences from host enzymes make it a lucrative target for studying its role in oxidative stress control and also to develop effective chemotherapeutics. One of the causes for drug resistance of trypanosomatids is due to their action of inducing oxidative stress which in turn activates repair mechanisms resulting in the development of drug resistance. Hence, studying oxidative stress mechanism of trypanosomatids gives insights into drug resistance, which is an impendence in attaining efficacious chemotherapy. In this chapter, we have tried to give an outline of the significance of redox stress and its role in different cellular metabolisms of trypanosomatids, with a special focus on trypanothione-trypanothione reductase (TR)-based redox system as a peculiar system to study trypanosomatids oxidative stress mechanism, also for drug designing.

Bioisosterism is a useful strategy in rational drug design to improve pharmacodynamic and pharmacokinetic properties of lead compounds. Imidazolidinones have been reported as potent kinase inhibitors and antileishmanial agents. In this study, bioisosteres of imidazolidinones (compounds 1-3) were evaluated for their antileishmanial properties. The modified imidazolidinones exhibited potent antileishmanial activity against extracellular as well as intracellular Leishmania donovani parasites in nanomolar concentrations. The selectivity index of these compounds on host cells was found to be more than 1000, emphasizing their specificity toward the parasite. Using SwissTargetPrediction software, we assessed the potential targets of these compounds and found MAPK as the most probable target. To in vitro validate, we developed a novel in vitro kinase assay that mimics the in vivo nature of the functional kinome. Compounds 1-3 displayed specific inhibition of parasite kinase activity accompanied by an increase in intracellular sodium levels in the parasites. This might be the effect of kinase inhibition that regulates sodium homeostasis through Na-ATPases. Finally, the compound-treated parasites underwent apoptosis-like death. This study represents bioisoterism as a novel approach for drug design to establish the structure-activity relationship, which in turn helps to improve the therapeutic activity of lead compounds.

Palmitoylation has been recently identified as an important post-translational rheostat for controlling protein function in eukaryotes. However, the molecular machinery underlying palmitoylation remains unclear in the neglected tropical parasite, Leishmania donovani. Herein, we have identified a catalog of 20 novel palmitoyl acyltransferases (PATs) and characterized the promastigote-specific PAT (LdPAT4) containing the canonical Asp-His-His-Cys (DHHC) domain. Immunofluorescence analysis using in-house generated LdPAT4-specific antibody demonstrated distinct expression of LdPAT4 in the flagellar pocket of promastigotes. Using metabolic labeling-coupled click chemistry method, the functionality of this recombinant enzyme could be authenticated in E. coli strain expressing LdPAT4-DHHC domain. This was evident by the cellular uptake of palmitic acid analogs, which could be successfully inhibited by 2-BMP, a PAT-specific inhibitor. Using CSS-Palm based in-silico proteomic analysis, we could predict up to 23 palmitoylated sites per protein in the promastigotes, and further identify distinctive palmitoylated protein clusters involved in microtubule assembly, flagella motility and vesicular trafficking. To highlight, proteins such as Flagellar Member proteins (FLAM1, FLAM5), Intraflagellar Transport proteins (IFT88), and flagellar motor assembly proteins including the Dynein family were found to be enriched. Furthermore, analysis of global palmitoylation in promastigotes using Acyl-biotin exchange purification identified a set of S-palmitoylated proteins overlapping with the in-silico proteomics data. The attenuation of palmitoylation using 2-BMP demonstrated several phenotypic alterations in the promastigotes including distorted morphology, reduced motility (flagellar loss or slow flagellar beating), and inefficient invasion of promastigotes to host macrophages. These analyses confirm the essential role of palmitoylation in promastigotes. In summary, the findings suggest that LdPAT4 acts as a functional acyltransferase that can regulate palmitoylation of proteins involved in parasite motility and invasion, thus, can serve as a potential target for designing chemotherapeutics in Visceral Leishmaniasis.

Plasmodium falciparum perforin like proteins (PfPLPs) are an important arsenal for the entry and exit of malaria parasites. These proteins bind and oligomerize on the membrane in calcium dependent manner and form an open pore. The calcium dependent pore forming activity of PLPs is usually conferred by their C2 like C-terminal domain. Here, we have tried to elucidate the calcium binding residues in the C-terminal domain of PfPLP1, a member of P. falciparum PLPs, playing a crucial role in calcium dependent egress of blood stage parasites. Through our in silico study, we have found that the C-terminal domain of all PfPLPs is rich in β-pleated sheets and is structurally similar to C2 domain of human perforin. Furthermore, homology search based on 3-D structure of PfPLP1 confirmed that it is structurally homologous to the calcium binding C2 domain of many proteins. On further elucidation of the calcium-binding pocket of the C2 like domain of PfPLP1 showed that it binds to two calcium molecules. The calcium-binding pocket could be a target of novel chemotherapeutics for studying functional role of PfPLPs in parasite biology as well as for limiting blood stage growth of malaria parasite.