Cloning, purification, and homology modeling of Histone deacetylase in Leishmania donovani.

Neglected diseases, such as leishmaniasis, are still a major health problem in poor countries. To date, there is a severe lack of effective, safe, and affordable treatment for leishmaniasis. Currently, there are very limited chemotherapeutic options, and the development of vaccines is still underway. Hence, novel therapeutic strategies need to be developed against leishmanial parasites. Histone deacetylases (HDACs), silent regulators of many critical pathways, have been validated as potential therapeutic targets in cancer and several parasitic diseases. In the present work, we have isolated and characterized biologically active Zn2+-dependent HDAC protein from leishmania that can be studied further as a potential anti-leishmanial drug target to develop new therapies against neglected diseases. The nucleotide sequence of the HDAC gene with no intervening sequence was amplified, cloned in a pET-28a vector, and later transformed into the BL21(DE3) competent E. coli bacterial cells. After transformation, the cells were cultured and induced with 0.6 mM of IPTG to express histidine-tagged HDAC protein (LD_HDAC), which was later purified using nickel affinity chromatography. The approximate protein size confirmed with the help of 10% SDS-PAGE was ~48.0 kDa. The enzymatic assay using the purified protein confirmed it as biologically active. A three dimensional structure of LD_HDAC was modeled using the crystal structure of HDAC2 protein of Homo sapiens (PDB ID: 6G3O). This protein can be utilized for the screening of Leishmania-specific HDAC inhibitors.

Synthesis, characterization, and mechanistic studies of a gold nanoparticle-amphotericin B covalent conjugate with enhanced antileishmanial efficacy and reduced cytotoxicity.

BACKGROUND: Amphotericin B (AmB) as a liposomal formulation of AmBisome is the first line of treatment for the disease, visceral leishmaniasis, caused by the parasite Leishmania donovani. However, nephrotoxicity is very common due to poor water solubility and aggregation of AmB. This study aimed to develop a water-soluble covalent conjugate of gold nanoparticle (GNP) with AmB for improved antileishmanial efficacy and reduced cytotoxicity. METHODS: Citrate-reduced GNPs (~39 nm) were functionalized with lipoic acid (LA), and the product GNP-LA (GL ~46 nm) was covalently conjugated with AmB using carboxyl-to-amine coupling chemistry to produce GNP-LA-AmB (GL-AmB ~48 nm). The nanoparticles were characterized by dynamic light scattering, transmission electron microscopy (TEM), and spectroscopic (ultraviolet-visible and infrared) methods. Experiments on AmB uptake of macrophages, ergosterol depletion of drug-treated parasites, cytokine ELISA, fluorescence anisotropy, flow cytometry, and gene expression studies established efficacy of GL-AmB over standard AmB. RESULTS: Infrared spectroscopy confirmed the presence of a covalent amide bond in the conjugate. TEM images showed uniform size with smooth surfaces of GL-AmB nanoparticles. Efficiency of AmB conjugation was ~78%. Incubation in serum for 72 h showed <7% AmB release, indicating high stability of conjugate GL-AmB. GL-AmB with AmB equivalents showed ~5-fold enhanced antileishmanial activity compared with AmB against parasite-infected macrophages ex vivo. Macrophages treated with GL-AmB showed increased immunostimulatory Th1 (IL-12 and interferon-γ) response compared with standard AmB. In parallel, AmB uptake was ~5.5 and ~3.7-fold higher for GL-AmB-treated (P<0.001) macrophages within 1 and 2 h of treatment, respectively. The ergosterol content in GL-AmB-treated parasites was ~2-fold reduced compared with AmB-treated parasites. Moreover, GL-AmB was significantly less cytotoxic and hemolytic than AmB (P<0.01). CONCLUSION: GNP-based delivery of AmB can be a better, cheaper, and safer alternative than available AmB formulations.