Exploring host epigenetic enzymes as targeted therapies for visceral leishmaniasis: in silico design and in vitro efficacy of KDM6B and ASH1L inhibitors.

In order to combat various infectious diseases, the utilization of host-directed therapies as an alternative to chemotherapy has gained a lot of attention in the recent past, since it bypasses the existing limitations of conventional therapies. The use of host epigenetic enzymes like histone lysine methyltransferases and lysine demethylases as potential drug targets has successfully been employed for controlling various inflammatory diseases like rheumatoid arthritis and acute leukemia. In our earlier study, we have already shown that the functional knockdown of KDM6B and ASH1L in the experimental model of visceral leishmaniasis has resulted in a significant reduction of organ parasite burden. Herein, we performed a high throughput virtual screening against KDM6B and ASH1L using > 53,000 compounds that were obtained from the Maybridge library and PubChem Database, followed by molecular docking to evaluate their docking score/Glide Gscore. Based on their docking scores, the selected inhibitors were later assessed for their in vitro anti-leishmanial efficacy. Out of all inhibitors designed against KDM6B and ASH1L, HTS09796, GSK-J4 and AS-99 particularly showed promising in vitro activity with IC50 < 5 µM against both extracellular promastigote and intracellular amastigote forms of L. donovani. In vitro drug interaction studies of these inhibitors further demonstrated their synergistic interaction with amphotericin-B and miltefosine. However, GSK-J4 makes an exception by displaying an in different mode of interaction with miltefosine. Collectively, our in silico and in vitro studies acted as a platform to identify the applicability of these inhibitors targeted against KDM6B and ASH1L for anti-leishmanial therapy.

Integrated machine learning-based virtual screening and biological evaluation for identification of potential inhibitors against cathepsin K.

Cathepsin K is a type of cysteine proteinase that is primarily expressed in osteoclasts and has a key role in the breakdown of bone matrix protein during bone resorption. Many studies suggest that the deficiency of cathepsin K is concomitant with a suppression of osteoclast functioning, therefore rendering the resorptive properties of cathepsin K the most prominent target for osteoporosis. This innovative work has identified a novel anti-osteoporotic agent against Cathepsin K by using a comparison of machine learning and deep learning-based virtual screening followed by their biological evaluation. Out of ten shortlisted compounds, five of the compounds (JFD02945, JFD02944, RJC01981, KM08968 and SB01934) exhibit more than 50% inhibition of the Cathepsin K activity at 0.1 µM concentration and are considered to have a promising inhibitory effect against Cathepsin K. The comprehensive docking, MD simulation, and MM/PBSA investigations affirm the stable and effective interaction of these compounds with Cathepsin K to inhibit its function. Furthermore, the compounds RJC01981, KM08968 and SB01934 are represented to have promising anti-osteoporotic properties for the management of osteoporosis owing to their significantly well predicted ADMET properties.

Identification of Potential Aldose Reductase Inhibitors Using Convolutional Neural Network-Based in Silico Screening.

Aldose reductase (ALR2) is a notable enzyme of the polyol pathway responsible for aggravating diabetic neuropathy complications. The first step begins when it catalyzes the reduction of glucose to sorbitol with NADPH as a coenzyme. Elevated concentrations of sorbitol damage the tissues, leading to complications like neuropathy. Though considerable effort has been pushed toward the successful discovery of potent inhibitors, its discovery still remains an elusive task. To this end, we present a 3D convolutional neural network (3D-CNN) based ALR2 inhibitor classification technique by dealing with snapshots of images captured from 3D chemical structures with multiple rotations as input data. The CNN-based architecture was trained on the 360 sets of image data along each axis and further prediction on the Maybridge library by each of the models. Subjecting the retrieved hits to molecular docking leads to the identification of the top 10 molecules with high binding affinity. The hits displayed a better blood-brain barrier penetration (BBB) score (90% with more than four scores) as compared to standard inhibitors (38%), reflecting the superior BBB penetrating efficiency of the hits. Followed by molecular docking, the biological evaluation spotlighted five compounds as promising ALR2 inhibitors and can be considered as a likely prospect for further structural optimization with medicinal chemistry efforts to improve their inhibition efficacy and consolidate them as new ALR2 antagonists in the future. In addition, the study also demonstrated the usefulness of scaffold analysis of the molecules as a method for investigating the significance of structurally diverse compounds in data-driven studies. For reproducibility and accessibility purposes, all of the source codes used in our study are publicly available.

Ligand-based in silico identification and biological evaluation of potential inhibitors of nicotinamide N-methyltransferase.

Nicotinamide N-methyltransferase (NNMT) is a protein coding gene, which methylates the nicotinamide (NA) (vitamin B3) to produce 1-methylnicotinamide (MNA). Several studies have suggested that the overexpression of NNMT is associated with different metabolic disorders like obesity and type-2 diabetes thereby making it an important therapeutic target for development of anti-diabetic agents. Here we describe a workflow for identification of new inhibitors of NNMT from a library of small molecules. In this study, we have hypothesized a four-point pharmacophore model based on the pharmacophoric features of reported NNMT inhibitors in the literature. The statistically significant pharmacophore hypothesis was used to explore the Maybridge compound library that resulted in mapping of 1330 hit compounds on the proposed hypothesis. Subsequently, a total of eight high scoring compounds, showing good protein-ligand interactions in the molecular docking study, were selected for biological evaluation of NNMT activity. Eventually, four compounds were found to show significant inhibitory activity for NNMT and can be further explored to design new derivatives around the identified scaffolds with improved activities as NNMT inhibitors.

Recent trends in artificial intelligence-driven identification and development of anti-neurodegenerative therapeutic agents.

Neurological disorders affect various aspects of life. Finding drugs for the central nervous system is a very challenging and complex task due to the involvement of the blood-brain barrier, P-glycoprotein, and the drug's high attrition rates. The availability of big data present in online databases and resources has enabled the emergence of artificial intelligence techniques including machine learning to analyze, process the data, and predict the unknown data with high efficiency. The use of these modern techniques has revolutionized the whole drug development paradigm, with an unprecedented acceleration in the central nervous system drug discovery programs. Also, the new deep learning architectures proposed in many recent works have given a better understanding of how artificial intelligence can tackle big complex problems that arose due to central nervous system disorders. Therefore, the present review provides comprehensive and up-to-date information on machine learning/artificial intelligence-triggered effort in the brain care domain. In addition, a brief overview is presented on machine learning algorithms and their uses in structure-based drug design, ligand-based drug design, ADMET prediction, de novo drug design, and drug repurposing. Lastly, we conclude by discussing the major challenges and limitations posed and how they can be tackled in the future by using these modern machine learning/artificial intelligence approaches.

ß-Sitosterol-D-Glucopyranoside Mimics Estrogenic Properties and Stimulates Glucose Utilization in Skeletal Muscle Cells.

Estrogenic molecules have been reported to regulate glucose homeostasis and may be beneficial for diabetes management. Here, we investigated the estrogenic effect of ß-sitosterol-3-O-D-glucopyranoside (BSD), isolated from the fruits of Cupressus sempervirens and monitored its ability to regulate glucose utilization in skeletal muscle cells. BSD stimulated ERE-mediated luciferase activity in both ERα and ERß-ERE luc expression system with greater response through ERß in HEK-293T cells, and induced the expression of estrogen-regulated genes in estrogen responsive MCF-7 cells. In silico docking and molecular interaction studies revealed the affinity and interaction of BSD with ERß through hydrophobic interaction and hydrogen bond pairing. Furthermore, prolonged exposure of L6-GLUT4myc myotubes to BSD raised the glucose uptake under basal conditions without affecting the insulin-stimulated glucose uptake, the effect associated with enhanced translocation of GLUT4 to the cell periphery. The BSD-mediated biological response to increase GLUT4 translocation was obliterated by PI-3-K inhibitor wortmannin, and BSD significantly increased the phosphorylation of AKT (Ser-473). Moreover, BSD-induced GLUT4 translocation was prevented in the presence of fulvestrant. Our findings reveal the estrogenic activity of BSD to stimulate glucose utilization in skeletal muscle cells via PI-3K/AKT-dependent mechanism.

Computational Design of Biologically Active Anticancer Peptides and Their Interactions with Heterogeneous POPC/POPS Lipid Membranes.

Over the last few decades, anticancer peptides (ACPs) have turned into potential warheads against cancer. Apart from small molecules and monoclonal antibodies, ACPs have been proven to be effective against cancer cells. ACPs are small cationic peptides that selectively bind to the negatively charged cancer cell membrane and kill them by various mechanisms. In the present study, we prepared a random scrambled library of 1200 peptides from the 100 known ACPs and virtually screened them for their anticancer properties. From in silico-predicted ACPs, 27 peptides were prioritized based on their support vector machine (SVM) score. Based on the SVM score and properties such as hydrophobicity, size, overall net charge, secondary structure, and synthetic feasibility, finally, four peptides were synthesized and screened for their biological activities. Cancer cell membrane-deforming potential of two most active peptides, peptide1 and peptide2 was assessed with molecular dynamics simulation. We found that peptide1 remains adsorbed to the membrane surface, while peptide2 has membrane penetration capability. The present study will be helpful in the computational design of ACPs and understanding their interaction with the cancerous cell's membrane.

Ammonium trichloro [1,2-ethanediolato-O,O']-tellurate cures experimental visceral leishmaniasis by redox modulation of Leishmania donovani trypanothione reductase and inhibiting host integrin linked PI3K/Akt pathway.

In an endeavor to search for affordable and safer therapeutics against debilitating visceral leishmaniasis, we examined antileishmanial potential of ammonium trichloro [1,2-ethanediolato-O,O']-tellurate (AS101); a tellurium based non toxic immunomodulator. AS101 showed significant in vitro efficacy against both Leishmania donovani promastigotes and amastigotes at sub-micromolar concentrations. AS101 could also completely eliminate organ parasite load from L. donovani infected Balb/c mice along with significant efficacy against infected hamsters (˃93% inhibition). Analyzing mechanistic details revealed that the double edged AS101 could directly induce apoptosis in promastigotes along with indirectly activating host by reversing T-cell anergy to protective Th1 mode, increased ROS generation and anti-leishmanial IgG production. AS101 could inhibit IL-10/STAT3 pathway in L. donovani infected macrophages via blocking α4ß7 integrin dependent PI3K/Akt signaling and activate host MAPKs and NF-κB for Th1 response. In silico docking and biochemical assays revealed AS101's affinity to form thiol bond with cysteine residues of trypanothione reductase in Leishmania promastigotes leading to its inactivation and inducing ROS-mediated apoptosis of the parasite via increased Ca2+ level, loss of ATP and mitochondrial membrane potential along with metacaspase activation. Our findings provide the first evidence for the mechanism of action of AS101 with excellent safety profile and suggest its promising therapeutic potential against experimental visceral leishmaniasis.

Molecular, biochemical characterization and assessment of immunogenic potential of cofactor-independent phosphoglycerate mutase against Leishmania donovani: a step towards exploring novel vaccine candidate.

Despite immense efforts, vaccine against visceral leishmaniasis has yet not been developed. Earlier our proteomic study revealed a novel protein, cofactor-independent phoshoglycerate mutase (LdiPGAM), an important enzyme in glucose metabolism, in T helper cells type 1 (Th1) stimulatory region of soluble Leishmania donovani antigen. In this study, LdiPGAM was biochemically and molecularly characterized and evaluated for its immunogenicity and prophylactic efficacy against L. donovani. Immunogenicity of recombinant LdiPGAM (rLdiPGAM) was initially assessed in naïve hamsters immunized with it by analysing mRNA expression of inducible nitric oxide (NO) synthase (iNOS) and other Th1/T helper cells type 2 cytokines, which revealed an upregulation of Th1 cytokines along with iNOS. Immunogenicity of rLdiPGAM was further evaluated in lymphocytes of treated Leishmania-infected hamsters and peripheral blood mononuclear cells of Leishmania patients in clinical remission by various parameters, viz. lymphoproliferation assay and NO production (hamsters and patients) and levels of various cytokines (patients). rLdiPGAM induced remarkable Lymphoproliferative response and NO production in treated Leishmania-infected hamsters as well as in patients and increase in interferon gamma (IFN-γ), interleukin-12 (IL-12p40) responses in Leishmania patients in clinical remission. Vaccination with rLdiPGAM exerted considerable prophylactic efficacy (73%) supported by increase in mRNA expression of iNOS, IFN-γ and IL-12p40 with decrease in transforming growth factor beta and interleukin-10. Above results indicate the importance of rLdiPGAM protein as a potential vaccine candidate against visceral leishmaniasis.

Polypeptide release factors and stop codon recognition in the apicoplast and mitochondrion of Plasmodium falciparum.

Correct termination of protein synthesis would be a critical step in translation of organellar open reading frames (ORFs) of the apicoplast and mitochondrion of the malaria parasite. We identify release factors (RFs) responsible for recognition of the UAA and UGA stop-codons of apicoplast ORFs and the sole UAA stop-codon that terminates translation from the three mitochondrial ORFs. A single nuclear-encoded canonical RF2, PfRF2Api , localizes to the apicoplast. It has a conserved tripeptide motif (SPF) for stop-codon recognition and is sufficient for peptidyl-tRNA hydrolysis (PTH) from both UAA and UGA. Two RF family proteins are targeted to the parasite mitochondrion; a canonical RF1, PfRF1Mit , with a variant codon-recognition motif (PxN instead of the conserved RF1 PxT) is the major peptidyl-hydrolase with specific recognition of the UAA codon relevant to mitochondrial ORFs. Mutation of the N residue of the PfRF1Mit PxN motif and two other conserved residues of the codon recognition domain lowers PTH activity from pre-termination ribosomes indicating their role in codon-recognition. The second RF imported by the mitochondrion is the non-canonical PfICT1 that functions as a dimer and mediates codon nonspecific peptide release. Our results help delineate a critical step in organellar translation in Plasmodium, which is an important target for anti-malarials.

Benzofuran-dihydropyridine hybrids: A new class of potential bone anabolic agents.

A series of novel benzofuran-dihydropyridine hybrids were designed by molecular hybridization approach and evaluated for bone anabolic activities. Among the screened library, ethyl 4-(7-(sec-butyl)-2-(4-methylbenzoyl)benzofuran-5-yl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (compound 21) significantly enhanced the ALP production and mineralized nodule formation, which are primary requisites in the process of in vitro osteogenesis. Oral administration of compound 21 at 10 mg.kg-1 day-1 for two weeks led to restoration of trabecular bone microarchitecture in drill hole fracture model by significantly increasing BV/TV and Tb.N. Furthermore, histological and molecular studies showed compound 21 triggering the new bone regeneration in a drill hole defect site by increasing BMP expression. Furthermore, molecular modeling studies were performed to gain insight into the binding approach, which revealed that both benzofuran and dihydropyridine moieties are essential to show similar binding interactions to fit into the active site of BMP2 receptor, an important target of the osteogenic agents. Our results suggest that compound 21 stimulates BMP2 synthesis in osteoblast cells that promotes new bone formation (∼40%) at the fracture site which helps in shorten the healing period.

Physico-chemical and in-silico analysis of a phytocystatin purified from Brassica juncea cultivar RoAgro 5444.

This study describes the isolation and purification of a phytocystatin from seeds of Brassica juncea (Indian mustard; cultivar RoAgro 5444), which is an important oilseed crop both agriculturally and economically. The protein was purified by gel filtration chromatography with 24.3% yield and 204-fold purification, and visualised by 2D gel electrophoresis. The 18.1 kDa mustard cystatin was highly specific for cysteine proteinases. The plant cystatin inhibited cathepsin B, confirming its role in conferring pest resistance. The inhibitor was highly stable over a pH range of 3-10 and retained significant inhibitory potential up to 70 °C. The stoichiometry of its interaction with papain, determined by isothermal calorimetry, suggests a 1:1 complex. Secondary structural elements calculated by far-UV circular dichroism (CD) spectroscopy show an 18.8% α-helical and 21% ß-sheet structure. The protein was a non-competitive inhibitor of thiol proteinases. The Stokes radius and frictional co-efficient were used to describe the shape and size of the protein. Homology modelling and docking studies proposed a prototype illustrating the Brassica phytocystatin mediated papain inhibition. Molecular dynamics (MD) study revealed the excellent stability of the papain-phytocystatin complex during a simulation for 100 ns. Detailed results identify the mustard cystatin as an important member of the phytocystatin family.

Virtual screening strategies: recent advances in the identification and design of anti-cancer agents.

Virtual screening (VS) is a well-established technique, which is now routinely employed in computer aided drug designing process. VS can be broadly classified into two categories, i.e., ligand-based and structure-based approach. In recent years, VS has emerged as a time saving and cost effective technique, capable of screening millions of compounds in a user friendly manner. In the area of cancer drug design, VS methods have been widely used and helped in identifying novel molecules as potential anti-cancer agents. Both ligand-based VS (LBVS) structure-based VS (SBVS) methods have been highly useful in the identification of a number of potential anti-cancer agents exhibiting activities in nanomolar range. In tune with the rapid progress in the enhancement of computational power, VS has witnessed significant change in terms of speed and hit rate and in future it is expected that VS will be a preferential alternative to high throughput screening (HTS). This review, discusses recent trends and contribution of VS in the area of anti-cancer drug discovery.

Recombinant Leishmania Rab6 (rLdRab6) is recognized by sera from visceral leishmaniasis patients.

Rab proteins form the largest branch of the Ras superfamily. Rab proteins are key regulators of intracellular vesicular transport and membrane trafficking. Although RabGTPases are well-recognized targets in human diseases but are under-explored therapeutically in the Leishmania parasite. Using a quantitative cytofluorimetric assay, we analyzed the composition and organization of Rab6GTPase protein which was found to be primarily localized on the parasite subpellicular membrane and flagellum due to its association with kinesin motor proteins in the cytoskeletal microtubules. Our aim was to also assess the diagnostic role of recombinant Rab6 protein from Leishmania donovani (rLdRab6) using sera/plasma of Indian visceral leishmaniasis (VL) patients. Receiver-operating characteristic (ROC) curve analysis indicated 100% sensitivity and 100% specificity for rLdRab6-based ELISA which was almost similar in comparison to recombinant K39-based ELISA (95.83% sensitivity and 100% specificity). Sera of patients from another intracellular pathogenic infection, Mycobacterium tuberculosis, did not contain any significant levels of anti-rLdRab6 antibody. Thus rLdRab6 accuracy in visceral leishmaniasis diagnosis makes it a promising antigen for clinical use.

Evolutionary comparison of prenylation pathway in kinetoplastid Leishmania and its sister Leptomonas.

BACKGROUND: Leptomonas is monogenetic kinetoplastid parasite of insects and is primitive in comparison to Leishmania. Comparative studies of these two kinetoplastid may share light on the evolutionary transition to dixenous parasitism in Leishmania. In order to adapt and survive within two hosts, Leishmania species must have acquired virulence factors in addition to mechanisms that mediate susceptibility/resistance to infection in the pathology associated with disease. Rab proteins are key mediators of vesicle transport and contribute greatly to the evolution of complexity of membrane transport system. In this study we used our whole genome sequence data of these two divergent kinetoplastids to analyze the orthologues/paralogues of Rab proteins. RESULTS: During change of lifestyle from monogenetic (Leptomonas) to digenetic (Leishmania), we found that the prenyl machinery remained unchanged. Geranylgeranyl transferase-I (GGTase-I) was absent in both Leishmania and its sister Leptomonas. Farnesyltransferase (FTase) and geranylgeranyl transferase-II (GGTase-II) were identified for protein prenylation. We predict that activity of the missing alpha-subunit (α-subunit) of GGTase-II in Leptomonas was probably contributed by the α-subunit of FTase, while beta-subunit (ß-subunit) of GGTase-II was conserved and indicated functional conservation in the evolution of these two kinetoplastids. Therefore the ß-subunit emerges as an excellent target for compounds inhibiting parasite activity in clinical cases of co-infections. We also confirmed that during the evolution to digenetic life style in Leishmania, the parasite acquired capabilities to evade drug action and maintain parasite virulence in the host with the incorporation of short-chain dehydrogenase/reductase (SDR/MDR) superfamily in Rab genes. CONCLUSION: Our study based on whole genome sequences is the first to build comparative evolutionary analysis and identification of prenylation proteins in Leishmania and its sister Leptomonas. The information presented in our present work has importance for drug design targeted to kill L. donovani in humans but not affect the human form of the prenylation enzymes.

Homology modeling of NAD+-dependent DNA ligase of the Wolbachia endosymbiont of Brugia malayi and its drug target potential using dispiro-cycloalkanones.

Lymphatic filarial nematodes maintain a mutualistic relationship with the endosymbiont Wolbachia. Depletion of Wolbachia produces profound defects in nematode development, fertility, and viability and thus has great promise as a novel approach for treating filarial diseases. NAD(+)-dependent DNA ligase is an essential enzyme of DNA replication, repair, and recombination. Therefore, in the present study, the antifilarial drug target potential of the NAD(+)-dependent DNA ligase of the Wolbachia symbiont of Brugia malayi (wBm-LigA) was investigated using dispiro-cycloalkanone compounds. Dispiro-cycloalkanone specifically inhibited the nick-closing and cohesive-end ligation activities of the enzyme without inhibiting human or T4 DNA ligase. The mode of inhibition was competitive with the NAD(+) cofactor. Docking studies also revealed the interaction of these compounds with the active site of the target enzyme. The adverse effects of these inhibitors were observed on adult and microfilarial stages of B. malayi in vitro, and the most active compounds were further monitored in vivo in jirds and mastomys rodent models. Compounds 1, 2, and 5 had severe adverse effects in vitro on the motility of both adult worms and microfilariae at low concentrations. Compound 2 was the best inhibitor, with the lowest 50% inhibitory concentration (IC50) (1.02 µM), followed by compound 5 (IC50, 2.3 µM) and compound 1 (IC50, 2.9 µM). These compounds also exhibited the same adverse effect on adult worms and microfilariae in vivo (P < 0.05). These compounds also tremendously reduced the wolbachial load, as evident by quantitative real-time PCR (P < 0.05). wBm-LigA thus shows great promise as an antifilarial drug target, and dispiro-cycloalkanone compounds show great promise as antifilarial lead candidates.

Substrate and stereocontrolled iodocycloetherification of highly functionalized enantiomerically pure allylic alcohols: application to synthesis of cytotoxic 2-epi jaspine B and its biological evaluation.

Stereoselectivities of electrophilic additions of molecular iodine to enantiomerically pure highly functionalized allylic alcohols with internal nucleophiles have been investigated. The intramolecular nucleophilic attack on the I2-π complex by an oxygen nucleophile to obtain tri- and tetrasubstituted THFs is highly regio-, stereoselective and substrate controlled. The application of this study has been shown by utilizing one of the THFs 4a as a key intermediate to complete the total synthesis of marine anti-cancer natural product 2-epi jaspine B.

Identification of Novel Inhibitors of Mycobacterium tuberculosis PknG Using Pharmacophore Based Virtual Screening, Docking, Molecular Dynamics Simulation, and Their Biological Evaluation.

PknG is a Ser/thr protein kinase that plays a crucial role in regulatory processes within the mycobacterial cell and signaling cascade of the infected host cell. The essentiality of PknG in mycobacterial virulence by blocking phagosome-lysosome fusion as well as its role in intrinsic antibiotic resistance makes it an attractive drug target. However, only very few compounds have been reported as Mycobacterium tuberculosis PknG (MtPknG) inhibitors so far. Therefore, in an effort to find potential inhibitors against MtPknG, we report here a sequential pharmacophore-based virtual screening workflow, 3-fold docking with different search algorithms, and molecular dynamic simulations for better insight into the predicted binding mode of identified hits. After detailed analysis of the results, six ligands were selected for in vitro analysis. Three of these molecules showed significant inhibitory activity against MtPknG. In addition, inhibitory studies of mycobacterial growth in infected THP-1 macrophages demonstrated considerable growth inhibition of M. bovis BCG induced through compound NRB04248 without any cytotoxic effect against host macrophages. Our results suggest that the compound NRB04248 can be explored for further design and optimization of MtPknG inhibitors.

3D-QSAR and molecular modeling studies on 2,3-dideoxy hexenopyranosid-4-uloses as anti-tubercular agents targeting alpha-mannosidase.

Ligand-based and structure-based methods were applied in combination to exploit the physicochemical properties of 2,3-dideoxy hex-2-enopyranosid-4-uloses against Mycobacterium tuberculosis H37Rv. Statistically valid 3D-QSAR models with good correlation and predictive power were obtained with CoMFA steric and electrostatic fields (r(2) = 0.797, q(2) = 0.589) and CoMSIA with combined steric, electrostatic, hydrophobic and hydrogen bond acceptor fields (r(2) = 0.867, q(2) = 0.570) based on training set of 33 molecules with predictive r(2) of 0.808 and 0.890 for CoMFA and CoMSIA respectively. The results illustrate the requirement of optimal alkyl chain length at C-1 position and acceptor groups along hydroxy methyl substituent of C-6 to enhance the anti-tubercular activity of the 2,3-dideoxy hex-2-enopyranosid-4-uloses while any substitution at C-3 position exert diminishing effect on anti-tubercular activity of these enulosides. Further, homology modeling of M. tuberculosis alpha-mannosidase followed by molecular docking and molecular dynamics simulations on co-complexed models were performed to gain insight into the rationale for binding affinity of selected inhibitors with the target of interest. The comprehensive information obtained from this study will help to better understand the structural basis of biological activity of this class of molecules and guide further design of more potent analogues as anti-tubercular agents.

Recycling factors for ribosome disassembly in the apicoplast and mitochondrion of Plasmodium falciparum.

The reduced genomes of the apicoplast and mitochondrion of the malaria parasite Plasmodium falciparum are actively translated and antibiotic-mediated translation inhibition is detrimental to parasite survival. In order to understand recycling of organellar ribosomes, a critical step in protein translation, we identified ribosome recycling factors (RRF) encoded by the parasite nuclear genome. Targeting of PfRRF1 and PfRRF2 to the apicoplast and mitochondrion respectively was established by localization of leader sequence-GFP fusions. Unlike any RRF characterized thus far, PfRRF2 formed dimers with disulphide interaction(s) and additionally localized in the cytoplasm, thus suggesting adjunct functions for the factor. PfRRF1 carries a large 108-amino-acid insertion in the functionally critical hinge region between the head and tail domains of the protein, yet complemented Escherichia coli RRF in the LJ14frr(ts) mutant and disassembled surrogate E. coli 70S ribosomes in the presence of apicoplast-targeted EF-G. Recombinant PfRRF2 bound E. coli ribosomes and could split monosomes in the presence of the relevant mitochondrial EF-G but failed to complement the LJ14frr(ts) mutant. Although proteins comprising subunits of P. falciparum organellar ribosomes are predicted to differ from bacterial and mitoribosomal counterparts, our results indicate that the essential interactions required for recycling are conserved in parasite organelles.