Heat Shock Protein 90 as a Drug Target against Protozoan Infections Biochemical characterization of HSP90 From plasmodium falciparum and trypanosoma evansi and evaluation of its inhibitor as a candidate drug

Using a pharmacological inhibitor of Hsp90 in cultured malarial parasite, we have previously implicated Plasmodium falciparum Hsp90 (PfHsp90) as a drug target against malaria. In this study, we have biochemically characterized PfHsp90 in terms of its ATPase activity and interaction with its inhibitor geldanamycin (GA) and evaluated its potential as a drug target in a preclinical mouse model of malaria. In addition, we have explored the potential of Hsp90 inhibitors as drugs for the treatment of Trypanosoma infection in animals. Our studies with full-length PfHsp90 showed it to have the highest ATPase activity of all known Hsp90s; its ATPase activity was 6 times higher than that of human Hsp90. Also, GA brought about more robust inhibition of PfHsp90 ATPase activity as compared with human Hsp90. Mass spectrometric analysis of PfHsp90 expressed in P. falciparum identified a site of acetylation that overlapped with Aha1 and p23 binding domain, suggesting its role in modulating Hsp90 multichaperone complex assembly. Indeed, treatment of P. falciparum cultures with a histone deacetylase inhibitor resulted in a partial dissociation of PfHsp90 complex. Furthermore, we found a well known, semisynthetic Hsp90 inhibitor, namely 17-(allylamino)-17-demethoxygeldanamycin, to be effective in attenuating parasite growth and prolonging survival in a mouse model of malaria. We also characterized GA binding to Hsp90 from another protozoan parasite, namely Trypanosoma evansi. We found 17-(allylamino)-17-demethoxygeldanamycin to potently inhibit T. evansi growth in a mouse model of trypanosomiasis. In all, our biochemical characterization, drug interaction, and animal studies supported Hsp90 as a drug target and its inhibitor as a potential drug against protozoan diseases.

PAMAM Dendrimer-Drug Interactions: Effect of pH on the Binding and Release Pattern

Understanding the dendrimer-drug interaction is of great importance to design and optimize the dendrimer-based drug delivery system. Using atomistic molecular dynamics (MD) simulations, we have analyzed the release pattern of four ligands (two soluble drugs, namely, salicylic acid (Sal), L-alanine (Ala), and two insoluble drugs, namely, phenylbutazone (Pbz) and primidone (Prim)), which were initially encapsulated inside the ethylenediamine (EDA) cored polyamidoamine (PAMAM) dendrimer using the docking method. We have computed the potential of mean force (PMF) variation with generation 5 (G5)-PAMAM dendrimer complexed with drug molecules using umbrella sampling. From our calculated PMF values, we observe that soluble drugs (Sal and Ala) have lower energy barriers than insoluble drugs (Pbz and Prim). The order of ease of release pattern for these drugs from G5 protonated PAMAM dendrimer was found to be Ala > Sal > Prim > Pbz. In the case of insoluble drugs (Prim and Pbz), because of larger size, we observe much nonpolar contribution, and thus, their larger energy barriers can be reasoned to van der Waals contribution. From the hydrogen bonding analysis of the four PAMAM drug complexes under study, we found intermolecular hydrogen bonding to show less significant contribution to the free energy barrier. Another interesting feature appears while calculating the PMF profile of G5NP (nonprotonated)-PAMAM Pbz and G5NP (nonprotonated)-PAMAM-Sal complex. The PMF was found to be less when the drug is bound to nonprotonated dendrimer compared to the protonated dendrimer. Our results suggest that encapsulation of the drug molecule into the host PAMAM dendrimer should be carried out at higher pH values (near pH 10). When such complex enters the human body, the pH is around 7.4 and at that physiological pH, the dendrimer holds the drug tightly. Hence the release of drug can occur at a controlled rate into the bloodstream. Thus, our findings provide a microscopic picture of the encapsulation and controlled release of drugs in the case of dendrimer-based host-guest systems.

Mechanism of interaction of the antileukemic drug cytosine arabinoside with aromatic peptides : role of sugar conformation and peptide backbone

Interaction of the antileukemic drugs, cytosine-arabinoside (Ara-C) and adenosine-arabinoside (Ara-A) and a structural analogue, cytidine, with aromatic dipeptides has been studied by fluorescence and NMR spectroscopy. Ara-C and cytidine bind tryptophanyl and histidyl dipeptides but not tyrosyl dipeptides, while Ara-A does not bind to any of them. Both studies indicate association involving stacking of aromatic moieties. NMR spectra also indicate a protonation of the histidine moiety by Ara-C. In case of cytidine, the chemical shifts observed on binding to His-Phe imply that the backbone protons of the dipeptide participate in the binding. The conformation of the sugar and the base seem to play a very important role in the binding phenomenon as three similar molecules, Ara-C, Ara-A and cytidine bind in totally different ways.

Molecule Modeling of Human Pentameric alpha(7) Neuronal Nicotinic Acetylcholine Receptor and Its Interaction with its Agonist and Competitive Antagonist

The nicotinic Acetylcholine Receptor (nAChR) is the major class of neurotransmitter receptors that is involved in many neurodegenerative conditions such as schizophrenia, Alzheimer's and Parkinson's diseases. The N-terminal region or Ligand Binding Domain (LBD) of nAChR is located at pre- and post-synaptic nervous system, which mediates synaptic transmission. nAChR acts as the drug target for agonist and competitive antagonist molecules that modulate signal transmission at the nerve terminals. Based on Acetylcholine Binding Protein (AChBP) from Lymnea stagnalis as the structural template, the homology modeling approach was carried out to build three dimensional model of the N-terminal region of human alpha(7)nAChR. This theoretical model is an assembly of five alpha(7) subunits with 5 fold axis symmetry, constituting a channel, with the binding picket present at the interface region of the subunits. alpha-netlrotoxin is a potent nAChR competitive antagonist that readily blocks the channel resulting in paralysis. The molecular interaction of alpha-Bungarotoxin, a long chain alpha-neurotoxin from (Bungarus multicinctus) and human alpha(7)nAChR seas studied. Agonists such as acetylcholine, nicotine, which are used in it diverse array of biological activities, such as enhancements of cognitive performances, were also docked with the theoretical model of human alpha(7)nAChR. These docked complexes were analyzed further for identifying the crucial residues involved i interaction. These results provide the details of interaction of agonists and competitive antagonists with three dimensional model of the N-terminal region of human alpha(7)nAChR and thereby point to the design of novel lead compounds.

Interaction of cationic amphiphilic drugs with lipid A: Implications for development of endotoxin antagonists

This report presents evidence for the interactions of several classes of cationic amphiphilic drugs including the phenothiazines, aminoquinolines, biguanides, and aromatic diamidines, with lipid A, the endotoxic principle of lipopolysaccharides. The interactions of the drugs were quantitatively assessed by fluorescence methods. The affinities of the drugs for lipid A parallel their endotoxin-antagonistic effects in the Limulus gelation assay. Dicationic compounds bind lipid A with greater affinity; the affinity of such molecules increases exponentially as a function of the distance between the basic moieties. The bis-amidine drug - pentamidine - examined in greater detail, binds lipid A with high affinity (apparent K-d: 0.12 mu M), and LPS, probably due to simultaneous interactions of the terminal amidine groups with the anionic phosphates on lipid A. The sequestration of endotoxin by pentamidine reduces its propensity to bind to cells, and the complex exhibits attenuated toxicity in biological assays. These results have implications in the development of therapeutic strategies against endotoxin-related disease states.

Interaction of heterocyclic thiols/thiones eliminated from cephalosporins with iodine and its biological implications

Hydrolysis of beta-lactam antibiotics by beta-lactamases (e. g., metallo-beta-lactamase, m beta l) is one of the major bacterial defense systems. These enzymes can catalyze the hydrolysis of a variety of antibiotics including the latest generation of cephalosporins, cephamycins and imipenem. It is shown in this paper that the thiol/thione moieties eliminated from certain cephalosporins by m beta l-mediated hydrolysis readily react with molecular iodine to produce ionic compounds having S-I bonds. While the reaction of MTT with iodine produced the corresponding disulfide, MDT and DMETT produced the charge-transfer complexes MDT-I-2 and DMETT-I-2, respectively. Addition of two equivalents of I-2 to MDT produced a novel cationic complex having an almost linear S-I+-S moiety and I-5(-) counter anion.However, this reaction appears to be highly solvent dependent. When the reaction of MDT with I2 was carried out in water, the reaction produced a monocation having I-5(-), indicating the reactivity of MDT toward I2 is very similar to that of the most commonly used antithyroid drug methimazole (MMI). In contrast to MMI, MDT and DMETT, the triazine-based compound MTDT acts as a weak donor toward iodine. (C)2010 Elsevier Ltd. All rights reserved.

Steric and rotational constraints in the X-ray structure of the animalarial drug amodiaquine

In the crystal structure of the antimalarial drug amodiaquine, the bonds linking the quinoline and the phenyl groups show partial double-bond character. The partial double-bond character of the two exocyclic bonds, together with stereochemical constraints, reduce flexibility of the two ring systems of the molecule. The dihedral angle between the two ring planes is lowest compared to those in the antileukaemic drug amsacrine and its derivatives. CPK-modelling studies suggest the way amodiaquine can bind to DNA. Stacking interaction between the quinoline and phenyl groups of independent molecules and the hydrogen-bond network stabilize the crystal structure.

DNA topoisomerase I from mycobacteria - A potential drug target

DNA topoisomerases are ubiquitous group of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA. The enzymes are classified based on the pattern of DNA cleavage. Type IA enzymes found in all bacteria nick the DNA and attach themselves covalently to the 5' side of the nick during the first transesterification reaction. Most of the information on this group of enzymes comes from studies with E. coli topoisomerase I and III. Members of type IA group are single subunit Zn++ metalloenzymes recognizing single stranded DNA without high degree of sequence specificity during relaxation reaction of negatively super coiled DNA. So far no inhibitors are known for this group of enzymes inspite of their important role in maintaining homeostasis of DNA topology. Molecular characterization of DNA topoisomerase I from mycobacteria has revealed some of the important features of type IA enzymes hitherto unknown and provide scope for identifying novel inhibitors. The present review describes the recent developments in the area summarizing the distinctive features of mycobacterial topoisomerase I. The enzyme has several properties not shared by either type IA or 113 enzymes with respect to DNA binding, recognition, sequence specificity and interaction pattern. The physiological basis of the unusual features is discussed. The unique properties described would aid in developing the enzyme as a target molecule in pharmaceutical design. In addition, the findings lead to address some fundamental questions on the intracellular role of topoisomerase I in the biology of mycobacteria which are one of the most formidable group of pathogenic organisms.

Investigation of biodegradable and biocompatible castor oil poly(mannitol-citric-sebacate) polyester as a drug carrier

Castor oil-based poly(mannitol-citric sebacate) was synthesized by simple, catalyst-free melt condensation process using monomers having potential to be metabolized in vivo. The polymer was characterized using various techniques and the tensile and hydration properties of the polymers were also determined. The biocompatibility of the polymer was tested using human foreskin fibroblasts cells. The in vitro degradation studies show that the time for complete degradation of the polymer was more than 21 days. The usage of castor oil polyester as a drug carrier was analysed by doping the polymer with 5-fluorouracil model drug and the release rate was studied by varying the percentage loading of drugs and the pH of the PBS solution medium. The cumulative drug-release profiles exhibited a biphasic release with an initial burst release and cumulative 100% release within 42 h. To understand the role of the polymer as a drug carrier in the release behaviour, drug-release studies were conducted with another drug, isoniazid. The release behaviour of isoniazid drug from the same polymer matrix followed an nth order kinetic model and 100% cumulative release was achieved after 12 days. The variation in the release behaviour for two model drugs from the same polymer matrix suggests a strong interaction between the polymer and the drug molecule.

Crowd Sourcing a New Paradigm for Interactome Driven Drug Target Identification in Mycobacterium tuberculosis

A decade since the availability of Mycobacterium tuberculosis (Mtb) genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative `Connect to Decode' (C2D) to generate the first and largest manually curated interactome of Mtb termed `3interactome pathway' (IPW), encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach.

Network approaches to drug discovery

Introduction: Advances in genomics technologies are providing a very large amount of data on genome-wide gene expression profiles, protein molecules and their interactions with other macromolecules and metabolites. Molecular interaction networks provide a useful way to capture this complex data and comprehend it. Networks are beginning to be used in drug discovery, in many steps of the modern discovery pipeline, with large-scale molecular networks being particularly useful for the understanding of the molecular basis of the disease. Areas covered: The authors discuss network approaches used for drug target discovery and lead identification in the drug discovery pipeline. By reconstructing networks of targets, drugs and drug candidates as well as gene expression profiles under normal and disease conditions, the paper illustrates how it is possible to find relationships between different diseases, find biomarkers, explore drug repurposing and study emergence of drug resistance. Furthermore, the authors also look at networks which address particular important aspects such as off-target effects, combination-targets, mechanism of drug action and drug safety. Expert opinion: The network approach represents another paradigm shift in drug discovery science. A network approach provides a fresh perspective of understanding important proteins in the context of their cellular environments, providing a rational basis for deriving useful strategies in drug design. Besides drug target identification and inferring mechanism of action, networks enable us to address new ideas that could prove to be extremely useful for new drug discovery, such as drug repositioning, drug synergy, polypharmacology and personalized medicine.

Dinuclear zinc bis(thiosemicarbazone) complexes: Synthesis, in vitro anticancer activity, cellular uptake and DNA interaction study

Four dinucleating bis(thiosemicarbazone) ligands and their zinc complexes have been synthesized and characterized by multinuclear NMR (H-1 and C-13), IR, UV-Vis, ESI-MS and fluorescence spectroscopic techniques. Their purity was assessed by elemental analysis. Cytotoxicity was tested against five human cancer cell lines using the sulphorhodamine B (SRB) assay, where one of the complexes, 1,3-bis{biacetyl-2'-(4 `'-N-pyrrolidinylthiosemicarbazone)-3'-(4 `'-N-pyrrolidinylthiosemicarbazone) zinc(II)} propane (6), was found to be quite cytotoxic against MCF-7 (breast cancer) and HepG2 (hepatoma cancer) cell lines, with a potency similar to that of the well known anticancer drug adriamycin. It is evident from the cellular uptake studies that the uptake is same for the active complex 6 and the inactive complex 8 (1,6-bis{biacetyl- 2'-(4 `'-N-pyrrolidinylthiosemicarbazone)-3'-(4 `'-N-pyrrolidinylthiosemicarbazone) zinc(II)} hexane) in MCF-7 and HepG2 cell lines. In vitro DNA binding and cleavage studies revealed that all complexes bind with DNA through electrostatic interaction, and cause no significant cleavage of DNA. (C) 2'13 Elsevier B. V. All rights reserved.

PLIC: protein-ligand interaction clusters

Most of the biological processes are governed through specific protein-ligand interactions. Discerning different components that contribute toward a favorable protein-ligand interaction could contribute significantly toward better understanding protein function, rationalizing drug design and obtaining design principles for protein engineering. The Protein Data Bank (PDB) currently hosts the structure of similar to 68 000 protein-ligand complexes. Although several databases exist that classify proteins according to sequence and structure, a mere handful of them annotate and classify protein-ligand interactions and provide information on different attributes of molecular recognition. In this study, an exhaustive comparison of all the biologically relevant ligand-binding sites (84 846 sites) has been conducted using PocketMatch: a rapid, parallel, in-house algorithm. PocketMatch quantifies the similarity between binding sites based on structural descriptors and residue attributes. A similarity network was constructed using binding sites whose PocketMatch scores exceeded a high similarity threshold (0.80). The binding site similarity network was clustered into discrete sets of similar sites using the Markov clustering (MCL) algorithm. Furthermore, various computational tools have been used to study different attributes of interactions within the individual clusters. The attributes can be roughly divided into (i) binding site characteristics including pocket shape, nature of residues and interaction profiles with different kinds of atomic probes, (ii) atomic contacts consisting of various types of polar, hydrophobic and aromatic contacts along with binding site water molecules that could play crucial roles in protein-ligand interactions and (iii) binding energetics involved in interactions derived from scoring functions developed for docking. For each ligand-binding site in each protein in the PDB, site similarity information, clusters they belong to and description of site attributes are provided as a relational database-protein-ligand interaction clusters (PLIC).

Role of surface chemistry in modulating drug release kinetics in titania nanotubes

Surface chemistry and the intrinsic porous architectures of porous substrates play a major role in the design of drug delivery systems. An interesting example is the drug elution characteristic from hydrothermally synthesised titania nanotubes with tunable surface chemistry. The variation in release rates of Ibuprofen (IBU) is largely influenced by the nature of the functional groups on titania nanotubes and pH of suspending medium. To elucidate the extent of interaction between the encapsulated IBU and the functional groups on titania nanotubes, the release profiles have been modelled with an empirical Hill equation. The analysis aided in establishing a probable mechanism for the release of IBU from the titania nanotubes. The study of controlled drug release from TiO2 has wider implication in the context of biomedical engineering. (C) 2014 Elsevier B.V. All rights reserved.

CANDO and the infinite drug discovery frontier

The Computational Analysis of Novel Drug Opportunities (CANDO) platform (http://protinfo.org/cando) uses similarity of compound-proteome interaction signatures to infer homology of compound/drug behavior. We constructed interaction signatures for 3733 human ingestible compounds covering 48,278 protein structures mapping to 2030 indications based on basic science methodologies to predict and analyze protein structure, function, and interactions developed by us and others. Our signature comparison and ranking approach yielded benchmarking accuracies of 12-25% for 1439 indications with at least two approved compounds. We prospectively validated 49/82 `high value' predictions from nine studies covering seven indications, with comparable or better activity to existing drugs, which serve as novel repurposed therapeutics. Our approach may be generalized to compounds beyond those approved by the FDA, and can also consider mutations in protein structures to enable personalization. Our platform provides a holistic multiscale modeling framework of complex atomic, molecular, and physiological systems with broader applications in medicine and engineering.