Unusual hydrate stabilization in the two-dimensional layered structure of quinacrinium bis(2-carboxy-4,5-dichlorobenzoate) tetrahydrate, a proton-transfer compound of the drug quinacrine

The crystal structure of the hydrated proton-transfer compound of the drug quinacrine [rac-N'-(6-chloro-2-methoxyacridin-9-yl)-N,N-diethylpentane-1,4-diamine] with 4,5-dichlorophthalic acid, C23H32ClN3O2+ . 2(C8H3Cl2O4-).4H2O (I), has been determined at 200 K. The four labile water molecules of solvation form discrete ...O--H...O--H... hydrogen-bonded chains parallel to the quinacrine side chain, the two N--H groups of which act as hydrogen-bond donors for two of the water acceptor molecules. The other water molecules, as well as the acridinium H atom, also form hydrogen bonds with the two anion species and extend the structure into two-dimensional sheets. Between these sheets there are also weak cation--anion and anion--anion pi-pi aromatic ring interactions. This structure represents only the third example of a simple quinacrine derivative for which structural data are available but differs from the other two in that it is unstable in the X-ray beam due to efflorescence, probably associated with the destruction of the unusual four-membered water chain structures.

Proton-transfer and non-transfer compounds of the multi-purpose drug dapsone [4-(4-Aminophenylsulfonyl)aniline] with 3,5-dinitrosalicylic acid and 5-nitroisophthalic acid

The structures of the compounds from the reaction of the drug dapsone [4-(4-aminophenylsulfonyl)aniline] with 3,5-dinitrosalicylic acid, the salt hydrate [4-(4-aminohenylsulfonyl)anilinium 2-carboxy-4,6-dinitrophenolate monohydrate] (1) and the 1:1 adduct with 5-nitroisophthalic acid [4-(4-aminophenylsulfonyl)aniline 5-nitrobenzene-1,3-dicarboxylic acid] (2) have been determined. Crystals of 1 are triclinic, space group P-1, with unit cell dimensions a = 8.2043(3), b = 11.4000(6), c = 11.8261(6)Å, α = 110.891(5), β = 91.927(3), γ = 98.590(4)deg. and Z = 4. Compound 2 is orthorhombic, space group Pbcn, with unit cell dimensions a = 20.2662(6), b = 12.7161(4), c = 15.9423(5)Å and Z = 8. In 1, intermolecular analinium N-H…O and water O-H…O and O-H…N hydrogen-bonding interactions with sulfone, carboxyl, phenolate and nitro O-atom and aniline N-atom acceptors give a two-dimensional layered structure. With 2, the intermolecular interactions involve both aniline N-H…O and carboxylic acid O-H…O and O-H…N hydrogen bonds to sulfone, carboxyl, nitro and aniline acceptors, giving a three-dimensional network structure. In both structures π--π aromatic ring associations are present.

The emerging role of extracellular vesicle-mediated drug resistance in cancers: Implications in advanced prostate cancer

Emerging evidence has shown that the extracellular vesicles (EVs) regulate various biological processes and can control cell proliferation and survival, as well as being involved in normal cell development and diseases such as cancers. In cancer treatment, development of acquired drug resistance phenotype is a serious issue. Recently it has been shown that the presence of multidrug resistance proteins such as Pgp-1 and enrichment of the lipid ceramide in EVs could have a role in mediating drug resistance. EVs could also mediate multidrug resistance through uptake of drugs in vesicles and thus limit the bioavailability of drugs to treat cancer cells. In this review, we discussed the emerging evidence of the role EVs play in mediating drug resistance in cancers and in particular the role of EVs mediating drug resistance in advanced prostate cancer. The role of EV-associated multidrug resistance proteins, miRNA, mRNA, and lipid as well as the potential interaction(s) among these factors was probed. Lastly, we provide an overview of the current available treatments for advanced prostate cancer, considering where EVs may mediate the development of resistance against these drugs.

Characterization of Ion-Exchange Fibers for Controlled Drug Delivery

Increasing attention has been focused on methods that deliver pharmacologically active compounds (e.g. drugs, peptides and proteins) in a controlled fashion, so that constant, sustained, site-specific or pulsatile action can be attained. Ion-exchange resins have been widely studied in medical and pharmaceutical applications, including controlled drug delivery, leading to commercialisation of some resin based formulations. Ion-exchangers provide an efficient means to adjust and control drug delivery, as the electrostatic interactions enable precise control of the ion-exchange process and, thus, a more uniform and accurate control of drug release compared to systems that are based only on physical interactions. Unlike the resins, only few studies have been reported on ion-exchange fibers in drug delivery. However, the ion-exchange fibers have many advantageous properties compared to the conventional ion-exchange resins, such as more efficient compound loading into and release from the ion-exchanger, easier incorporation of drug-sized compounds, enhanced control of the ion-exchange process, better mechanical, chemical and thermal stability, and good formulation properties, which make the fibers attractive materials for controlled drug delivery systems. In this study, the factors affecting the nature and strength of the binding/loading of drug-sized model compounds into the ion-exchange fibers was evaluated comprehensively and, moreover, the controllability of subsequent drug release/delivery from the fibers was assessed by modifying the conditions of external solutions. Also the feasibility of ion-exchange fibers for simultaneous delivery of two drugs in combination was studied by dual loading. Donnan theory and theoretical modelling were applied to gain mechanistic understanding on these factors. The experimental results imply that incorporation of model compounds into the ion-exchange fibers was attained mainly as a result of ionic bonding, with additional contribution of non-specific interactions. Increasing the ion-exchange capacity of the fiber or decreasing the valence of loaded compounds increased the molar loading, while more efficient release of the compounds was observed consistently at conditions where the valence or concentration of the extracting counter-ion was increased. Donnan theory was capable of fully interpreting the ion-exchange equilibria and the theoretical modelling supported precisely the experimental observations. The physico-chemical characteristics (lipophilicity, hydrogen bonding ability) of the model compounds and the framework of the fibrous ion-exchanger influenced the affinity of the drugs towards the fibers and may, thus, affect both drug loading and release. It was concluded that precisely controlled drug delivery may be tailored for each compound, in particularly, by choosing a suitable ion-exchange fiber and optimizing the delivery system to take into account the external conditions, also when delivering two drugs simultaneously.

Structural Basis of Drug Resistance by Genetic Variants of HIV Type 1 Clade C Protease from India

Using computer modeling of three-dimensional structures and structural information available on the crystal structures of HIV-1 protease, we investigated the structural effects of mutations, in treatment-naive and treatment-exposed individuals from India and postulated mechanisms of resistance in clade C variants. A large number of models (14) have been generated by computational mutation of the available crystal structures of drug bound proteases. Localized energy minimization was carried out in and around the sites of mutation in order to optimize the geometry of interactions present. Most of the mutations result in structural differences at the flap that favors the semiopen state of the enzyme. Some of the mutations were also found to confer resistance by affecting the geometry of the active site. The E35D mutation affects the flap structure in clade B strains and E35N and E35K mutation, seen in our modeled strains, have a more profound effect. Common polymorphisms at positions 36 and 63 in clade C also affected flap structure. Apart from a few other residues Gln-58, Asn-83, Asn-88, and Gln-92 and their interactions are important for the transition from the closed to the open state. Development of protease inhibitors by structure-based design requires investigation of mechanisms operative for clade C to improve the efficacy of therapy.

On the functional ordering of biomembranes : Implications for drug binding

Cells are packed with membrane structures, defining the inside and outside, and the different subcellular compartments. These membranes consisting mainly of phospholipids have a variety of functions in addition to providing a permeability barrier for various compounds. These functions involve cellular signaling, where lipids can act as second messengers, or direct regulation of membrane associating proteins. The first part of this study focuses on relating some of the physicochemical properties of membrane lipids to the association of drug compounds to membranes. A fluorescence based method is described allowing for determination of the membrane association of drugs. This method was subsequently applied to a novel drug, siramesine, previously shown to have anti-cancer activity. Siramesine was found to associate with anionic lipids. Especially interesting is its strong affinity for a second messenger lipid phosphatidic acid. This is the first example of a small molecule drug compound specifically interacting with a cellular lipid. Phosphatidic acid in cells is required for the activation of many signaling pathways mediating growth and proliferation. This provides an intriguing possibility for a simple molecular mechanism of the observed anti-cancer activity of siramesine. In the second part the thermal behavior and self assembly of charged and uncharged membrane assemblies was studied. Strong inter-lamellar co-operativity was observed for multilamellar DPPC vesicles using fluorescence techniques together with calorimetry. The commonly used membrane models, large unilamellar vesicles (LUV) and multilamellar vesicles (MLV) were found to possess different biophysical properties as interlamellar interactions of MLVs drive segregation of a pyrene labeled lipid analogue into clusters. The effect of a counter-ion lattice on the self assembly of a cationic gemini surfactant was studied. The presence of NaCl strongly influenced the thermal phase behavior of M-1 vesicles, causing formation of giant vesicles upon exceeding a phase transition temperature, followed by a subsequent transition into a more homogenous dispersion. Understanding the underlying biophysical aspects of cellular membranes is of fundamental importance as the complex picture of the structure and function of cells is evolving. Many of the cellular reactions take place on membranes and membranes are known to regulate the activity of many peripheral and intergral membrane associating proteins. From the point of view of drug design and gene technology, membranes can provide an interesting target for future development of drugs, but also a vehicle sensitive for environmental changes allowing for encapsulating drugs and targeting them to the desired site of action.

Computer-Aided Drug Design of Pyranopyrazoles and Related Compounds for Checkpoint Kinase-1

Checkpoint-1 kinase plays an important role in the G(2)M cell cycle control, therefore its inhibition by small molecules is of great therapeutic interest in oncology. In this paper, we have reported the virtual screening of an in-house library of 2499 pyranopyrazole derivatives against the ATP-binding site of Chk1 kinase using Glide 5.0 program, which resulted in six hits. All these ligands were docked into the site forming most crucial interactions with Cys87, Glu91 and Leu15 residues. From the observed results these ligands are suggested to be potent inhibitors of Chk1 kinase with sufficient scope for further elaboration.

Inhibition of CYP1A2-mediated drug metabolism in vitro and in humans : With special emphasis on rofecoxib and other NSAIDs

The cytochrome P450 1A2 (CYP1A2) is one of the major metabolizing enzymes. The muscle relaxant tizanidine is a selective substrate of CYP1A2, and the non-steroidal anti-inflammatory drug (NSAID) rofecoxib was thought to modestly in-hibit it. Cases suggesting an interaction between tizanidine and rofecoxib had been reported, but the mechanism was unknown. Also other NSAIDs are often used in combination with muscle relaxants. The aims of this study were to investigate the effect of rofecoxib, several other NSAIDs and female sex steroids on CYP1A2 ac-tivity in vitro and in vivo, and to evaluate the predictability of in vivo inhibition based on in vitro data. In vitro, the effect of several NSAIDs, female sex steroids and model inhibitors on CYP1A2 activity was studied in human liver microsomes, without and with preincubation. In placebo controlled, cross-over studies healthy volunteers ingested a single dose of tizanidine after a pretreament with the inhibitor (rofecoxib, tolfenamic acid or celecoxib) or placebo. Plasma (and urine) concentrations of tizanidine and its metabolites were measured, and the pharmacodynamic effects were recorded. A caffeine test was also performed. In vitro, fluvoxamine, tolfenamic acid, mefenamic acid and rofecoxib potently in-hibited CYP1A2. Ethinylestradiol, celecoxib, desogestrel and zolmitriptan were moderate, and etodolac, ciprofloxacin, etoricoxib and gestodene were weak inhibi-tors of CYP1A2. At 100 µM, other tested NSAIDs and steroids inhibited CYP1A2 less than 35%. Rofecoxib was found to be a mechanism-based inhibitor of CYP1A2. In vivo, rofecoxib greatly increased the plasma concentrations (over ten-fold) and the pharmacodynamic effects of tizanidine. Also the metabolism of caf-feine was impaired by rofecoxib. Despite the relatively strong in vitro CYP1A2 inhibitory effects, tolfenamic acid and celecoxib did not have a significant effect on tizanidine and caffeine concentrations in humans. Competitive inhibition model and the free plasma concentration of the inhibitor predicted well the effect of fluvoxam-ine and the lack of effect of tolfenamic acid and celecoxib on tizanidine concentra-tions in humans, and mechanism-based inhibition model explained the effects of rofecoxib. However, the effects of ciprofloxacin and oral contraceptives were un-derestimated from the in vitro data. Rofecoxib is a potent mechanism-based inhibitor of CYP1A2 in vitro and in vivo. This mechanism may be involved in the adverse cardiovascular effects of rofecoxib. Tolfenamic acid and celecoxib seem to be safe in combination with tizanidine, but mefenamic acid might have some effect on tizanidine concentrations in vivo. Con-sidering the mechanism of inhibition, and using the free plasma concentration of the inhibitor, many but not all CYP1A2 interactions can be predicted from in vitro data.

Three-dimensional structure of heat shock protein 90 from Plasmodium falciparum: molecular modelling approach to rational drug design against malaria

We have recently implicated heat shock protein 90 from Plasmodium falciparum (PfHsp90) as a potential drug target against malaria. Using inhibitors specific to the nucleotide binding domain of Hsp90, we have shown potent growth inhibitory effects on development of malarial parasite in human erythrocytes. To gain better understanding of the vital role played by PfHsp90 in parasite growth, we have modeled its three dimensional structure using recently described full length structure of yeast Hsp90. Sequence similarity found between PfHsp90 and yeast Hsp90 allowed us to model the core structure with high confidence. The superimposition of the predicted structure with that of the template yeast Hsp90 structure reveals an RMSD of 3.31 angstrom. The N-terminal and middle domains showed the least RMSD (1.76 angstrom) while the more divergent C-terminus showed a greater RMSD (2.84 angstrom) with respect to the template. The structure shows overall conservation of domains involved in nucleotide binding, ATPase activity, co-chaperone binding as well as inter-subunit interactions. Important co-chaperones known to modulate Hsp90 function in other eukaryotes are conserved in malarial parasite as well. An acidic stretch of amino acids found in the linker region, which is uniquely extended in PfHsp90 could not be modeled in this structure suggesting a flexible conformation. Our results provide a basis to compare the overall structure and functional pathways dependent on PfHsp90 in malarial parasite. Further analysis of differences found between human and parasite Hsp90 may make it possible to design inhibitors targeted specifically against malaria.

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.

Rationalization and prediction of drug resistant mutations in targets for clinical anti-tubercular drugs

Resistance to therapy limits the effectiveness of drug treatment in many diseases. Drug resistance can be considered as a successful outcome of the bacterial struggle to survive in the hostile environment of a drug-exposed cell. An important mechanism by which bacteria acquire drug resistance is through mutations in the drug target. Drug resistant strains (multi-drug resistant and extensively drug resistant) of Mycobacterium tuberculosis are being identified at alarming rates, increasing the global burden of tuberculosis. An understanding of the nature of mutations in different drug targets and how they achieve resistance is therefore important. An objective of this study is to first decipher sequence as well as structural bases for the observed resistance in known drug resistant mutants and then to predict positions in each target that are more prone to acquiring drug resistant mutations. A curated database containing hundreds of mutations in the 38 drug targets of nine major clinical drugs, associated with resistance is studied here. Mutations have been classified into those that occur in the binding site itself, those that occur in residues interacting with the binding site and those that occur in outer zones. Structural models of the wild type and mutant forms of the target proteins have been analysed to seek explanations for reduction in drug binding. Stability analysis of an entire array of 19 mutations at each of the residues for each target has been computed using structural models. Conservation indices of individual residues, binding sites and whole proteins are computed based on sequence conservation analysis of the target proteins. The analyses lead to insights about which positions in the polypeptide chain have a higher propensity to acquire drug resistant mutations. Thus critical insights can be obtained about the effect of mutations on drug binding, in terms of which amino acid positions and therefore which interactions should not be heavily relied upon, which in turn can be translated into guidelines for modifying the existing drugs as well as for designing new drugs. The methodology can serve as a general framework to study drug resistant mutants in other micro-organisms as well.

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.

Crystal structures and binding studies of atovaquone and its derivatives with cytochrome bc(1): a molecular basis for drug design

Crystal structure of trans-atovaquone (antimalarial drug), its polymorph and its stereoisomer (cis) along with five other derivatives with different functional groups have been analyzed. Based on the conformational features of these compounds and the characteristics of the nature of intermolecular interactions, valuable insights into the atomistic details of protein-inhibitor interactions have been derived by docking studies. Atovaquone and its derivatives pack in the crystal lattice using intermolecular O-H center dot center dot center dot O hydrogen bond dimer motifs supported by surrogate weak interactions including C-H center dot center dot center dot O and C-H center dot center dot center dot Cl hydrogen bonds. The docking results of these molecules with cytochrome bc(1) show preferences to form N-H center dot center dot center dot O, O-H center dot center dot center dot O and O-H center dot center dot center dot Cl hydrogen bonds. The involvement of halogen atoms in the binding pocket appears to be significant and is contrary to the theoretically predicted mechanism of protein-ligand docking reported earlier based on mimicking experimental binding results of stigmatellin with cytochrome bc(1). The significance of subtle energy factors controlled by weak intermolecular interactions appears to play a major role in drug binding.

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.

Differential Cocrystallization Behavior of Isomeric Pyridine Carboxamides toward Antitubercular Drug Pyrazinoic Acid

Pyrazinoic acid, the active form of the antitubercular pro-drug Pyrazinamide, is an amphiprotic molecule containing carboxylic acid and pyridine groups and therefore can form both salts and cocrystals with relevant partner molecules. Cocrystallization of pyrazinoic acid with isomeric pyridine carboxamide series resulted in a dimorphic mixed-ionic complex with isonicotinamide and in eutectics with nicotinamide and picolinamide, respectively. It is observed that with alteration of the carboxamide position, steric and electrostatic compatibility issues between molecules of the combination emerge and affect intermolecular interactions and supramolecular growth, thus leading to either cocrystal or eutectic for different pyrazinoic acid-pyridine carboxamide combinations. Intermolecular interaction energy calculations have been performed to understand the role of underlying energetics on the formation of cocrystal/eutectic in different combinations. On the other hand, two molecular salts with piperazine and cytosine and a gallic acid cocrystal of the drug were obtained, and their X-ray crystal structures were also determined in this work.