Engineering of the E. coli Outer Membrane Protein FhuA to overcome the Hydrophobic Mismatch in Thick Polymeric Membranes

Background

There is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity.

Results

The cucumarioside A₂-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times.

Conclusions

We propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.     

Lipid-induced changes in protein conformation as a means to regulate the immunogenicity of antigens incorporated in tubular immunostimulating complexes

Nanoparticles of tubular immunostimulating complexes (TI-complexes), which consist of the glycolipid monogalactosyldiacylglycerol (MGDG) from marine macrophytes (macroalgae and seagrasses), the triterpene glycoside cucumarioside A₂-2 from the holothurian Cucumaria japonica, and cholesterol, are a promising adjuvant carrier of antigens for modern subunit vaccines. MGDG provides a lipid matrix for the antigen incorporated in TI-complexes. This paper discusses the manner in which the physicochemical properties of MGDGs isolated from different marine macrophyte species affect the conformation of two model protein antigens (Yersinia pseudotuberculosis OmpF-like porin (YOmpF) and recombinant influenza virus hemagglutinin) incorporated in TI-complexes and how the modulating effect of MGDG may be used to improve the efficacy of vaccine preparations.     

ISCOM-based vaccines: the second decade

The immunostimulating complex or 'iscom' was first described 20 years ago as an antigen delivery system with powerful immunostimulating activity. Iscoms are cage-like structures, typically 40 nm in diameter, that are comprised of antigen, cholesterol, phospholipid and saponin. ISCOM-based vaccines have been shown to promote both antibody and cellular immune responses in a variety of experimental animal models. This review focuses on the evaluation of ISCOM-based vaccines in animals over the past 10 years, as well as examining the progress that has been achieved in the development of human vaccines based on ISCOM adjuvant technology.     

The influence of monogalactosyldiacylglycerols from different marine macrophytes on immunogenicity and conformation of protein antigen of tubular immunostimulating complex

The tubular immunostimulating complex (TI-complex) is a novel nanoparticulate antigen delivery system consisting of cholesterol, triterpene glycoside cucumarioside A(2)-2, and glycolipid monogalactosyldiacylglycerol (MGDG) isolated from marine macrophytes. MGDG is crucial for the formation of a lipid matrix for the protein antigen incorporated in TI-complexes. Fatty acid composition and the physical state of this glycolipid depend on the taxonomic position of marine macrophytes. Therefore, the aim of the present work was to study the capacity of MGDGs, isolated from five species of marine macrophytes, to influence conformation and to enhance immunogenicity of porin from Yersinia pseudotuberculosis (YOmpF) as a model antigen of subunit vaccine based on TI-complexes. The trimeric porin was chosen for these experiments, because it was approximately two times more immunogenic than monomeric porin incorporated in TI-complexes. Immunization of mice with YOmpF within TI-complexes, comprised of different MGDGs, revealed a dependence of the immunostimulating effect of TI-complexes on the microvicosity of this glycolipid. TI-complexes comprising MGDGs from Sargassum pallidum and Ulva fenestrata with medium microviscosity induced maximal levels of anti-porin antibodies (four times higher when compared with those induced by pure porin). The adjuvant effect of TI-complexes based on other MGDGs varied by 2.8, 2.3 and 1.3 times for TI-complexes comprised of MGDGs from Zostera marina, Ahnfeltia tobuchiensis, and Laminaria japonica, respectively. MGDGs are also able to influence cytokine mechanisms of immunological regulation. DSC and spectroscopic studies showed that maximal immunostimulating effect of TI-complexes correlated with a moderate stabilizing influence of MGDGs from S. pallidum and U. fenestrata on the conformation of porin. The results obtained suggest lipid "nanofluidics" as a novel strategy for optimizing the immune response to protein antigens within lipid particulate systems.     

An Experimental Tool to Estimate the Probability of a Nucleotide Presence in the Crystal Structures of the Nucleotide–Protein Complexes

A correlation between the ligand–protein affinity and the identification of the ligand in the experimental electron density maps obtained by X-ray crystallography has been tested for a number of RNA-binding proteins. Bacterial translation regulators ProQ, TRAP, Rop, and Hfq together with their archaeal homologues SmAP have been used. The equilibrium dissociation constants for the N-methyl-anthraniloyl-labelled adenosine and guanosine monophosphates titrated by the proteins have been determined by the fluorescent anisotropy measurements. The estimated stability of the nucleotide–protein complexes has been matched with a presence of the nucleotides in the structures of the proposed nucleotide–protein complexes. It has been shown that the ribonucleotides can be definitely identified in the experimental electron density maps at equilibrium dissociation constant <10 μM. At K_D of 20–40 μM, long incubation of the protein crystals in the nucleotide solution is required to obtain the structures of the complexes. The complexes with K_D value higher than 50 μM are not stable enough to survive in crystallization conditions.     

Induction of humoral antibody response by soluble polysaccharide of Cryptococcus neoformans

Mice were immunized with purifiedCryptococcus neoformans soluble polysaccharide, or with the polysaccharide coupled to methylated bovine serum albumin or methylated bovine gamma globulin. The polysaccharide-methylated protein complexes were no more immunogenic than the purified polysaccharide when used without Freund's incomplete adjuvant; however, the methylated protein complexes induced higher levels of antibody than purified polysaccharide when emulsified in the adjuvant. Sera from mice were titrated by passive hemagglutination, and maximum antibody titers were observed 14 to 21 days after immunization. Antibody titers declined rapidly after 14 to 21 days in mice immunized with antigen alone; whereas, animals immunized with cryptococcal antigen emulsified in adjuvant remained at peak levels throughout a six week experimental period. All antigens were immunogenic over a wider dosage range when contained in adjuvant. Individual mice immunized with an adjuvant emulsion of purified polysaccharide varied widely in the amount of antibody produced, with some of the animals producing no detectable antibody.     

Immunostimulatory characteristics of a novel adjuvant on the basis of cucumarioside A<Subscript>2</Subscript>-2 and monogalactosyldiacylgycerol

A novel antigen carrier has been formulated on the basis of a cucumarioside-A₂-2 triterpene glycoside (CD) complex with cholesterol and monogalactosyldiacylglycerol from Ahnfeltia tobuchiensis (MGDGAt) and Ulva fenestrate (MGDGUf). Morphological and immunostimulative characteristics of the carrier were studied. Electron microscopy experiments demonstrated the formation of homogeneous tubular structures in a mixture of CD, cholesterol, and MGDG in molar ratio of 1: 2: 3. In animals immunized by the carrier bearing pore forming protein monomer of pseudotuberculosis agent CD and MGDG synergically affected synthesis of specific antibodies, interleukin-2, and γ-interferon and delayed hypersensitivity reaction when compared to Freund’s complete adjuvant or to immunostimulatory complexes between Quillaja saponaria saponins and phosphatidylcholine from egg yolk. The immunostimulatory effect depends upon the composition of polyunsaturated fatty acids of MGDG. The new tubular adjuvant carrier is a competitive adjuvant, as it includes CD obtained from far-eastern sea cucumber commercial species Cucumaria japonica, and MGDG from seaweed.     

Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths

A somatic protein from adult Haemonchus contortus (Hc23), the most abundant component in a low molecular weight fraction with known immunizing effect against experimental haemonchosis, has been purified by immunochromatography. The immunoprophylactic value of Hc23 was tested in groups of 5–6 months old Assaf lambs using Al(OH)₃ or Escherichia coli lipopolysaccharide + inactivated Propionibacterium acnes as adjuvant and the results compared with uninfected control, uninfected and challenged or infected and challenged lambs. Immunization with Hc23 in either adjuvant elicited significant reductions in fecal egg counts after challenge with 15,000 L3s (70.67%–85.64%, respectively) and reduced (67.1% and 86%) abomasal worm counts (45 days post-challenge). Immunized lambs displayed higher peripheral eosinophil counts, were less anaemic and had weight gains than challenged controls. The results suggest that the Hc23 antigen can induce a partially protective response against haemonchosis in lambs.     

Theoretical studies on DNA-binding, DNA-photocleavage and spectral properties of Co(III) complexes [Co(phen)2(L)] (L = pip, hpip, hnaip)

Theoretical studies on the DNA-binding, DNA-photocleavage and spectral properties of Co(III) polypyridyl complexes [Co(phen)₂(L)]³⁺ (L = pip, hpip, hnaip) have been carried out, using the density functional theory (DFT), Hartree-Fock (HF) and configuration interaction singles (CIS) methods. The optimized geometric structures of these Co(III) complexes in aqueous solution are more close to experimental data than those in vacuo at the B3LYP/LanL2DZ level. Based on the optimized geometric structures in solution, the electronic structures of these Co(III) complexes were analyzed and the trend in the DNA-binding constants (K_b) was reasonably explained. In particular, via the analysis of natural charges of the complexes in ground state and excited state, it is very interesting to find the following: under UV or visible light irradiation, the Co(Ш) polypyridyl complexes undergo an intra-molecular electron transfer from S₀ state to T₁ state, and the positive charges on the main-ligand in the T₁ state are greatly increased, so as to form a radical cation with strong oxidation ability. Meanwhile, the change in geometry of the complexes under light irradiation also helps to the radical cation easily approaching and further oxidating DNA-base-pairs. These results offer the theoretical explanation for the photo-induced oxidation-reduction mechanism which was experimentally proposed on DNA-photocleavage by Co(Ш) polypyridyl complexes. In addition, the electronic absorption spectra of these complexes were calculated and simulated in aqueous solution using the time dependent DFT (TDDFT) method, in satisfying agreement with experimental results, and the properties of experimental absorption bands have been theoretically explained in detail.     

Different respiratory syncytial virus and Quillaja saponin formulations induce murine peritoneal cells to express different proinflammatory cytokine profiles

The recognition of a pathogen or a vaccine antigen formulation by cells in the innate immune system leads to production of proinflammatory cytokines, which will determine the ensuing acquired immune response quantitatively and qualitatively. Tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6 are the first set of cytokines produced upon such an encounter, which have roles both in protective immunity and immunopathogenesis evident with respiratory syncytial virus (RSV). RSV antigens in different physical adjuvant-vaccine formulations were analysed for their capacity to provoke cultured murine peritoneal cells to produce these three proinflammatory cytokines. RSV immunostimulating complex (ISCOM), i.e. both antigen and adjuvant are incorporated in the same particle, induced high levels of IL-1alpha being of the same magnitude or higher than those of live RSV and lipopolysaccharide (LPS). Live virus and LPS induced higher levels of IL-6 and TNF-alpha than ISCOM and so did non-adjuvanted UV-inactivated RSV but only at high doses. ISCOM-Matrix, i.e. ISCOM without antigens, admixed as a separate entity to inactivated RSV, downregulated or blocked the cytokine response to the inactivated RSV in contrast to ISCOM. Kinetic studies showed that ISCOM induced cytokine production first detected at hours 1, 2, 4 for TNF-alpha, IL-6 and IL-1alpha respectively, which was earlier than for the other antigen formulations containing corresponding doses of antigen and/or Quillaja adjuvant. Peak values for production of TNF-alpha and IL-6 were at 8 h and for IL-1alpha at 72 h following stimulation with ISCOM. The delayed appearance of IL-1alpha may reflect the cell-bound nature of this cytokine.     

Structure of immune stimulating complex matrices and immune stimulating complexes in suspension determined by small-angle x-ray scattering

Immune stimulating complex (ISCOM) particles consisting of a mixture of Quil-A, cholesterol, and phospholipids were structurally characterized by small-angle x-ray scattering (SAXS). The ISCOM particles are perforated vesicles of very well-defined structures. We developed and implemented a novel (to our knowledge) modeling method based on Monte Carlo simulation integrations to describe the SAXS data. This approach is similar to the traditional modeling of SAXS data, in which a structure is assumed, the scattering intensity is calculated, and structural parameters are optimized by weighted least-squares methods when the model scattering intensity is fitted to the experimental data. SAXS data from plain ISCOM matrix particles in aqueous suspension, as well as those from complete ISCOMs (i.e., with an antigen (tetanus toxoid) incorporated) can be modeled as a polydisperse distribution of perforated bilayer vesicles with icosahedral, football, or tennis ball structures. The dominating structure is the tennis ball structure, with an outer diameter of 40 nm and with 20 holes 5-6 nm in diameter. The lipid bilayer membrane is 4.6 nm thick, with a low-electron-density, 2.0-nm-thick hydrocarbon core. Surprisingly, in the ISCOMs, the tetanus toxoid is located just below the membrane inside the particles.     

A role for MCP-1/CCR2 in interstitial lung disease in children

Background

Particulate matter (PM) can exacerbate allergic airway diseases. Although health effects of PM with a diameter of less than 100 nm have been focused, few studies have elucidated the correlation between the sizes of particles and aggravation of allergic diseases. We investigated the effects of nano particles with a diameter of 14 nm or 56 nm on antigen-related airway inflammation.

Methods

ICR mice were divided into six experimental groups. Vehicle, two sizes of carbon nano particles, ovalbumin (OVA), and OVA + nano particles were administered intratracheally. Cellular profile of bronchoalveolar lavage (BAL) fluid, lung histology, expression of cytokines, chemokines, and 8-hydroxy-2'-deoxyguanosine (8-OHdG), and immunoglobulin production were studied.

Results

Nano particles with a diameter of 14 nm or 56 nm aggravated antigen-related airway inflammation characterized by infiltration of eosinophils, neutrophils, and mononuclear cells, and by an increase in the number of goblet cells in the bronchial epithelium. Nano particles with antigen increased protein levels of interleukin (IL)-5, IL-6, and IL-13, eotaxin, macrophage chemoattractant protein (MCP)-1, and regulated on activation and normal T cells expressed and secreted (RANTES) in the lung as compared with antigen alone. The formation of 8-OHdG, a proper marker of oxidative stress, was moderately induced by nano particles or antigen alone, and was markedly enhanced by antigen plus nano particles as compared with nano particles or antigen alone. The aggravation was more prominent with 14 nm of nano particles than with 56 nm of particles in overall trend. Particles with a diameter of 14 nm exhibited adjuvant activity for total IgE and antigen-specific IgG₁ and IgE.

Conclusion

Nano particles can aggravate antigen-related airway inflammation and immunoglobulin production, which is more prominent with smaller particles. The enhancement may be mediated, at least partly, by the increased local expression of IL-5 and eotaxin, and also by the modulated expression of IL-13, RANTES, MCP-1, and IL-6.     

Effects of nano particles on antigen-related airway inflammation in mice

Background

Particulate matter (PM) can exacerbate allergic airway diseases. Although health effects of PM with a diameter of less than 100 nm have been focused, few studies have elucidated the correlation between the sizes of particles and aggravation of allergic diseases. We investigated the effects of nano particles with a diameter of 14 nm or 56 nm on antigen-related airway inflammation.

Methods

ICR mice were divided into six experimental groups. Vehicle, two sizes of carbon nano particles, ovalbumin (OVA), and OVA + nano particles were administered intratracheally. Cellular profile of bronchoalveolar lavage (BAL) fluid, lung histology, expression of cytokines, chemokines, and 8-hydroxy-2''-deoxyguanosine (8-OHdG), and immunoglobulin production were studied.

Results

Nano particles with a diameter of 14 nm or 56 nm aggravated antigen-related airway inflammation characterized by infiltration of eosinophils, neutrophils, and mononuclear cells, and by an increase in the number of goblet cells in the bronchial epithelium. Nano particles with antigen increased protein levels of interleukin (IL)-5, IL-6, and IL-13, eotaxin, macrophage chemoattractant protein (MCP)-1, and regulated on activation and normal T cells expressed and secreted (RANTES) in the lung as compared with antigen alone. The formation of 8-OHdG, a proper marker of oxidative stress, was moderately induced by nano particles or antigen alone, and was markedly enhanced by antigen plus nano particles as compared with nano particles or antigen alone. The aggravation was more prominent with 14 nm of nano particles than with 56 nm of particles in overall trend. Particles with a diameter of 14 nm exhibited adjuvant activity for total IgE and antigen-specific IgG₁ and IgE.

Conclusion

Nano particles can aggravate antigen-related airway inflammation and immunoglobulin production, which is more prominent with smaller particles. The enhancement may be mediated, at least partly, by the increased local expression of IL-5 and eotaxin, and also by the modulated expression of IL-13, RANTES, MCP-1, and IL-6.     

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1 I. Spectral properties and redox potentials

1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl₁ which lacks cytochrome aa₃. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied.2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the α peaks of b-type cytochrome(s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two α peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochrome c₁. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl₁ in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa₃ was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm.3. Cytochrome aa₃ was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl₁. Cytochrome aa₃ was more susceptible to heat than cytochromes b and c,c₁.4. CO difference spectra at 77 K revealed two different Co-cytochrome complexes. The first, found only in wild type mitochondria, was a typical CO-cytochrome a₃ complex characterized by peaks at 596 and 435 nm and troughs at 613 and 450 nm. The second, found both in mitochondria of the wild type and the mutant, was a CO-cytochrome b complex with peaks at 567, 539 and 420 nm and a trough at 558-549 nm. Both complexes are photo-dissociable.5. Spectral evidence was obtained for interaction of cyanide with the a-type cytochrome (shift of the α peak at 77 K from 608 to 605 nm), but not with the b-type cytochrome.6. The mid-point potentials of the different cytochromes at neutral pH are as follows: cytochrome aa₃ 235 and 395 mV, cytochrome c,c₁ 233 mV, cytochromes b 120 mV.     

Comparison of protection from homologous cell-free vs cell-associated SIV challenge afforded by inactivated whole SIV vaccines

This study attempted to determine if SIV vaccines could protect against challenge with peripheral blood mononuclear cells (PBMCs) from an SIV infected rhesus monkey. Mature Macaca mulatta were vaccinated four times with formalin inactivated SIV_mac32H administered in MDP adjuvant (n = 8) or SIV_mac32H ISCOM vaccine (n = 8). Controls included animals vaccinated with measles virus in MDP adjuvant (n = 4) or ISCOM (n = 4) preparations. Of each group, half were challenged intravenously (IV) with ten MID₅₀ of the cell-free SIV_mac32H (11-88) SIV stock and half were challenged with ten MID₃₀ of PBMCs from the SIV_mac32H infected macaque 1XC. All SIV vaccinated animals challenged with the 11-88 cell free stock of SIV_mac32H were protected, whereas only half of the SIV vaccinated monkeys receiving the same infectious dose of the 1XC cell stock were protected.     

Fluorescence kinetics of PSII crystals containing Ca2 + or Sr2 + in the oxygen evolving complex

Photosystem II (PSII) is the pigment–protein complex which converts sunlight energy into chemical energy by catalysing the process of light-driven oxidation of water into reducing equivalents in the form of protons and electrons. Three-dimensional structures from x-ray crystallography have been used extensively to model these processes. However, the crystal structures are not necessarily identical to those of the solubilised complexes. Here we compared picosecond fluorescence of solubilised and crystallised PSII core particles isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The fluorescence of the crystals is sensitive to the presence of artificial electron acceptors (K₃Fe(CN)₃) and electron transport inhibitors (DCMU). In PSII with reaction centres in the open state, the picosecond fluorescence of PSII crystals and solubilised PSII is indistinguishable. Additionally we compared picosecond fluorescence of native PSII with PSII in which Ca² in the oxygen evolving complex (OEC) is biosynthetically replaced by Sr^2 +. With the Sr^2 + replaced OEC the average fluorescence decay slows down slightly (81 ps to 85 ps), and reaction centres are less readily closed, indicating that both energy transfer/trapping and electron transfer are affected by the replacement.     

Substitution of trans ligands in μ-oxo-bis(μ-acetato)diruthenium(III) complexes: Synthesis and kinetic studies

[Ru₂O(L)₆(acetate)₂](PF₆)₂ {L = pyridine 1; 4-picoline 2} undergo aquation in acetone-water (60:40 v/v) mixed solvent to form diaquo complexes in solution as shown by proton NMR studies. Ligands trans to the μ-oxo group are substituted. These diaquo complexes react with substituted pyridines and imidazoles to form respective disubstituted complexes. Rate constants for aquation and complexation under pseudo first order conditions of ligand are reported. Rate constants increase with increase in the basicity of incoming ligand. Disubstituted complexes proposed to be formed in solution have been isolated and characterized by elemental analyses, visible spectra, proton NMR. Single crystal X-ray structures of 4-picoline and 4-methylimidazole disubstituted complexes are reported. All the isolated complexes exhibit a strong peak between 570 and 585 nm in their visible absorption spectra. λ_max varies linearly with ∑pk_a of terminal ligands. In disubstituted complexes of 1 with 2-methyl and 4-methyl imidazole deprotonation of N(1)H of methylimidazoles takes place in solution.     

Substrate and inhibitor complexes of dihydrofolate reductase from amethopterin-resistant L1210 cells.

Dihydrofolate reductase (EC 1.5.1.3), purified to homogeneity from an amethopterin-resistant subline (R6) of cultured L1210 murine leukemia cells, has been used to study enzyme-substrate and enzyme-inhibitor complexes. NADPH, NADP⁺acid-modified NADPH (λ_max at 265 nm, elevated absorbance at 290 nm), 2′-phosphoadenosine-5′-diphosphate ribose, dihydrofolate, and amethopterin formed binary complexes with the enzyme. Ternary complexes could be formed by admixing the enzyme with: (a) NADPH and amethopterin; (b) NADP⁺ and tetahydrofolate; and (c) acid-modified NADPH and dihydrofolate. All of these complexes migrated as stable well-defined bands on polyacrylamide gel electrophoresis at pH 8.3. The bands could be visualized by staining both for enzyme activity and for protein. These binary and ternary complexes were also stable to extensive dialysis. Spectra of the dialyzed enzyme complexes indicated that each ligand was present at an equimolar ratio with the enzyme.     

Measurement of protein by spectrophotometry at 205 nm

A method is described for the measurement of protein concentration by using the peptide bond absorption at 205 nm. ?₂₀₅ is estimated, allowing for the absorption due to Trp and Tyr residues, by measuring the absorbance at 280 nm as well as at 205 nm. The estimated ?₂₀₅ is compared with the actual ?₂₀₅ for a number of proteins, the mean error being less than 2%. This is about three times better than using an average ?₂₀₅^{1 mg/ml} of 31 and approaches the range of experimental error inherent in any method of protein estimation.     

Updates to Binding MOAD (Mother of All Databases): Polypharmacology Tools and Their Utility in Drug Repurposing

The goal of Binding MOAD is to provide users with a data set focused on high-quality x-ray crystal structures that have been solved with biologically relevant ligands bound. Where available, experimental binding affinities (K_a, K_d, K_i, IC₅₀) are provided from the primary literature of the crystal structure. The database has been updated regularly since 2005, and this most recent update has added nearly 7000 new structures (growth of 21%). MOAD currently contains 32,747 structures, composed of 9117 protein families and 16,044 unique ligands. The data are freely available on www.BindingMOAD.org. This paper outlines updates to the data in Binding MOAD as well as improvements made to both the website and its contents. The NGL viewer has been added to improve visualization of the ligands and protein structures. MarvinJS has been implemented, over the outdated MarvinView, to work with JChem for small molecule searching in the database. To add tools for predicting polypharmacology, we have added information about sequence, binding-site, and ligand similarity between entries in the database. A main premise behind polypharmacology is that similar binding sites will bind similar ligands. The large amount of protein–ligand information available in Binding MOAD allows us to compute pairwise ligand and binding-site similarities. Lists of similar ligands and similar binding sites have been added to allow users to identify potential polypharmacology pairs. To show the utility of the polypharmacology data, we detail a few examples from Binding MOAD of drug repurposing targets with their respective similarities.