Structural and Biochemical Characterization of the Human Angiogenin-Proliferating Cell Nuclear Antigen Interaction.

The molecular details of the interaction between human angiogenin (hAng) and proliferating cell nuclear antigen (PCNA) have been investigated by isothermal titration calorimetry (ITC), mutagenesis, and NMR spectroscopy. The two proteins were shown to interact directly through immunoprecipitation studies of hAng with PCNA in vitro, and their interaction was quantified by ITC, obtaining information on stoichiometry, enthalpy, entropy, and binding kinetics of the association. The hAng-PCNA association is strong, with a Kd value of 126 nM. The interaction surface was mapped by NMR spectroscopy, indicating participating residues. A structural model for the PCNA-hAng complex was constructed by docking and molecular dynamics simulations based on NMR data. The model was validated by mutating the hAng residues Arg5 and Arg101, which seem critical for the complex formation, to glutamate. ITC experiments showed that the angiogenin variants R5E and R5ER101E displayed 6.5 and 7.8 times higher Kd values, respectively, than that of the native protein, indicating the correctness of the model. The hAng S28AT36AS37A and hAng S28AT36AS37AS87A variants were also tested as positive controls, further supporting the validity of the model. The crystal structures of the hAng variants S28AT36AS37A and S28AT36AS37AS87A showed that the mutations did not cause any significant conformational change. This study presents evidence for the structural mode of the hAng-PCNA interaction, revealing valuable information about the angiogenin and PCNA biological roles in the cytoplasm.

Structure-Based Discovery of Receptor Activator of Nuclear Factor-κB Ligand (RANKL)-Induced Osteoclastogenesis Inhibitors.

Receptor activator of nuclear factor-κB ligand (RANKL) has been actively pursued as a therapeutic target for osteoporosis, given that RANKL is the master mediator of bone resorption as it promotes osteoclast differentiation, activity and survival. We employed a structure-based virtual screening approach comprising two stages of experimental evaluation and identified 11 commercially available compounds that displayed dose-dependent inhibition of osteoclastogenesis. Their inhibitory effects were quantified through TRAP activity at the low micromolar range (IC50 < 5 µΜ), but more importantly, 3 compounds displayed very low toxicity (LC50 > 100 µΜ). We also assessed the potential of an N-(1-aryl-1H-indol-5-yl)aryl-sulfonamide scaffold that was based on the structure of a hit compound, through synthesis of 30 derivatives. Their evaluation revealed 4 additional hits that inhibited osteoclastogenesis at low micromolar concentrations; however, cellular toxicity concerns preclude their further development. Taken together with the structure-activity relationships provided by the hit compounds, our study revealed potent inhibitors of RANKL-induced osteoclastogenesis of high therapeutic index, which bear diverse scaffolds that can be employed in hit-to-lead optimization for the development of therapeutics against osteolytic diseases.

In vitro and in vivo assessment of vitamin A encapsulation in a liposome-protein delivery system.

Vitamin A (VA) is an essential nutrient needed in small amounts by humans and supports a wide range of biological actions. Retinol, the most common and most biologically active form of VA has also been found to inhibit peroxidation processes in membranes and it has been widely used as an ingredient with pharmaceutical and nutritional applications. VA is a lipophilic molecule, sensitive to air, oxidizing agents, ultraviolet light and low pH levels. For these reasons, it is necessary for VA to be protected against oxidation. Another disadvantage in the application of VA is its low solubility in aqueous media. Both issues (sensitivity and solubility) can be solved by employing encapsulation techniques. Liposomes can efficiently encapsulate lipid-soluble materials, such as VA. The encapsulated materials are protected from environmental and chemical changes. A new liposome/ß-lactoglobulin formulation has been developed as a stable delivery system for VA. The aim of this study was the encapsulation of VA into ß-lactoglobulin-liposome complexes, recently developed in our laboratory. The in vivo bioavailability characterization of VA was tested after administration in laboratory animals (mice). In this report, we demonstrate that VA could be efficiently entrapped and delivered in a phospholipid-sterol-protein membrane resembling system, a newly synthesized promising carrier. Based on this finding, the phospholipid-sterol-protein membrane resembling system may be one of the promising approaches to enhance VA absorption and to overcome the formulation difficulties associated with lipophilic means. The carrier system described here has huge potential in food fortification applications to treat VA deficiency.

Cheminformatics-aided discovery of small-molecule Protein-Protein Interaction (PPI) dual inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL).

We present an in silico drug discovery pipeline developed and applied for the identification and virtual screening of small-molecule Protein-Protein Interaction (PPI) compounds that act as dual inhibitors of TNF and RANKL through the trimerization interface. The cheminformatics part of the pipeline was developed by combining structure-based with ligand-based modeling using the largest available set of known TNF inhibitors in the literature (2481 small molecules). To facilitate virtual screening, the consensus predictive model was made freely available at: http://enalos.insilicotox.com/TNFPubChem/. We thus generated a priority list of nine small molecules as candidates for direct TNF function inhibition. In vitro evaluation of these compounds led to the selection of two small molecules that act as potent direct inhibitors of TNF function, with IC50 values comparable to those of a previously-described direct inhibitor (SPD304), but with significantly reduced toxicity. These molecules were also identified as RANKL inhibitors and validated in vitro with respect to this second functionality. Direct binding of the two compounds was confirmed both for TNF and RANKL, as well as their ability to inhibit the biologically-active trimer forms. Molecular dynamics calculations were also carried out for the two small molecules in each protein to offer additional insight into the interactions that govern TNF and RANKL complex formation. To our knowledge, these compounds, namely T8 and T23, constitute the second and third published examples of dual small-molecule direct function inhibitors of TNF and RANKL, and could serve as lead compounds for the development of novel treatments for inflammatory and autoimmune diseases.

A comparison of statistical approaches used for the optimization of soluble protein expression in Escherichia coli.

During a discovery project of potential inhibitors for three proteins, TNF-α, RANKL and HO-1, implicated in the pathogenesis of rheumatoid arthritis, significant amounts of purified proteins were required. The application of statistically designed experiments for screening and optimization of induction conditions allows rapid identification of the important factors and interactions between them. We have previously used response surface methodology (RSM) for the optimization of soluble expression of TNF-α and RANKL. In this work, we initially applied RSM for the optimization of recombinant HO-1 and a 91% increase of protein production was achieved. Subsequently, we slightly modified a published incomplete factorial approach (called IF1) in order to evaluate the effect of three expression variables (bacterial strains, induction temperatures and culture media) on soluble expression levels of the three tested proteins. However, soluble expression yields of TNF-α and RANKL obtained by the IF1 method were significantly lower (<50%) than those obtained by RSM. We further modified the IF1 approach by replacing the culture media with induction times and the resulted method called IF-STT (Incomplete Factorial-Stain/Temperature/Time) was validated using the three proteins. Interestingly, soluble expression levels of the three proteins obtained by IF-STT were only 1.2-fold lower than those obtained by RSM. Although RSM is probably the best approach for optimization of biological processes, the IF-STT is faster, it examines the most important factors (bacterial strain, temperature and time) influencing protein soluble expression in a single experiment, and can be used in any recombinant protein expression project as a starting point.

Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones.

Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred to as monomeric in this work) in a straightforward and facile manner will obviate protein--production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove.

Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review.

The supply of many valuable proteins that have potential clinical or industrial use is often limited by their low natural availability. With the modern advances in genomics, proteomics and bioinformatics, the number of proteins being produced using recombinant techniques is exponentially increasing and seems to guarantee an unlimited supply of recombinant proteins. The demand of recombinant proteins has increased as more applications in several fields become a commercial reality. Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, producing soluble proteins in E. coli is still a major bottleneck for structural biology projects. One of the most challenging steps in any structural biology project is predicting which protein or protein fragment will express solubly and purify for crystallographic studies. The production of soluble and active proteins is influenced by several factors including expression host, fusion tag, induction temperature and time. Statistical designed experiments are gaining success in the production of recombinant protein because they provide information on variable interactions that escape the "one-factor-at-a-time" method. Here, we review the most important factors affecting the production of recombinant proteins in a soluble form. Moreover, we provide information about how the statistical design experiments can increase protein yield and purity as well as find conditions for crystal growth.

ß-Lactoglobulin improves liposome's encapsulation properties for vitamin E delivery.

Vitamin E (VE) or α-tocopherol is the major fat-soluble antioxidant in the human body. It is a sensitive, easily oxidized in the air, molecule, so it must be protected from pro-oxidant elements which could affect its physiological benefits. Encapsulation constitutes a promising approach to maintain VE native properties over time and increase its concentration in aqueous media. Liposomes have been studied as sustained delivery systems, being biodegradable, non-toxic and non-immunogenic. A new liposome/ß-lactoglobulin (ß-Lg) formulation has been developed and characterized as a possible stable delivery system for VE. ß-Lg has been selected due to its property to bind a variety of hydrophobic molecules. The aim of this study was the preparation of ß-Lg-liposome formulation and the determination of VE encapsulation efficiency, in order to develop a new more efficient carrier for VE in aqueous media.

Rationally designed less toxic SPD-304 analogs and preliminary evaluation of their TNF inhibitory effects.

SPD-304 was discovered as a promising tumor necrosis factor alpha (TNF) antagonist that promotes dissociation of TNF trimers and therefore blocks the interaction of TNF and its receptor. However, SPD-304 contains a potentially toxic 3-alkylindole moiety, which can be bioactivated to a reactive electrophilic intermediate. A series of SPD-304 analogs was synthesized with the aim to diminish its toxicophore groups while maintaining the binding affinity for TNF. Incorporation of electron-withdrawing substituents at the indole moiety, in conjunction with elimination of the 6'-methyl group of the 4-chromone moiety, led to a significantly less toxic and equally potent TNF inhibitor.

A statistical approach for optimization of RANKL overexpression in Escherichia coli: purification and characterization of the protein.

Receptor activator of nuclear factor-κB (RANK) and its cognate ligand (RANKL) is a member of the TNF superfamily of cytokines which is essential in osteobiology and its overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Therefore, RANKL is considered a major therapeutic target for the suppression of bone resorption in bone metabolic diseases such as rheumatoid arthritis and cancer metastasis. To evaluate the inhibitory effect of potential RANKL inhibitors a sufficient amount of protein is required. In this work RANKL was cloned for expression at high levels in Escherichia coli with the interaction of changing cultures conditions in order to produce the protein in a soluble form. In an initial step, the effect of expression host on soluble protein production was investigated and BL21(DE3) pLysS was the most efficient one found for the production of RANKL. Central composite design experiment in the following revealed that cell density before induction, IPTG concentration, post-induction temperature and time as well as their interactions had a significant influence on soluble RANKL production. An 80% increase of protein production was achieved after the determination of the optimum induction conditions: OD600nm before induction 0.55, an IPTG concentration of 0.3mM, a post-induction temperature of 25°C and a post-induction time of 6.5h. Following RANKL purification the thermal stability of the protein was studied. The interaction of RANKL with SPD304, a patented small-molecule inhibitor of TNF-α, was also studied in a fluorescence binding assay resulting in a Kd value of 14.1 ± 0.5 µM.

Strong Binding of C-Glycosylic1,2-Thiodisaccharides to Galectin-3─Enthalpy-Driven Affinity Enhancement by Water-Mediated Hydrogen Bonds.

Galectin-3 is involved in multiple pathways of many diseases, including cancer, fibrosis, and diabetes, and it is a validated pharmaceutical target for the development of novel therapeutic agents to address unmet medical needs. Novel 1,2-thiodisaccharides with a C-glycosylic functionality were synthesized by the photoinitiated thiol-ene click reaction of O-peracylated 1-C-substituted glycals and 1-thio-glycopyranoses. Subsequent global deprotection yielded test compounds, which were studied for their binding to human galectin-3 by fluorescence polarization and isothermal titration calorimetry to show low micromolar Kd values. The best inhibitor displayed a Kd value of 8.0 µM. An analysis of the thermodynamic binding parameters revealed that the binding Gibbs free energy (ΔG) of the new inhibitors was dominated by enthalpy (ΔH). The binding mode of the four most efficient 1,2-thiodisaccharides was also studied by X-ray crystallography that uncovered the unique role of water-mediated hydrogen bonds in conferring enthalpy-driven affinity enhancement for the new inhibitors. This 1,2-thiodisaccharide-type scaffold represents a new lead for galectin-3 inhibitor discovery and offers several possibilities for further development.

Optimization of TNF-α overexpression in Escherichia coli using response surface methodology: Purification of the protein and oligomerization studies.

Tumor necrosis factor-α (TNF-α) is responsible for many autoimmune disorders including rheumatoid arthritis, psoriasis, Chron's disease, stroke, and atherosclerosis. Thus, inhibition of TNF-α is a major challenge in drug discovery. However, a sufficient amount of purified protein is needed for the in vitro screening of potential TNF-α inhibitors. In this work, induction conditions for the production of human TNF-α fusion protein in a soluble form by recombinant Escherichia coli BL21(DE3) pLysS were optimized using response surface methodology based on the central composite design. The induction conditions included cell density prior induction (OD(600nm)), post-induction temperature, IPTG concentration and post-induction time. Statistical analysis of the results revealed that all variables and their interactions had significant impact on production of soluble TNF-α. An 11% increase of TNF-α production was achieved after determination of the optimum induction conditions: OD(600nm) prior induction 0.55, a post induction temperature of 25°C, an IPTG concentration of 1mM and a post-induction time of 4h. We have also studied TNF-α oligomerization, the major property of this protein, and a K(d) value of 0.26nM for protein dimerization was determined. The concentration of where protein trimerization occurred was also detected. However, we failed to determine a reliable K(d) value for protein trimerization probably due to the complexibility of our model.

Discovery of Small-Molecule Inhibitors of Receptor Activator of Nuclear Factor-κB Ligand with a Superior Therapeutic Index.

Receptor activator of nuclear factor-κB ligand (RANKL) constitutes the master mediator of osteoclastogenesis, while its pharmaceutical inhibition by a monoclonal antibody has been approved for the treatment of postmenopausal osteoporosis. To date, the pursuit of pharmacologically more favorable approaches using low-molecular-weight inhibitors has been hampered by low specificity and high toxicity issues. This study aimed to discover small-molecule inhibitors targeting RANKL trimer formation. Through a systematic screening of 39 analogues of SPD-304, a dual inhibitor of tumor necrosis factor (TNF) and RANKL trimerization, we identified four compounds (1b, 3b, 4a, and 4c) that selectively inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner, without affecting TNF activity or osteoblast differentiation. Based on structure-activity observations extracted from the most potent and less toxic inhibitors of RANKL-induced osteoclastogenesis, we synthesized a focused set of compounds that revealed three potent inhibitors (19a, 19b, and 20a) with remarkably low cell-toxicity and improved therapeutic indexes as shown by the LC50 to IC50 ratio. These RANKL-selective inhibitors are an excellent starting point for the development of small-molecule therapeutics against osteolytic diseases.

Design and Synthesis of Type-IV Inhibitors of BRAF Kinase That Block Dimerization and Overcome Paradoxical MEK/ERK Activation.

Despite the clinical success of BRAF inhibitors like vemurafenib in treating metastatic melanoma, resistance has emerged through "paradoxical MEK/ERK signaling" where transactivation of one protomer occurs as a result of drug inhibition of the other partner in the activated dimer. The importance of the dimerization interface in the signaling potential of wild-type BRAF in cells expressing oncogenic Ras has recently been demonstrated and proposed as a site of therapeutic intervention in targeting cancers resistant to adenosine triphosphate competitive drugs. The proof of concept for a structure-guided approach targeting the dimerization interface is described through the design and synthesis of macrocyclic peptides that bind with high affinity to BRAF and that block paradoxical signaling in malignant melanoma cells occurring through this drug target. The lead compounds identified are type-IV kinase inhibitors and represent an ideal framework for conversion into next-generation BRAF inhibitors through macrocyclic drug discovery.

Thermodynamic, crystallographic and computational studies of non-mammalian fatty acid binding to bovine ß-Lactoglobulin.

The milk protein ß-lactoglobulin has been widely studied since its discovery, both as a purified protein and in mixtures with other milk proteins, where its effect on the processing properties is of importance to the dairy industry. The protein can bind a variety of small hydrophobic molecules, which may allow its use as an oral delivery vehicle. In the present study we have examined the binding of odd-numbered fatty acids by isothermal calorimetry (ITC), X-ray crystallography and computer modelling to provide a clearer picture of the extent and variability of the central binding pocket. The Kd values for the fatty acids C13, C15, C16, C17 and C19 as determined by ITC are 1.93, 2.91, 3.05, 4.11 and 8.67 × 10-7 M, respectively. The molecular structures revealed the ligands bound in the central cavity with generally well ordered lipophilic tails but significant positional variation at the carboxyl group end. In silico docking analyses identified the lipophilic interactions within the central cavity as the main driving force for binding with electrostatic interactions and H-bonds playing a minor role.

Aqueous Solubility Enhancement for Bioassays of Insoluble Inhibitors and QSPR Analysis: A TNF-α Study.

The aim of this study is to improve the aqueous solubility of a group of compounds without interfering with their bioassay as well as to create a relevant prediction model. A series of 55 potential small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α; SPD304 and 54 analogues), many of which cannot be bioassayed because of their poor solubility, was used for this purpose. The solubility of many of the compounds was sufficiently improved to allow measurement of their respective dissociation constants (Kd). Parameters such as dissolution time, initial state of the solute (solid/liquid), co-solvent addition (DMSO and PEG3350), and sample filtration were evaluated. Except for filtration, the remaining parameters affected aqueous solubility, and a solubilization protocol was established according to these. The aqueous solubility of the 55 compounds in 5% DMSO was measured with this protocol, and a predictive quantitative structure property relationship model was developed and fully validated based on these data. This classification model separates the insoluble from the soluble compounds and predicts the solubility of potential small-molecule inhibitors of TNF-α in aqueous solution (containing 5% DMSO as co-solvent) with an accuracy of 81.2%. The domain of applicability of the model indicates the type of compounds for which estimation of aqueous solubility can be confidently predicted.

Current Status and Future Prospects of Small-molecule Protein-protein Interaction (PPI) Inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL).

The overexpression of Tumor Necrosis Factor (TNF) is directly related to the development of several autoimmune diseases, such as rheumatoid and psoriatic arthritis, inflammatory bowel disease, Crohn's disease, refractory asthma, and multiple sclerosis. Receptor Activator of Nuclear Factor Kappa- B Ligand (RANKL) belongs to the TNF family and is the primary mediator of osteoclast-induced bone resorption through interaction with its receptor RANK. The function of RANKL is physiologically inhibited by the action of osteoprotegerin (OPG), which is a decoy receptor that binds to RANKL and prevents the process of osteoclastogenesis. Malfunction among RANK/RANKL/OPG can also result in bone loss diseases, including postmenopausal osteoporosis, rheumatoid arthritis, bone metastasis and multiple myeloma. To disrupt the unwanted functions of TNF and RANKL, current attempts focus on blocking TNF and RANKL binding to their receptors. In this review, we present the research efforts toward the development of low-molecular-weight pharmaceuticals that directly block the detrimental actions of TNF and RANKL.

Structure-based discovery of a family of synthetic cyclophilin inhibitors showing a cyclosporin-A phenotype in Caenorhabditis elegans.

Cyclophilins, which are found in all cellular compartments and with diverse biological roles, are now drug targets for a number of diseases including HIV infection, malaria and ischaemia. We used the database-mining program LIDAEUS and in silico screening to discover the dimedone family of inhibitors which show a conserved 'ball and socket' binding mode with a dimethyl group in the hydrophobic binding pocket of human cyclophilin A (CypA) mimicking a key interaction of the natural inhibitor cyclosporin A (CsA). The most potent derivative binds CypA with a K(d) of 11.2+/-9.2 microM and an IC50 for activity against Caenorhabditis elegans (C. elegans) of 190 microM compared to 28 microM for CsA. These dimedone analogues provide a new scaffold for the synthesis of families of peptidomimetic molecules with potential activity against HIV, malaria, and helminth parasite infections.

Catch the kinase conformer.

Protein kinases exist in inactive and active states, but little attention has been paid to which state is or should be the target in drug discovery efforts. In this issue of Chemistry & Biology, Okram et al. tackle this issue and show that inhibitors can be designed specifically to bind to inactive Abl.

Differential binding of inhibitors to active and inactive CDK2 provides insights for drug design.

The cyclin-dependent kinases (CDKs) have been characterized in complex with a variety of inhibitors, but the majority of structures solved are in the inactive form. We have solved the structures of six inhibitors in both the monomeric CDK2 and binary CDK2/cyclinA complexes and demonstrate that significant differences in ligand binding occur depending on the activation state. The binding mode of two ligands in particular varies substantially in active and inactive CDK2. Furthermore, energetic analysis of CDK2/cyclin/inhibitors demonstrates that a good correlation exists between the in vitro potency and the calculated energies of interaction, but no such relationship exists for CDK2/inhibitor structures. These results confirm that monomeric CDK2 ligand complexes do not fully reflect active conformations, revealing significant implications for inhibitor design while also suggesting that the monomeric CDK2 conformation can be selectively inhibited.