Improved Binding Properties Research Articles

Abstract 3125: TCX-101, a novel antibody-drug conjugate targeting a tumor-associated carbohydrate antigen for the treatment of solid tumors

Abstract Tumor-associated carbohydrate antigens (TACAs) are short oligosaccharides expressed on the surface of cancer cells and play key roles in cellular communication, immune evasion, apoptosis and metastasis. TACAs are therefore interesting targets for antibody-based therapies in cancer. We have generated an antibody (TCX-101) against a TACA structure with improved binding affinity compared to other monoclonal antibodies (mAbs) against the same target as shown by surface plasmon resonance (~ 10x superior KD), as well as high level of specificity against glycan species related to the target, as accessed by glycan array, flow cytometry, and immunocytochemistry or immunofluorescence. Immunohistochemistry studies on fresh-frozen tissue microarrays showed high expression of the target in various solid tumors (e.g. gastrointestinal, lung, and breast) and only low expression in normal tissues. In vitro, TCX-101 has ~ 4x higher maximum binding, ~ 2x faster binding kinetics, ~ 4x faster cellular internalization and induces ~ 2x higher antibody-dependent cellular cytotoxicity than a reference antibody. Monomethyl auristatin E (MMAE) was used in a drug-antibody ratio (DAR) of 4 to generate an antibody-drug conjugate (ADC). This ADC was effective in in vitro toxicity assays against murine B16 melanoma cells induced to express the target (measurable effect on WT cells), as well as human breast cancer MCF7 WT cells (~ 25x less activity on target-knockout MCF7 cells, Table 1). When tested in vivo, TCX-101 was highly effective in two different xenograft mouse models (B16, MCF7) with more than 95% tumor growth inhibition compared to vehicle, and no signs of toxicity (no weight loss, no histophatological findings). A fully humanized version of TCX-101 with improved binding properties (EC50 6.27 nM vs. 8.13 nM) was generated and will be evaluated in further in vivo experiments. In conclusion, TCX-101 is a promising ADC for the treatment of various solid tumors. IC50 [nM] TCX-101-MMAE 4 DAR B16 KI 7.64 B16 WT no killing observed MCF7 WT 1.97 MCF7 KO 45.97 Citation Format: Fausto G. Gomes, Francesco Muraca, Gustavo M. Schmidt Garcia Moreira, Kathrin Rösch, Maria Bräutigam, Peter Sondermann, Matthias Ocker. TCX-101, a novel antibody-drug conjugate targeting a tumor-associated carbohydrate antigen for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3125.

MolOptimizer: A Molecular Optimization Toolkit for Fragment-Based Drug Design.

MolOptimizer is a user-friendly computational toolkit designed to streamline the hit-to-lead optimization process in drug discovery. MolOptimizer extracts features and trains machine learning models using a user-provided, labeled, and small-molecule dataset to accurately predict the binding values of new small molecules that share similar scaffolds with the target in focus. Hosted on the Azure web-based server, MolOptimizer emerges as a vital resource, accelerating the discovery and development of novel drug candidates with improved binding properties.

Enhanced binding of methyl alkylammonium cations through preorganization of a water-soluble calix[4]pyrrole.

We describe the synthesis of two tetra-α aryl-extended calix[4]pyrroles (C[4]Ps) 4a-b bearing four terminal carboxylic groups in their meso-propyl chains defining the lower rims. The synthesized C[4]Ps became soluble (1-3 mM) in water at pD = 10. We probed the interaction of 4a towards tetra-methylammonium (G1) chloride in water using 1H NMR spectroscopy. The C[4]P 4a includes G1 in the shallow aromatic cavity defined by the pyrrole rings in cone conformation forming a 1 : 1 complex G1⊂4a. Pyridine-N-oxide (PNO) binding in the larger polar aromatic cavity of 4a results in the quantitative self-assembly of the supramolecular receptor PNO@4a featuring the pyrrole rings preorganized in cone conformation. The PNO@4a receptor displays improved binding properties towards G1 than the parent C[4]P 4a. We thermodynamically characterized (1H NMR titrations and ITC experiments) the 1 : 1 complexes of PNO@4a with a series of tetra-alkylammonium salts, including biologically relevant examples. The PNO@4a supramolecular receptor displays significant affinity (log K = 3-4) but lacks selectivity in water binding of methyl trialkyl ammonium cations. Cation-π and coulombic interactions are the main intermolecular forces stabilizing the complexes. We also performed DFT calculations to gain some insights into the complexes' structures.

Flowability, binding and release property of “self-lubricating” microcrystalline cellulose

Microcrystalline cellulose (MCC) has been widely used as an excipient in tablets. Silicified microcrystalline cellulose (SMCC) has more application than MCC by using silicon dioxide (SiO2) as a lubricant. However, findings show that the silicon dioxide (SiO2) nanoparticle component is a physiological hazard. In our research, “self-lubricating” microcrystalline cellulose (MCC) was developed by a ball-milling treatment. Commercial MCC and undried MCC with 60% or 80% moisture content were ball-milled for 12 or 24 h. After ball milling, ellipsoid-like particles, with wrinkled surfaces, and a broader particle size distribution resulted in a balance between the flowability and tabletability of the treated MCC. FTIR and XRD analysis showed that cellulose Ⅱ was formed in the undried 60% moisture content MCC after ball milling for 12 or 24 h, which improved the burst disintegration ability of the tablets. In addition, the controlled-released MCC has also been developed. AFM patterns and the angle of repose showed a cohesion phenomenon and some bridges formed between particles of undried 80% moisture content MCC ball-milled for 12 h. Without adding SiO2, our MCC gained improved binding properties, a balance between flowability and tabletability, and a more useful controlled-released ability compared with commercial MCC and SMCC.

A third dose of the unmodified COVID-19 mRNA vaccine CVnCoV enhances quality and quantity of immune responses

A third vaccine dose is often required to achieve potent, long-lasting immune responses. We investigated the effect of three 8-μg doses of CVnCoV, CureVac’s severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine candidate containing sequence-optimized unmodified mRNA encoding the spike (S) glycoprotein, administered at 0, 4, and 28 weeks, on immune responses in rhesus macaques. After the third dose, S-specific binding and neutralizing antibodies increased 50-fold compared with post-dose 2 levels, with increased responses also evident in the lower airways and against the SARS-CoV-2 B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) variants. Enhanced binding affinity of serum antibodies after the third dose correlated with higher somatic hypermutation in S-specific B cells, corresponding with improved binding properties of monoclonal antibodies expressed from isolated B cells. Administration of low-dose mRNA led to fewer cells expressing antigen in vivo at the injection site and in the draining lymph nodes compared with a 10-fold higher dose, possibly reducing engagement of precursor cells with the antigen and resulting in the suboptimal response observed after two-dose vaccination schedules in phase IIb/III clinical trials of CVnCoV. However, when immune memory is established, a third dose efficiently boosts the immunological responses and improves antibody affinity and breadth.

A review on current progress of graphene-based ternary nanocomposites in the removal of anionic and cationic inorganic pollutants

The current review aims to summarize the ongoing advances in high-performing graphene-based ternary nanocomposites for removing cationic and anionic inorganic pollutants. Graphene derivatives are extensively utilized for the development of composites due to their high synergism with co-functional materials, rational design, flexible surface chemistry, high mobile charge carriers, improved binding properties, and many more. The past ten years have witnessed progressive research on graphene-based ternary nanocomposites in a multitude of pollution remediation applications. Therefore, the focus falls on understanding how these ternary nanocomposites are tailored to capture the inorganic cationic and anionic contaminants with particular emphasis on graphene derivatives as base matrix and filler. The review investigates the synthesis, categorization, and characterization techniques of graphene-based ternary composites. Besides, the study broadens the understanding of the binding mechanism of the pollutants onto graphene ternary composites. The review also assesses the separation and recycling efficacy of the composites in detail. The future prospects in improving the practical application of the ternary systems also have been discussed. The comprehensive review on graphene based ternary systems detailing their structural and functional aspects, as well as their performance as inorganic decontaminants can provide deep insights for researchers in improvising wastewater treatment technologies.

Multiple-Allele MHC Class II Epitope Engineering by a Molecular Dynamics-Based Evolution Protocol.

Epitopes that bind simultaneously to all human alleles of Major Histocompatibility Complex class II (MHC II) are considered one of the key factors for the development of improved vaccines and cancer immunotherapies. To engineer MHC II multiple-allele binders, we developed a protocol called PanMHC-PARCE, based on the unsupervised optimization of the epitope sequence by single-point mutations, parallel explicit-solvent molecular dynamics simulations and scoring of the MHC II-epitope complexes. The key idea is accepting mutations that not only improve the affinity but also reduce the affinity gap between the alleles. We applied this methodology to enhance a Plasmodium vivax epitope for multiple-allele binding. In vitro rate-binding assays showed that four engineered peptides were able to bind with improved affinity toward multiple human MHC II alleles. Moreover, we demonstrated that mice immunized with the peptides exhibited interferon-gamma cellular immune response. Overall, the method enables the engineering of peptides with improved binding properties that can be used for the generation of new immunotherapies.

Amyloid and Tau Positron Emission Tomography Imaging in Alzheimer's Disease and Other Tauopathies.

The detection and staging of Alzheimer’s disease (AD) using non-invasive imaging biomarkers is of substantial clinical importance. Positron emission tomography (PET) provides readouts to uncover molecular alterations in the brains of AD patients with high sensitivity and specificity. A variety of amyloid-β (Aβ) and tau PET tracers are already available for the clinical diagnosis of AD, but there is still a lack of imaging biomarkers with high affinity and selectivity for tau inclusions in primary tauopathies, such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and Pick’s disease (PiD). This review aims to provide an overview of the existing Aβ and tau PET imaging biomarkers and their binding properties from in silico, in vitro, and in vivo assessment. Imaging biomarkers for pathologic proteins are vital for clinical diagnosis, disease staging and monitoring of the potential therapeutic approaches of AD. Off-target binding of radiolabeled tracers to white matter or other neural structures is one confounding factor when interpreting images. To improve binding properties such as binding affinity and to eliminate off-target binding, second generation of tau PET tracers have been developed. To conclude, we further provide an outlook for imaging tauopathies and other pathological features of AD and primary tauopathies.

Novel Variant Ig Domain (vIgD) Proteins Generated Via Directed Evolution of Igsf Domains Have Therapeutic Efficacy in Animal Models of Graft Versus Host Disease

The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins extensively targeted for treatment of cancers and autoimmune diseases. Most of the therapeutic strategies targeting this family have focused on high affinity antibodies binding a single receptor. Wild-type IgSF receptors typically exhibit low affinities for their counter-structures, limiting their utility in therapeutic modulation of immune responses. We have developed a novel variant Ig domain™ (vIgD™) directed evolution platform to affinity mature human IgSF extracellular domains. In this platform, libraries of mutagenized IgSF domains are selected for altered affinity to specific recombinant protein counterstructures. Fc fusion proteins incorporating the resulting engineered IgSF domains are then tested in vitro for their ability to either agonize or antagonize T cell responses.We successfully engineered the IgSF protein ICOSL, which binds to the costimulatory molecule ICOS on T cells and triggers T cell activation. We used our directed evolution platform to engineer a first-in-class single vIgD domain with higher affinity for ICOS and high affinity binding to the costimulatory molecule CD28, which ICOSL does not normally bind. Following the creation of dual ICOS/CD28 antagonism, the resulting domains were used to generate individual constructs encoding Fc-fusion proteins. These enhanced ICOS/CD28 dual antagonist Fc-fusion proteins were transfected into HEK-293 cells and protein produced by these cells was purified by protein A affinity chromatography. The resultant purified proteins were then tested in a FACS based binding assay on cells transfected with each costimulatory receptor showing up to a 2-3-fold increase in ICOS binding and high affinity binding to CD28. Various ICOS/CD28 dual antagonist Fc-fusion proteins were then tested in soluble format over a range of concentrations for functional activity in mixed lymphocyte reaction cultures (MLR) where allogeneic dendritic cells (DC) were used to stimulate purified human T cells. Most Variants showed significant ability to inhibit the MLR response as read out measuring proliferation and cytokine production. The potency of the Fc-fusion proteins varied somewhat depending on their relative affinities for CD28 and ICOS, with all being superior to wild type ICOSL protein and many showing superiority to the CD28 blocking reagent belatacept (CTLA4-Fc). The level of inhibition correlated well with binding of the Fc-fusion proteins to the T cells in culture.To assess the function of the ICOS/CD28 dual antagonist Fc-fusion proteins in vivo, a subset were tested in the human PBMC-NSG™ GvHD mouse model. This model monitors graft versus host disease generated by xenogeneic responses of T cells in human PBMCs transferred into NSG™. Administration of the dual ICOS/CD28 antagonist Fc-fusion proteins significantly protected mice from the effects of xenogeneic T cell activation in vivo with treated animals showing dramatically enhanced survival and greatly reduced disease scores. Interestingly, the level of protection correlated with the potency of the molecules in the in vitro MLR assay with less potent molecules in MLR assays failing to protect while more potent molecules protected well. Belatacept was also effective in protecting animals from GvHD, but not as well as the ICOS/CD28 dual antagonists produced by our vIgD platform. Collectively, these data demonstrate the vIgD directed evolution platform can generate IgSF proteins with altered affinities and improved binding properties. Variants engineered for desired immunological properties can effectively perturb T cell costimulation and affect T cell responses both in vitro and in vivo with the in vitro potency of the proteins correlating to in vivo potency. The dual ICOS/CD28 antagonists produced with the vIgD platform can be used therapeutically in disease settings where attenuation of T cell responses is beneficial, including GvHD and potentially other autoimmune diseases. Phase I enabling studies are in progress. [Display omitted] DisclosuresLevin:Alpine Immune Sciences: Employment, Equity Ownership. Evans:Alpine Immune Sciences: Employment, Equity Ownership. Rickel:Alpine Immune Sciences: Employment, Equity Ownership. Wolfson:Alpine Immune Sciences: Employment, Equity Ownership. Dillon:Alpine Immune Sciences: Employment, Equity Ownership. Kornacker:Alpine Immune Sciences: Employment, Equity Ownership, Patents & Royalties. Swanson:Alpine Immune Sciences: Employment, Equity Ownership, Patents & Royalties. Peng:Alpine Immune Sciences: Employment, Equity Ownership.

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode.

Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion.

The systematic evolution of ligands by exponential enrichment (SELEX) process enables the isolation of aptamers from random oligonucleotide libraries. However, it is generally difficult to identify the best aptamer from the resulting sequences, and the selected aptamers often exhibit suboptimal affinity and specificity. Post-SELEX aptamer engineering can improve aptamer performance, but current methods exhibit inherent bias and variable rates of success or require specialized instruments. Here, we describe a generalizable method that utilizes exonuclease III and exonuclease I to interrogate the binding properties of small-molecule-binding aptamers in a rapid, label-free assay. By analyzing an ochratoxin-binding DNA aptamer and six of its mutants, we determined that ligand binding alters the exonuclease digestion kinetics to an extent that closely correlates with the aptamer's ligand affinity. We then utilized this assay to enhance the binding characteristics of a DNA aptamer which binds indiscriminately to ATP, ADP, AMP, and adenosine. We screened 13 mutants derived from this aptamer against all these analogues and identified two new high-affinity aptamers that solely bind to adenosine. We incorporated these two aptamers directly into an electrochemical aptamer-based sensor, which achieved a detection limit of 1 μM adenosine in 50% serum. We also confirmed the generality of our method to characterize target-binding affinities of protein-binding aptamers. We believe our approach is generalizable for DNA aptamers regardless of sequence, structure, and length and could be readily adapted into an automated format for high-throughput engineering of small-molecule-binding aptamers to acquire those with improved binding properties suitable for various applications.

Design and Characterisation of Inhibitory Peptides against Bleg1_2478, an Evolutionary Divergent B3 Metallo-β-lactamase.

Previously, a hypothetical protein (HP) termed Bleg1_2437 (currently named Bleg1_2478) from Bacillus lehensis G1 was discovered to be an evolutionary divergent B3 subclass metallo-β-lactamase (MBL). Due to the scarcity of clinical inhibitors for B3 MBLs and the divergent nature of Bleg1_2478, this study aimed to design and characterise peptides as inhibitors against Bleg1_2478. Through in silico docking, RSWPWH and SSWWDR peptides with comparable binding energy to ampicillin were obtained. In vitro assay results showed RSWPWH and SSWWDR inhibited the activity of Bleg1_2478 by 50% at concentrations as low as 0.90 µM and 0.50 µM, respectively. At 10 µM of RSWPWH and 20 µM of SSWWDR, the activity of Bleg1_2478 was almost completely inhibited. Isothermal titration calorimetry (ITC) analyses showed slightly improved binding properties of the peptides compared to ampicillin. Docked peptide–protein complexes revealed that RSWPWH bound near the vicinity of the Bleg1_2478 active site while SSWWDR bound at the center of the active site itself. We postulate that the peptides caused the inhibition of Bleg1_2478 by reducing or blocking the accessibility of its active site from ampicillin, thus hampering its catalytic function.

Structure of an affinity-matured inhibitory recombinant fab against urokinase plasminogen activator reveals basis of potency and specificity

Affinity maturation of U33, a recombinant Fab inhibitor of uPA, was used to improve the affinity and the inhibitory effect compared to the parental Fab. Arginine scanning of the six CDR loops of U33 was done to identify initial binding determinants since uPA prefers arginine in its primary substrate binding pocket. Two CDR loops were selected to create an engineered affinity maturation library of U33 that was diversified around ArgL91 (CDR L3) and ArgH52 (CDR H2). Biopanning of the randomized U33 library under stringent conditions resulted in eight Fabs with improved binding properties. One of the most potent inhibitors, AB2, exhibited a 13-fold decrease in IC50 when compared to U33 largely due to a decrease in its off rate. To identify contributions of interfacial residues that might undergo structural rearrangement upon interface formation we used X-ray footprinting and mass spectrometry (XFMS). Four residues showed a pronounced decrease in solvent accessibility, and their clustering suggests that AB2 targets the active site and also engages residues in an adjacent pocket unique to human uPA. The 2.9 Å resolution crystal structure of AB2-bound to uPA shows a binding mode in which the CDR L1 loop inserts into the active site cleft and acts as a determinant of inhibition. The selectivity determinant of this binding mode is unlike previously identified inhibitory Fabs against uPA related serine proteases, MTSP-1, HGFA and FXIa. CDRs H2 and L3 loops aid in interface formation and provide critical salt-bridges to remodel loops surrounding the active site of uPA providing specificity and further evidence that antibodies can be potent and selective inhibitors of proteolytic enzymes.

Computational engineering the binding affinity of Adalimumab monoclonal antibody for designing potential biosimilar candidate

Amongst the anti-TNF-α therapy for rheumatoid arthritis and other autoimmune diseases, Adalimumab mAb is one of the best candidates. However, several risk factors are found to be associated with higher doses. Improvement of the binding properties will therefore significantly increase its therapeutic efficacy, reduce the dosage requirements, and ultimately the associated toxicity and treatment cost. Here, we proposed a systematic in silico approach of finding newer mAb variants with improved binding properties. Using various bioinformatics tools, we have identified the significant amino acid residues on Adalimumab mAb. Next, we searched for the suitability of the other residues for mutating the significant residues and from the combinations of suitable mutations, variants were designed. To find the most significant ones, binding properties of the variants were compared with the wild type Adalimumab mAb using molecular docking scrutiny and molecular dynamics simulation. Finally, structural properties between the variant and wild type were analyzed. We have identified the six most significant residues on Adalimumab mAb involved in the antigen-antibody interactions. Using the suitable mutations replacing each of these residues, we have modeled 143 variants. From several docking analyses, we have found five significant variants and after molecular dynamics simulation, one most significant variant with improved binding affinity was identified whose structural properties are similar to the wild type Adalimumab mAb. Designed variant from this study, may provide newer insights on the structure-based affinity improvements of monoclonal antibodies and likewise modifications of the Fc region will also improve the therapeutic effector functions of antibodies too.

Fidelity of Al2(SO4)3 for the Discharge of Pectin during the Pretreatment of Apocynum Venetum

ABSTRACT During the extraction of fibers from the bast of A. venetum, there is an issue of the existence of undesirable macromolecules, for example, pectins. Separation of these compounds is mostly done by pretreatment with H2SO4 and then by NaOH boiling in the traditional method (Fiber-C). The use of Sulfuric acid for pretreatment may cause harm to both fibers and the environment. In order to overcome these limitations, attempts were made to find a new pretreatment (Fiber-N) method. The hypothetical premise of the new pretreatment strategy dependent on the use of Al2(SO4)3 is presented. Four model solutions were investigated using a method of measuring zeta potential. Furthermore, fibers obtained by pretreatment methods along with the raw fibers of A. venetum had been characterized by Electron spin resonance spectroscopy (ESR), X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transforms infrared spectroscopy (FT-IR), and water retention value (WRV). Results showed that pure salts of Al2(SO4)3 presented improved binding properties with the pectin and causes better swelling and eventually helps to remove pectin than other pretreatment processes. Ultimately, Donnan equilibrium, metal ion complex formation, and Gouy–Chapman–Stern (GCS) model were used to theoretically elucidate the discharging mechanism of pectin macromolecules from A. venetum in the presence of Al3+ ions during pretreatment.

Structure-based virtual screening of influenza virus RNA polymerase inhibitors from natural compounds: Molecular dynamics simulation and MM-GBSA calculation

The resistances of matrix protein 2 (M2) protein inhibitors and neuraminidase inhibitors for influenza virus have attracted much attention and there is an urgent need for new drug. The antiviral drugs that selectively act on RNA polymerase are less prone to resistance and possess fewer side effects on the patient. Therefore, there is increased interest in screening compounds that can inhibit influenza virus RNA polymerase. Three natural compounds were found by using molecular docking-based virtual screening, which could bind tightly within the polymerase acidic protein-polymerase basic protein 1 (PA-PB1) subunit of influenza virus polymerase. Firstly, their drug likeness properties were evaluated, which showed that the hepatotoxicity values of all the three compounds indicating they had less or no hepatotoxicity, and did not have the plasma protein biding (PPB) ability, the three compounds needed to be modified in some aspects, like bulky molecular size. The stability of the complexes of PA-hits was validated through molecular dynamics (MD) simulation, revealing compound 2 could form more stable complex with PA subunit. The torsional conformations of each rotatable bond of the ligands in PA subunit were also monitored, to investigate variation in the ligand properties during the simulation, compound 3 had fewer rotatable bonds, indicating that the molecule had stronger rigidity. The bar charts of protein–ligand contacts and contacts over the course of trajectory showed that four key residues (Glu623, Lys643, Asn703 and Trp706) of PA subunit that participated in hydrogen-bond, water bridge and hydrophobic interactions with the hit compounds. Finally, the binding free energy and contributed energies were calculated by using MM-GBSA method. Out of the three compounds, compound 1 showed the lowest total binding free energy. Among all the interactions, the contribution of the covalent binding and the van der Waals energy were more than other items, compound 1 formed more stable hydrogen bonds with the residues of PA subunit binding pocket. This study smoothed the path for the development of novel lead compounds with improved binding properties, high drug likeness, and low toxicity to humans for the treatment of influenza, which provided a good basis for further research on novel and effective influenza virus PA-PB1 interaction inhibitors.

(R)-[18F]NEBIFQUINIDE: A promising new PET tracer for TSPO imaging

Positron emission tomography (PET) imaging of the 18 kDa translocator protein (TSPO), has a high diagnostic potential in neurodegenerative disorders and cancer. However, TSPO is considered a challenge for molecular imaging due to the poor availability of suitable radiotracers with adequate pharmacokinetic properties. Here, we describe the development of a radiofluorinated pyridinyl isoquinoline analogue of the established TSPO PET tracer (R)-[11C]PK11195 with improved binding properties in all known human TSPO phenotypes. We conducted a complete preclinical evaluation using in vitro, in vivo and ex vivo methods to assess the performance of this novel radiotracer and observed high specific binding of the radiotracer to TSPO, as well as high metabolic stability. Therefore, we propose this radiolabeled compound for further evaluation in animal models as well as in clinical trials.

Dimerization of a ubiquitin variant leads to high affinity interactions with a ubiquitin interacting motif.

We previously described structural and functional characterization of the first ubiquitin variant (UbV), UbV.v27.1, engineered by phage display to bind with high affinity to a specific ubiquitin interacting motif (UIM). We identified two substitutions relative to ubiquitin (Gly10Val/His68Tyr) that were critical for enhancing binding affinity but could only rationalize the mechanism of action of the Tyr68 substitution. Here, we extend our characterization and uncover the mechanism by which the Val10 substitution enhances binding affinity. We show that Val10 in UbV.v27.1 drives UbV dimerization through an intermolecular β-strand exchange. Dimerization serves to increase the contact surface between the UIM and UbV and also affords direct contacts between two UIMs through an overall 2:2 binding stoichiometry. Our identification of the role of Val10 in UbV dimerization suggests a general means for the development of dimeric UbVs with improved affinity and specificity relative to their monomeric UbV counterparts. Statement: Previously, we used phage display to engineer a UbV that bound tightly and specifically to a UIM. Here, we discovered that tight binding is partly due to the dimerization of the UbV, which increases the contact surface between the UbV and UIM. We show that UbV dimerization is dependent on the Gly10Val substitution, and posit that dimerization may provide a general means for engineering UbVs with improved binding properties.

Identification of novel PI3Kδ inhibitors by docking, ADMET prediction and molecular dynamics simulations

BackgroundPhosphoinositide-3-kinase Delta (PI3Kδ) plays a key role in B-cell signal transduction and inhibition of PI3Kδ is confirmed to have clinical benefit in certain types of activation of B-cell malignancies. Virtual screening techniques have been used to discover new molecules for developing novel PI3Kδ inhibitors with little side effects. MethodComputer aided drug design method were used to rapidly screen optimal PI3Kδ inhibitors from the Asinex database. Virtual screening based molecular docking was performed to find novel and potential lead compound targeting PI3Kδ, at first. Subsequently, drug likeness studies were carried out on the retrieved hits to evaluate and analyze their drug like properties such as absorption, distribution, metabolism, excretion, and toxicity (ADMET) for toxicity prediction. Three least toxic compounds were selected for the molecular dynamics (MD) simulations for 30 ns in order to validate its stability inside the active site of PI3Kδ receptor. ResultsBased on the present in silico analysis, two molecules have been identified which occupied the same binding pocket confirming the selection of active site. ASN 16296138 (Glide score: −12.175 kcal/mol, cdocker binding energy: −42.975 kcal/mol and ΔGbind value: −90.457 kcal/mol) and BAS 00227397 (Glide score: −10.988 kcal/mol, cdocker binding energy: −39.3376 kcal/mol and ΔGbind value: −81.953 kcal/mol) showed docking affinities comparatively much stronger than those of already reported known inhibitors against PI3Kδ. These two ligand’s behaviors also showed consistency during the simulation of protein-ligand complexes for 30000 ps respectively, which is indicative of its stability in the receptor pocket. ConclusionCompound ASN 16296138 and BAS 00227397 are potential candidates for experimental validation of biological activity against PI3Kδ in future drug discovery studies. This study smoothes the path for the development of novel leads with improved binding properties, high drug likeness, and low toxicity to humans for the treatment of cancer.

Radiosynthesis and in vivo evaluation of a fluorine-18 labeled pyrazine based radioligand for PET imaging of the adenosine A2B receptor

On the basis of a pyrazine core structure, three new adenosine A2B receptor ligands (7a–c) were synthesized containing a 2-fluoropyridine moiety suitable for 18F-labeling. Compound 7a was docked into a homology model of the A2B receptor based on X-ray structures of the related A2A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A1 receptor, 7a was radiolabeled as the most suitable candidate (Ki(A2B) = 4.24 nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [18F]7a with a standardized uptake value (SUV) of ≈1 at 5 min post injection followed by a fast wash out. Metabolism studies of [18F]7a in mice revealed the formation of a blood–brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo.