Enhancing breadth and durability of humoral immune responses in non-human primates with an adjuvanted group 1 influenza hemagglutinin stem antigen.

Seasonal influenza vaccines must be updated annually and suboptimally protect against strains mismatched to the selected vaccine strains. We previously developed a subunit vaccine antigen consisting of a stabilized trimeric influenza A group 1 hemagglutinin (H1) stem protein that elicits broadly neutralizing antibodies. Here, we further optimized the stability and manufacturability of the H1 stem antigen (H1 stem v2, also known as INFLUENZA G1 mHA) and characterized its formulation and potency with different adjuvants in vitro and in animal models. The recombinant H1 stem antigen (50 µg) was administered to influenza-naïve non-human primates either with aluminum hydroxide [Al(OH)3] + NaCl, AS01B, or SLA-LSQ formulations at week 0, 8 and 34. These SLA-LSQ formulations comprised of varying ratios of the synthetic TLR4 agonist 'second generation synthetic lipid adjuvant' (SLA) with liposomal QS-21 (LSQ). A vaccine formulation with aluminum hydroxide or SLA-LSQ (starting at a 10:25 µg ratio) induced HA-specific antibodies and breadth of neutralization against a panel of influenza A group 1 pseudoviruses, comparable with vaccine formulated with AS01B, four weeks after the second immunization. A formulation with SLA-LSQ in a 5:2 µg ratio contained larger fused or aggregated liposomes and induced significantly lower humoral responses. Broadly HA stem-binding antibodies were detectable for the entire period after the second vaccine dose up to week 34, after which they were boosted by a third vaccine dose. These findings inform about potential adjuvant formulations in clinical trials with an H1 stem-based vaccine candidate.

RSV A2-Based Prefusion F Vaccine Candidates Induce RSV A and RSV B Cross Binding and Neutralizing Antibodies and Provide Protection against RSV A and RSV B Challenge in Preclinical Models.

RSV is divided into two antigenic subtypes, RSV A and RSV B, which is largely based on the variation in the G protein, while the fusion protein F is more conserved and a target for antibody-mediated neutralization. Here we evaluate the breadth of the protective immune responses across RSV A and RSV B subtypes, induced by vaccines based on the RSV A-based fusion protein, stabilized in the prefusion conformation (preF) in preclinical models. Immunization of naïve cotton rats with preF subunit or preF encoded by a replication incompetent Adenoviral 26, induced antibodies capable of neutralizing recent RSV A and RSV B clinical isolates, as well as protective efficacy against a challenge with RSV A and RSV B strains. Similarly, induction of cross-neutralizing antibodies was observed after immunization with Ad26-encoded preF, preF protein or a mix of both (Ad26/preF protein) in RSV pre-exposed mice and African Green Monkeys. Transfer of serum of human subjects immunized with Ad26/preF protein into cotton rats provide protection against challenges with both RSV A and RSV B, with complete protection against both strains observed in the lower respiratory tract. In contrast, almost no protection against RSV A and B infection was observed after the transfer of a human serum pool isolated pre-vaccination. These results collectively show that the RSV A-based monovalent Ad26/preF protein vaccine induced neutralizing antibodies, as well as protection against both RSV A and RSV B subtypes in animals, including by passive transfer of human antibodies alone, suggesting that clinical efficacy against both subtypes can be achieved.

Universal stabilization of the influenza hemagglutinin by structure-based redesign of the pH switch regions.

For an efficacious vaccine immunogen, influenza hemagglutinin (HA) needs to maintain a stable quaternary structure, which is contrary to the inherently dynamic and metastable nature of class I fusion proteins. In this study, we stabilized HA with three substitutions within its pH-sensitive regions where the refolding starts. An X-ray structure reveals how these substitutions stabilize the intersubunit ß-sheet in the base and form an interprotomeric aliphatic layer across the stem while the native prefusion HA fold is retained. The identification of the stabilizing substitutions increases our understanding of how the pH sensitivity is structurally accomplished in HA and possibly other pH-sensitive class I fusion proteins. Our stabilization approach in combination with the occasional back mutation of rare amino acids to consensus results in well-expressing stable trimeric HAs. This repair and stabilization approach, which proves broadly applicable to all tested influenza A HAs of group 1 and 2, will improve the developability of influenza vaccines based on different types of platforms and formats and can potentially improve efficacy.

Stabilizing the closed SARS-CoV-2 spike trimer.

The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, we report the structure-based design of soluble S trimers with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. We identify regions critical for stability: the heptad repeat region 1, the SD1 domain and position 614 in SD2. We combine a minimal selection of mostly interprotomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.

Drug Discovery and Repurposing Inhibits a Major Gut Pathogen-Derived Oncogenic Toxin.

Objective: The human intestinal microbiome plays an important role in inflammatory bowel disease (IBD) and colorectal cancer (CRC) development. One of the first discovered bacterial mediators involves Bacteroides fragilis toxin (BFT, also named as fragilysin), a metalloprotease encoded by enterotoxigenic Bacteroides fragilis (ETBF) that causes barrier disruption and inflammation of the colon, leads to tumorigenesis in susceptible mice, and is enriched in the mucosa of IBD and CRC patients. Thus, targeted inhibition of BFT may benefit ETBF carrying patients. Design: By applying two complementary in silico drug design techniques, drug repositioning and molecular docking, we predicted potential BFT inhibitory compounds. Top candidates were tested in vitro on the CRC epithelial cell line HT29/c1 for their potential to inhibit key aspects of BFT activity, being epithelial morphology changes, E-cadherin cleavage (a marker for barrier function) and increased IL-8 secretion. Results: The primary bile acid and existing drug chenodeoxycholic acid (CDCA), currently used for treating gallstones, cerebrotendinous xanthomatosis, and constipation, was found to significantly inhibit all evaluated cell responses to BFT exposure. The inhibition of BFT resulted from a direct interaction between CDCA and BFT, as confirmed by an increase in the melting temperature of the BFT protein in the presence of CDCA. Conclusion: Together, our results show the potential of in silico drug discovery to combat harmful human and microbiome-derived proteins and more specifically suggests a potential for retargeting CDCA to inhibit the pro-oncogenic toxin BFT.

Structural basis for recognition of the central conserved region of RSV G by neutralizing human antibodies.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly, and yet there remains no effective treatment or vaccine. The surface of the virion is decorated with the fusion glycoprotein (RSV F) and the attachment glycoprotein (RSV G), which binds to CX3CR1 on human airway epithelial cells to mediate viral attachment and subsequent infection. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region of G have been shown to neutralize both subtypes of RSV and to protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated two human antibodies directed against the central conserved region of RSV G and demonstrated that they neutralize RSV infection of human bronchial epithelial cell cultures in the absence of complement. Moreover, the antibodies protected cotton rats from severe RSV disease. Both antibodies bound with high affinity to a secreted form of RSV G as well as to a peptide corresponding to the unglycosylated central conserved region. High-resolution crystal structures of each antibody in complex with the G peptide revealed two distinct conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with the structure of fractalkine (CX3CL1) alone or in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. Collectively, these results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide the development of new vaccines and antibody-based therapies for RSV.

Steering Siglec-Sialic Acid Interactions on Living Cells using Bioorthogonal Chemistry.

Sialic acid sugars that terminate cell-surface glycans form the ligands for the sialic acid binding immunoglobulin-like lectin (Siglec) family, which are immunomodulatory receptors expressed by immune cells. Interactions between sialic acid and Siglecs regulate the immune system, and aberrations contribute to pathologies like autoimmunity and cancer. Sialic acid/Siglec interactions between living cells are difficult to study owing to a lack of specific tools. Here, we report a glycoengineering approach to remodel the sialic acids of living cells and their binding to Siglecs. Using bioorthogonal chemistry, a library of cells with more than sixty different sialic acid modifications was generated that showed dramatically increased binding toward the different Siglec family members. Rational design reduced cross-reactivity and led to the discovery of three selective Siglec-5/14 ligands. Furthermore, glycoengineered cells carrying sialic acid ligands for Siglec-3 dampened the activation of Siglec-3+ monocytic cells through the NF-κB and IRF pathways.

3D-e-Chem-VM: Structural Cheminformatics Research Infrastructure in a Freely Available Virtual Machine.

3D-e-Chem-VM is an open source, freely available Virtual Machine ( http://3d-e-chem.github.io/3D-e-Chem-VM/ ) that integrates cheminformatics and bioinformatics tools for the analysis of protein-ligand interaction data. 3D-e-Chem-VM consists of software libraries, and database and workflow tools that can analyze and combine small molecule and protein structural information in a graphical programming environment. New chemical and biological data analytics tools and workflows have been developed for the efficient exploitation of structural and pharmacological protein-ligand interaction data from proteomewide databases (e.g., ChEMBLdb and PDB), as well as customized information systems focused on, e.g., G protein-coupled receptors (GPCRdb) and protein kinases (KLIFS). The integrated structural cheminformatics research infrastructure compiled in the 3D-e-Chem-VM enables the design of new approaches in virtual ligand screening (Chemdb4VS), ligand-based metabolism prediction (SyGMa), and structure-based protein binding site comparison and bioisosteric replacement for ligand design (KRIPOdb).

Molecular interaction fingerprint approaches for GPCR drug discovery.

Protein-ligand interaction fingerprints (IFPs) are binary 1D representations of the 3D structure of protein-ligand complexes encoding the presence or absence of specific interactions between the binding pocket amino acids and the ligand. Various implementations of IFPs have been developed and successfully applied for post-processing molecular docking results for G Protein-Coupled Receptor (GPCR) ligand binding mode prediction and virtual ligand screening. Novel interaction fingerprint methods enable structural chemogenomics and polypharmacology predictions by complementing the increasing amount of GPCR structural data. Machine learning methods are increasingly used to derive relationships between bioactivity data and fingerprint descriptors of chemical and structural information of binding sites, ligands, and protein-ligand interactions. Factors that influence the application of IFPs include structure preparation, binding site definition, fingerprint similarity assessment, and data processing and these factors pose challenges as well possibilities to optimize interaction fingerprint methods for GPCR drug discovery.

Involvement of a Hydrophobic Pocket and Helix†11 in Determining the Modes of Action of Prenylated Flavonoids and Isoflavonoids in the Human Estrogen Receptor.

Six prenylated (iso)flavonoids were purified from a licorice root extract and subjected to competition experiments with six commercially available (iso)flavonoids. The agonistic and antagonistic activities of these compounds towards both hERα (human estrogen receptor alpha) and hERß were determined. Differences in the modes of action (agonist or antagonist) were observed for the various compounds tested. In general, each compound had the same mode of action towards both ERs. In silico modeling was performed in order to study the differences in estrogenicity observed between the compounds. It is suggested that prenyl chains fit into a hydrophobic pocket present in the hER, resulting in an increased agonistic activity. In addition, it was shown that an increase in length (≈1.7 Å) of pyran prenylated isoflavonoids resulted in an antagonistic mode of action. This might be caused by collision of the pyran ring with helix 11 in the ligand binding cavity of the hER.

Statin-Induced Myopathy Is Associated with Mitochondrial Complex III Inhibition.

Cholesterol-lowering statins effectively reduce the risk of major cardiovascular events. Myopathy is the most important adverse effect, but its underlying mechanism remains enigmatic. In C2C12 myoblasts, several statin lactones reduced respiratory capacity and appeared to be strong inhibitors of mitochondrial complex III (CIII) activity, up to 84% inhibition. The lactones were in general three times more potent inducers of cytotoxicity than their corresponding acid forms. The Qo binding site of CIII was identified as off-target of the statin lactones. These findings could be confirmed in muscle tissue of patients suffering from statin-induced myopathies, in which CIII enzyme activity was reduced by 18%. Respiratory inhibition in C2C12 myoblasts could be attenuated by convergent electron flow into CIII, restoring respiration up to 89% of control. In conclusion, CIII inhibition was identified as a potential off-target mechanism associated with statin-induced myopathies.

Mitochondrial ADP/ATP exchange inhibition: a novel off-target mechanism underlying ibipinabant-induced myotoxicity.

Cannabinoid receptor 1 (CB1R) antagonists appear to be promising drugs for the treatment of obesity, however, serious side effects have hampered their clinical application. Rimonabant, the first in class CB1R antagonist, was withdrawn from the market because of psychiatric side effects. This has led to the search for more peripherally restricted CB1R antagonists, one of which is ibipinabant. However, this 3,4-diarylpyrazoline derivative showed muscle toxicity in a pre-clinical dog study with mitochondrial dysfunction. Here, we studied the molecular mechanism by which ibipinabant induces mitochondrial toxicity. We observed a strong cytotoxic potency of ibipinabant in C2C12 myoblasts. Functional characterization of mitochondria revealed increased cellular reactive oxygen species generation and a decreased ATP production capacity, without effects on the catalytic activities of mitochondrial enzyme complexes I-V or the complex specific-driven oxygen consumption. Using in silico off-target prediction modelling, combined with in vitro validation in isolated mitochondria and mitoplasts, we identified adenine nucleotide translocase (ANT)-dependent mitochondrial ADP/ATP exchange as a novel molecular mechanism underlying ibipinabant-induced toxicity. Minor structural modification of ibipinabant could abolish ANT inhibition leading to a decreased cytotoxic potency, as observed with the ibipinabant derivative CB23. Our results will be instrumental in the development of new types of safer CB1R antagonists.

Statin Lactonization by Uridine 5'-Diphospho-glucuronosyltransferases (UGTs).

Statins are cholesterol-lowering drugs that have proven to be effective in lowering the risk of major cardiovascular events. Although well tolerated, statin-induced myopathies are the most common side effects. Compared to their pharmacologically active acid form, statin lactones are more potent inducers of toxicity. They can be formed by glucuronidation mediated by uridine 5'-diphospho-glucuronosyltransferases (UGTs), but a systematic characterization of subtype specificity and kinetics of lactonization is lacking. Here, we demonstrate for six clinically relevant statins that only UGT1A1, 1A3, and 2B7 contribute significantly to their lactonization. UGT1A3 appeared to have the highest lactonization capacity with marked differences in statin conversion rates: pitavastatin ≫ atorvastatin > cerivastatin > lovastatin > rosuvastatin (simvastatin not converted). Using in silico modeling we could identify a probable statin interaction region in the UGT binding pocket. Polymorphisms in these regions of UGT1A1, 1A3, and 2B7 may be a contributing factor in statin-induced myopathies, which could be used in personalization of statin therapy with improved safety.

Snooker structure-based pharmacophore model explains differences in agonist and blocker binding to bitter receptor hTAS2R39.

The human bitter taste receptor hTAS2R39 can be activated by many dietary (iso)flavonoids. Furthermore, hTAS2R39 activity can be blocked by 6-methoxyflavanones, 4'-fluoro-6-methoxyflavanone in particular. A structure-based pharmacophore model of the hTAS2R39 binding pocket was built using Snooker software, which has been used successfully before for drug design of GPCRs of the rhodopsin subfamily. For the validation of the model, two sets of compounds, both of which contained actives and inactives, were used: (i) an (iso)flavonoid-dedicated set, and (ii) a more generic, structurally diverse set. Agonists were characterized by their linear binding geometry and the fact that they bound deeply in the hTAS2R39 pocket, mapping the hydrogen donor feature based on T5.45 and N3.36, analogues of which have been proposed to play a key role in activation of GPCRs. Blockers lack hydrogen-bond donors enabling contact to the receptor. Furthermore, they had a crooked geometry, which could sterically hinder movement of the TM domains upon receptor activation. Our results reveal characteristics of hTAS2R39 agonist and bitter blocker binding, which might facilitate the development of blockers suitable to counter the bitterness of dietary hTAS2R39 agonists in food applications.

Discovery of small molecule CD40-TRAF6 inhibitors.

The CD154-CD40 receptor complex plays a pivotal role in several inflammatory pathways. Attempts to inhibit the formation of this complex have resulted in systemic side effects. Downstream inhibition of the CD40 signaling pathway therefore seems a better way to ameliorate inflammatory disease. To relay a signal, the CD40 receptor recruits adapter proteins called tumor necrosis factor receptor-associated factors (TRAFs). CD40-TRAF6 interactions are known to play an essential role in several inflammatory diseases. We used in silico, in vitro, and in vivo experiments to identify and characterize compounds that block CD40-TRAF6 interactions. We present in detail our drug docking and optimization pipeline and show how we used it to find lead compounds that reduce inflammation in models of peritonitis and sepsis. These compounds appear to be good leads for drug development, given the observed absence of side effects and their demonstrated efficacy for peritonitis and sepsis in mouse models.

Influence of azide incorporation on binding affinity by small papain inhibitors.

In order to develop affinity-based biosensor platforms, appropriate ligands with a functional handle for immobilization onto a biosensor surface are required. To this end, a library of papain inhibitors was designed and synthesized, containing different azide linkers for subsequent immobilization by 'click' chemistry, in this particular case by copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC). Furthermore, a molecular docking study was performed to obtain a better insight as to at which position such azide handles could be tolerated without affecting binding affinity. Although the azide moiety is small, in some cases its introduction strongly influenced the binding affinity. For one class of inhibitors a swapped binding mode was proposed to explain the results. In addition, a specific site for linker introduction was identified, which did not significantly affect the binding affinity.

In silico identification and in vitro validation of potential cholestatic compounds through 3D ligand-based pharmacophore modeling of BSEP inhibitors.

Drug-induced cholestasis is a frequently observed side effect of drugs and is often caused by an unexpected interaction with the bile salt export pump (BSEP/ABCB11). BSEP is the key membrane transporter responsible for the transport of bile acids from hepatocytes into bile. Here, we developed a pharmacophore model that describes the molecular features of compounds associated with BSEP inhibitory activity. To generate input and validation data sets, in vitro experiments with membrane vesicles overexpressing human BSEP were used to assess the effect of compounds (50 µM) on BSEP-mediated (3)H-taurocholic acid transport. The model contains two hydrogen bond acceptor/anionic features, two hydrogen bond acceptor vector features, four hydrophobic/aromatic features, and exclusion volumes. The pharmacophore was validated against a set of 59 compounds, including registered drugs. The model recognized 9 out of 12 inhibitors (75%), which could not be identified based on general parameters, such as molecular weight or SlogP, alone. Finally, the model was used to screen a virtual compound database. A number of compounds found via virtual screening were tested and displayed statistically significant BSEP inhibition, ranging from 13 ± 1% to 67 ± 7% of control (P < 0.05). In conclusion, we developed and validated a pharmacophore model that describes molecular features found in BSEP inhibitors. The model may be used as an in silico screening tool to identify potentially harmful drug candidates at an early stage in drug development.

High-affinity inhibitors of Zymomonas mobilis tRNA-guanine transglycosylase through convergent optimization.

The tRNA-modifying enzyme tRNA-guanine transglycosylase (TGT) has been recognized as a drug target for the treatment of the foodborne illness shigellosis. The active site of TGT consists of three pockets: the central guanine/preQ1 recognition site and the ribose-33 and ribose-34 pockets. In previous work, lin-benzoguanines and lin-benzohypoxanthines, which differ by the presence of an exocyclic NH2 group in the former and its absence in the latter, were used as central scaffolds that bind to the guanine/preQ1 recognition site and allow suitable functionalization along exit vectors targeting the two ribose pockets. The substituents for both of these two pockets have been optimized individually. Here, a series of bifunctionalized inhibitors that occupy both ribose pockets are reported for the first time. Dissociation constants Kd down to the picomolar range were measured for the bifunctionalized lin-benzoguanine-based ligands and Kd values in the nanomolar range were measured for the corresponding lin-benzohypoxanthine-based ligands. The binding mode of all inhibitors was elucidated by X-ray crystal structure analysis. A remarkable influence of the crystallization protocol on the solvation pattern in the solid state and the residual mobility of the bound ligands was observed.