Biolayer Interferometry Technology Research Articles

Human serum albumin promotes interactions between HSA-IL-2 fusion protein and CD122 for enhancing immunotherapy

Interleukin 2 (IL-2) is a multifunctional cytokine that is crucial for T-lymphocytes proliferation and differentiation. However, IL-2 binds to IL-2Rα (CD25) subunit preferentially and tends to stimulate regulatory T cells (Tregs), which express high-affinity trimeric receptors (IL-2Rαβγ), resulting in immunosuppressive effects. Therefore, development of methods that enhance IL-2/CD122 interactions and activate immune responses without affecting therapeutic efficacy of IL-2 may be desirable. In this work, we constructed a recombinant IL-2 fusion protein (HSA-IL-2), comprising human serum albumin (HSA) and IL-2, there was a new interaction interface between HSA domain and CD122 in HSA-IL-2 fusion protein predicted by AlphaFold2, and followed by determining binding affinity between HSA-IL-2 and CD122 through ForteBio’s Bio-Layer Interferometry technology. Strikingly, HSA did promoted interactions between HSA-IL-2 fusion protein and CD122 compared with wild-type IL-2. In vivo experiments, HSA-IL-2 fusion protein had capacity to promote CD8+ T cells infiltration while reducing Treg cells infiltration for boosting immunotherapeutic efficacy. Furthermore, it facilitated synergistic therapeutic effect with α-PD-L1 to inhibit tumor growth. Overall, our research unveiled an enhanced binding affinity method underlying interactions between IL-2 and CD122 via fusing albumin, and propose a promising therapeutic strategy to facilitate IL-2 administration and broaden its clinical use.

Glycyrrhetinic acid blocks SARS-CoV-2 infection by activating the cGAS-STING signalling pathway.

Traditional Chinese medicine (TCM) played an important role in controlling the COVID-19 pandemic, but the scientific basis and its active ingredients are still weakly studied. This study aims to decipher the underlying anti-SARS-CoV-2 mechanisms of glycyrrhetinic acid (GA). GA's anti-SARS-CoV-2 effect was verified both in vitro and in vivo. Homogeneous time-resolved fluorescence assays, biolayer interferometry technology, and molecular docking were employed to examine interactions of GA with human stimulator of interferon genes (hSTING). Immunofluorescence staining, western blot, and RT-qPCR were used to investigate nuclear translocation of interferon regulatory factor 3 (IRF3) and levels of STING target genes. Pharmacokinetics of GA was studied in mice. GA could directly bind to Ser162 and Tyr240 residues of hSTING, thus up-regulating downstream targets and activation of the STING signalling pathway. Such activation is crucial for limiting the replication of SARS-CoV-2 Omicron in Calu-3 cells and protecting against lung injury induced by SARS-CoV-2 Omicron infection in K18-ACE2 transgenic mice. Immunofluorescence staining and western blot indicated that GA increased levels of phosphorylated STING, phosphorylated TANK-binding kinase-1, and cyclic GMP-AMP synthase (cGAS). Importantly, GA increased nuclear translocation of IRF3. Pharmacokinetic analysis of GA in mice indicated it can be absorbed into circulation and detected in the lung at a stable level. Activation of the cGAS-STING pathway through the GA-STING-IRF3 axis is essential for the antiviral activity of GA in mice, providing new insights into the potential translation of GA for treating SARS-CoV-2 in patients.

Penthorum chinense Pursh inhibits ferroptosis in cellular and Caenorhabditis elegans models of Alzheimer's disease

Background: Ferroptosis, a unique type of cell death triggered by iron-dependent lipid peroxidation, plays a critical role in the pathogenesis of Alzheimer's disease (AD), a debilitating condition marked by memory loss and cognitive impairment due to the accumulation of beta-amyloid (Aβ) and hyperphosphorylated Tau protein. Increasing evidence suggests that inhibitors of ferroptosis could be groundbreaking in the treatment of AD. Method: In this study, we established in vitro ferroptosis using erastin-, RSL-3-, hemin-, and iFSP1-induced PC-12 cells. Using MTT along with Hoechst/PI staining, we assessed cell viability and death. To determine various aspects of ferroptosis, we employed fluorescence probes, including DCFDA, JC-1, C11 BODIPY, Mito-Tracker, and PGSK, to measure ROS production, mitochondrial membrane potential, lipid peroxidation, mitochondrial morphology, and intracellular iron levels. Additionally, Western blotting, biolayer interferometry technology, and shRNA were utilized to investigate the underlying molecular mechanisms. Furthermore, p-CAX APP Swe/Ind- and pRK5-EGFP-Tau P301L overexpressing PC-12 cells, along with Caenorhabditis elegans (C. elegans) strains CL4176, CL2331, and BR5270, were employed to examine ferroptosis in AD models. Results: Here, we conducted a screening of our natural medicine libraries and identified the ethanol extract of Penthorum chinense Pursh (PEE), particularly its ethyl acetate fraction (PEF), displayed inhibitory effects on ferroptosis in cells. Specifically, PEF inhibited the generation of ROS, lipid peroxidation, and intracellular iron levels. Furthermore, PEF demonstrated protective effects against H2O2-induced cell death, ROS production, and mitochondrial damage. Mechanistic investigations unveiled PEF's modulation of intracellular iron accumulation, GPX4 expression and activity, and FSP1 expression. In p-CAX APP Swe/Ind and pRK5-EGFP-Tau P301L overexpressing PC-12 cells, PEF significantly reduced cell death, as well as ROS and lipid peroxidase production. Moreover, PEF ameliorated paralysis and slowing rate in Aβ and Tau transgenic C. elegans models, while inhibiting ferroptosis, as evidenced by decreased DHE intensity, lipid peroxidation levels, iron accumulation, and expression of SOD-3 and gst-4. Conclusion: Our findings highlight the suppressive effects of PEF on ferroptosis in AD cellular and C. elegans models. This study helps us better understand how ferroptosis affects AD and emphasizes the potential of PCP as a candidate for AD intervention.

Efficient Hapten-Specific Biopanning Strategy Based on the Fe3O4@ENR-Functionalized Core-Shell Magnetic Nanoparticles.

The biopanning of target-specific phages is one of the most critical steps in the preparation of single-domain antibodies. In the traditional biopanning of haptens, the nonspecific binding of library phages to macromolecular proteins is one of the most challenging problems in preparing single-domain antibodies. In this research, Fe3O4@ENR-functionalized core-shell magnetic nanoparticles (FMNPs) were silylated and aminated by tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane, and target enrofloxacin was coupled onto the surface by the carbodiimide method. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, particle size distribution, zeta potential, transmission electron microscopy observation, and indirect enzyme-linked immunosorbent assay (ELISA). A biopanning strategy based on Fe3O4@ENR FMNPs was then established to solve the problem in the traditional solid-phase biopanning process. The results showed that a considerable number of enrofloxacin (ENR)-positive phages were screened by only one round of biopanning. Finally, two ENR-specific shark-derived single-domain genes were identified and validated by monoclonal phage ELISA, gene sequencing, and biolayer interferometry technology. Our study provides a new biopanning strategy based on Fe3O4@ENR FMNPs for efficiently providing phages specific to haptens.

Biolayer Interferometry Technology to Detect Interactions between Ligand and Analyte

Biolayer Interferometry Technology to Detect Interactions between Ligand and Analyte

Screening and Identification of a Novel Anti-Siglec-15 Human Antibody 3F1 and Relevant Antitumor Activity.

Sialic acid-binding Ig-like lectin-15 is an important immunosuppressive molecule considered to be a key target in next-generation tumor immunotherapy. In this study, we screened 22 high-affinity antibodies that specifically recognize human Siglec-15 by using a large human phage antibody library, and five representative sequences were selected for further study. The results showed the binding activity of five antibodies to Siglec-15 (EC50 ranged from 0.02368 μg/mL to 0.07949 μg/mL), and in two Siglec-15-overexpressed cell lines, three antibodies had the strongest binding activity, so the two clones were discarded for further study. Subsequently, the affinity of three antibodies were measured by bio-layer interferometry technology (5-9 × 10E-09M). As the reported ligands of Siglec-15, the binding activity of Siglec-15 and sialyl-Tn, cluster of differentiation 44, myelin-associated glycoprotein, and leucine-rich repeat-containing protein 4C can be blocked by three of the antibodies. Among these, 3F1 had a competitive advantage. Then, the antibody 3F1 showed an obvious antibody-dependent cell-mediated cytotoxicity effect (EC50 was 0.85 μg/mL). Further, antibody 3F1 can reverse the inhibitory effect of Siglec-15 on lymphocyte proliferation (especially CD4+T and CD8+T) and cytokine release Interferon-γ. Given the above results, 3F1 was selected as a candidate for the in vivo pharmacodynamics study. In the tumor model of Balb/c Nude mice, 3F1 (10 mg/kg) showed certain antitumor effects [tumor growth inhibition (TGI) was 31.5%], while the combination of 3F1 (5 mg/kg) and Erbitux (5 mg/kg) showed significant antitumor effects (TGI was 48.7%) compared with the PBS group. In conclusion, novel human antibody 3F1 has antitumor activity and is expected to be an innovative candidate drug targeting Siglec-15 for tumor immunotherapy. SIGNIFICANCE STATEMENT: Siglec-15 is considered as an important target in the next generation of tumor immunotherapy. 3F1 is expected to be the most promising potential candidate for targeting Siglec-15 for cancer treatment and could provide a reference for the development of antitumor drugs.

Esculentoside A alleviates cognitive deficits and amyloid pathology through peroxisome proliferator-activated receptor γ-dependent mechanism in an Alzheimer's disease model

BackgroundAlzheimer's disease (AD) is characterized clinically by cognitive deficits and pathologically by amyloid-β (Aβ) deposition and tau aggregation, as well as the brain atrophy. Esculentoside A (EsA), a neuroprotective saponin, is isolated from Phytolacca esculenta and shows potent health-promoting effects in a variety of experimental models. However, there are minimal reports on the effects of EsA on triple transgenic AD mice. PurposeThe current research aimed at investigating the protective effects and underlying mechanisms of EsA on the mitigation of cognitive deficits and pathology in triple transgenic AD mice. MethodsTriple transgenic AD mice (3 × Tg-AD) of 8 months old received intraperitoneal treatment of 5 or 10 mg/kg EsA for 8 consecutive weeks. Morris water maze test and open field test were made to evaluate the cognitive function and degree of anxiety of the mice. Liquid chromatography with tandem mass spectrometry analysis was performed to characterize and to quantify EsA in the blood and brain of mice. Immunofluorescence assay and Western blot were adopted to measure the levels of peroxisome proliferator-activated receptor gamma (PPARγ) and key proteins in Aβ pathology, ER stress- and apoptosis-associated pathways. The combination of EsA with PPARγ were theoretically calculated by molecular docking programs and experimentally confirmed by the bio-layer interferometry technology. ResultsSupplemental EsA could improve the cognitive deficits of 3 × Tg-AD mice. EsA penetrated the brain-blood barrier to exert a strong effect on AD mice, evidenced as decreasing Aβ generation, reducing the degrees of oxidative and ER stress, and mitigating neuronal apoptosis through the increase of PPARγ expression. In the culture of primary neurons, addition of PPARγ inhibitor GW9662 eliminated the effects of EsA on AD pathologies. Direct combination of EsA with PPARγ were demonstrated by molecular docking programs and bio-layer interferometry technology. ConclusionsFor the first time, these outcomes revealed that EsA could penetrate the brain-blood barrier to exert a strong effect on ameliorating cognitive deficits in 3 × Tg-AD mice and exert neuroprotective effects toward AD pathology via PPARγ-dependent mechanism.

Tylosin Inhibits Streptococcus suis Biofilm Formation by Interacting With the O-acetylserine (thiol)-lyase B CysM.

Streptococcus suis (S. suis) can decrease its virulence or modify local conditions through biofilm formation, which promotes infection persistence in vivo. Biofilm formation is an important cause of chronic drug-resistant S. suis infection. The aim of this study was to evaluate whether tylosin effectively inhibits S. suis biofilm formation by interacting with O-acetylserine (thiol)-lyase B (CysM), a key enzymatic regulator of cysteine synthesis. Biofilm formation of the mutant (ΔcysM) strain was significantly lower compared to the wild-type ATCC 700794 strain. Tylosin inhibited cysM gene expression, decreased extracellular matrix contents, and reduced cysteine, homocysteine, and S-adenosylmethionine levels, indicating its potential value as an effective inhibitor of S. suis biofilm formation. Furthermore, using biolayer interferometry technology and fourier-transform infrared spectroscopy, we found that tylosin and CysM could be combined directly. Overall, these results provide evidence that tylosin inhibits S. suis biofilm formation by interacting with CysM.

Rapid and sensitive detection of Staphylococcus aureus using biolayer interferometry technology combined with phage lysin LysGH15

Staphylococcus aureus (S. aureus), considered as a common foodborne pathogenic microorganism, usually causes food poisoning and various infectious diseases. Therefore, development of rapid and accurate bacterial detection method is the key to preventing food poisoning and achieving early diagnosis and treatment of various infectious diseases caused by S. aureus. Biolayer interferometry (BLI) technology is a novel technique of label-free optical analysis for real-time monitoring of biomolecular interactions. The C54A mutation induced the lytic activity loss of phage lysin LysGH15 but retained the capacity for specific recognizing and binding S. aureus. In this study, a novel method for the detection of S. aureus was established using the C54A mutant LysGH15 as the receptor in combination with BLI. Using this BLI-based method, S. aureus whole cells could be directly assayed and the limit of detection was 13 CFU/mL with a binding time of 12 min. Because the C54A mutant LysGH15 recognizes S. aureus with very high specificity, the method can exclude potential interference from other bacterial species. In addition, this method could also distinguish between viable and dead S. aureus. Moreover, S. aureus was successfully detected in ice cubes and light soy sauce by using this method. Collectively, these results indicate that the LysGH15-based BLI method can be used as an efficient and reliable diagnostic tool in the field of food safety and other related fields for the rapid, sensitive, label-free, and real-time detection of S. aureus.

Development of a BLI assay-based method for detecting LptA/LptC interaction

In Gram-negative bacteria, lipopolysaccharide transport (Lpt) protein LptA and LptC form a complex to transport LPS from the inner membrane (IM) to the outer membrane (OM). Blocking the interaction between LptA and LptC will lead to the defect of OM and cell death. Therefore, Lpt protein interaction could be used as a target to screen new drugs for killing Gram-negative bacteria. Here we used biolayer interferometry (BLI) assay to detect the interaction between LptA and LptC, with the aim to develop a method for screening the LptA/LptC interaction blockers in vitro. Firstly, LptC and LptA with or without signal peptide (LptAfull or LptAno signal) were expressed in E. coli BL21(DE3). The purified proteins were then labeled with biotin and the super streptavidin (SSA) biosensor was blocked with diluent. The biotin labeled protein sample was mixed with the sensor, and then the binding of the protein with a series of diluted non biotinylated protein was detected. At the same time, non-biotinylated protein was used as a control. The binding of biotinylated protein to a small molecule IMB-881 and the blocking of interaction were also detected by the same method. In the blank control, the biosensor without biotinylated protein was used to detect the serially diluted samples. The signal response constant was calculated by using steady analysis. The results showed that biotinylated LptC had a good binding activity with LptAfull and LptAno signal with KD value 2.9e⁻⁷±7.9e⁻⁸ and 6.0e⁻⁷±2.8e⁻⁸, respectively; biotinylated LptAno signal had a good binding activity with LptC, with a KD value of 9.6e⁻⁷±7.2e⁻⁸. All binding curves showed obvious fast binding and fast dissociation morphology. The small molecule compound IMB-881 can bind to LptA to block the interaction between LptA and LptC, but has no binding activity with LptC. In summary, we developed a method for detecting the LptA/LptC interaction based on the BLI technology, and confirmed that this method can be used to evaluate the blocking activity of small molecule blockers, providing a new approach for the screening of LptA/LptC interaction blockers.

Study on Anti-Inflammatory Activity of Niloticin by Targeting MD-2 Based on Computer-Aided Drug Design and Biolayer Interferometry

Niloticin is an active compound from Cortex phellodendri, but its antiinflammatory activity has not yet been explored. The aim of the present study was to assess the drug potential of niloticin and to study the MD-2-targeting mechanism of its anti-inflammatory activity. Niloticin’s drug potential was analyzed using the Traditional Chinese Medicine Systems Pharmacology Database. Molecular docking and biolayer interferometry technology were used to explore the anti-inflammatory mechanism of niloticin by targeting myeloid differentiation protein 2 (MD-2), which mediates a series of Toll-Like Receptor (TLR) 4-dependent inflammatory responses. The cytokines involved in the LPSTLR4/ MD-2-NF-κB pathway were evaluated by ELISA, RT-PCR, and western blot. The results showed that niloticin has drug potential and could bind to MD- 2. Niloticin had no impact on cell viability. Niloticin could significantly decrease the levels of NO, IL-6, TNF-a, and IL-1β (P<0.01) induced by LPS. IL-1β, IL-6, iNOS, TNF-a, and COX-2 mRNA expression levels were decreased by niloticin (all P<0.01). Compared with the control group, TLR4, p65, MyD88, p-p65, and iNOS expression levels induced by LPS were suppressed by niloticin (all P<0.01). In conclusion, niloticin is a potential MD-2 antagonist. It might interact with MD-2 to play an anti-inflammatory role by suppressing the activation of the LPS-TLR4/MD-2-NF-κB signaling pathway.

Application of innovative technology of biolayer interferometry in basic research in the development of new vaccines

One of the effective methods for real-time protein analysis is based on the phenomenon of light interference and is called "biolayer interferometry". The patented Bio-Layer Interferometry (BLI) technology, which underlies ForteBio Octet platform, provides surface interaction analysis for disposable fiber optic biosensors. The article presents the technology of interferometry of the biolayer of BLI molecules, which can be used to determine the kinetics and affinity, for the specific quantitative determination of molecules bound to the biosensor. The purpose of the review was to summarize scientific data on the use of the interferometry technology of the biolayer of BLI molecules in determining the concentration of proteins and studying the kinetics of interaction using Octet systems when creating new vaccines in many ways. BLI biolayer interferometry technology allows protein concentration measurements to be performed without using of labels or auxiliary reagents, even in untreated media. It is characterized by a sensitivity up to several ng/cm3, provides accurate quantitative changes in a few seconds, and not in many hours like traditional methods (ELISA, HPL Coder). The affinity, concentration, and binding kinetics of the studied proteins can be measured in 4- ml drop samples directly on the laboratory table. The technology of interferometry of the biolayer of BLI molecules can be used by researchers to select the most immunogenic epitopes in the molecular structure of a pathogen, it is also possible to conduct studies on the characterization and recognition, diversity and distribution of pathogens, affinity for antibodies, it is possible to characterize the immune response of the host, to study molecular interactions between the pathogen and the host, and to carry out therapeutic and clinical research. Currently, the technology of interferometry of the biolayer of BLI molecules has already been used in fundamental studies of pathogens that cause HIV, herpes, Ebola, influenza A H7N9, Dengue, malaria, Zika virus, diphtheria, tuberculosis, listeriosis, gastroenteritis, respiratory pathology, including the COVID outbreak. -19.

Methods to Validate Binding and Kinetics of "Proximity-Inducing" Covalent Immune-Recruiting Molecules.

The emergence of covalent inhibitors and chemoproteomic probes in translational chemical biology research requires the development of robust biophysical and analytical methods to characterize their complex interactions with target biomolecules. Importantly, these methods must efficiently assess target selectivity and accurately discern noncovalent binding from the formation of resultant covalent adducts. One recently reported covalent chemical tool used in tumor immune oncology, covalent immune recruiters (CIRs), increases the proximity of immune cells and cancer cells, promoting immune recognition and response. Herein we describe biolayer interferometry (BLI) biosensor, flow cytometry, and solution fluorescence-based assay approaches to characterize CIR:antibody binding and CIR-antibody covalent-labeling kinetics. BLI technology, akin to surface plasmon resonance, provides the unique opportunity to investigate molecular binding and labeling kinetics both on a solid surface (Basic Protocol 1) and in solution (Alternate Protocol 1). Here, recruitment of mass-containing proteins to the BLI probe via CIR is measured with high sensitivity and is used as a readout of CIR labeling activity. Further, CIR technology is used to label antibodies with a fluorescent handle. In this system, labeling is monitored via SDS-PAGE with a fluorescence gel imager, where increased fluorescence intensity of a sample reflects increased labeling (Basic Protocol 2). Analysis of CIR:antibody target-specific immune activation is demonstrated with a flow cytometry-based antibody-dependent cellular phagocytosis (ADCP) assay (Basic Protocol 3). This ADCP protocol may be further used to discern CIR:antibody binding from covalent adduct formation (Alternate Protocol 3). For the protocols described, each method may be used to analyze characteristics of any covalent-tagging or antibody-recruiting small molecule or protein-based technology. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Determining "on-probe" reaction kinetics of CIR1/CIR4 via biolayer interferometry with Octet RED96 Alternate Protocol 1: Determining "in-solution" reaction kinetics of prostate-specific membrane antigen targeting CIR (CIR3) via biolayer interferometry with Octet RED96 Basic Protocol 2: Reaction kinetics of covalently labeled antibodies via fluorescence SDS-PAGE Basic Protocol 3: Small molecule-directed antibody-dependent cellular phagocytosis on live human cells measured via flow cytometry Alternate Protocol 2: Kinetic analysis of CIR3:antibody labeling via antibody-dependent cellular phagocytosis on flow cytometry Support Protocol 1: Activation of U937 monocytes with interferon γ Support Protocol 2: Labeling streptavidin beads with biotinylated prostate-specific membrane antigen receptor.

Highly Efficient Separation of Methylated Peptides Utilizing Selective Complexation between Lysine and 18-Crown-6.

Protein methylation is one of the most common and important post-translational modifications, and it plays vital roles in epigenetic regulation, signal transduction, and chromatin metabolism. However, due to the diversity of methylation forms, slight difference between methylated sites and nonmodified ones, and ultralow abundance, it is extraordinarily challenging to capture and separate methylated peptides from biological samples. Here, we introduce a simple and highly efficient method to separate methylated and nonmethylated peptides using 18-crown-6 as a mobile phase additive in high-performance liquid chromatography. Selective complexation between lysine and 18-crown-6 remarkably increases the retention of the peptides on a C18 stationary phase, leading to an excellent baseline separation between the lysine methylated and nonmethylated peptides. A possible binding mechanism is verified by nuclear magnetic resonance titration, biolayer interferometry technology, and quantum chemistry calculation. Through establishment of a simple enrichment methodology, a good selectivity is achieved and four methylated peptides with greatly improved signal-to-noise (S/N) ratios are successfully separated from a complex peptide sample containing 10-fold bovine serum albumin tryptic digests. By selecting rLys N as an enzyme to digest histone, methylation information in the histone could be well identified based on our enrichment method. This study will open an avenue and provide a novel insight for selective enrichment of lysine methylated peptides in post-translational modification proteomics.

Development of anti-Müllerian hormone immunoassay based on biolayer interferometry technology.

Anti-Müllerian hormone (AMH) is a biomarker for the assessment of female fertility. The accurate measurement of the concentration of AMH is relevant for the success of assisted reproductive therapies and diagnosis of clinical cases. In this study, we show that cytokines such as fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), granulocyte-macrophage colony-stimulating factor (GM-CSF), and β2-microglobulin (β2M) significantly enhance the immune response against AMH. Two anti-AMH monoclonal antibodies (mAbs) with high affinity were selected by biolayer interferometry (BLI) technology for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect AMH in the range of 0.125~20ngmL-1 with a detection limit of 0.099ngmL-1. This immunoassay showed high specificity with no cross-reaction with structurally related proteins and some of the other members of the TGF-β super family, such as inhibin A, activin A, follicle-stimulating hormone, and luteinizing hormone. The average recovery rates of three different batches were 100.19%, 102.72%, and 103.59%, respectively, with coefficients of variation of less than 12%. The developed assay was applied in the detection of AMH in 69 serum samples from randomly selected patients. Our data showed a high correlation with those obtained using commercially available ELISA kits (correlation coefficient, 0.9831). Hence, we suggest that this immunoassay could find application in the development of POCT for the diagnosis of AMH in clinical samples. Graphical abstract.

Binding affinity of TWIST2 wild type and mutant proteins and their potential role in the Barber‐Say Syndrome

Barber Say Syndrome (BSS) is a rare autosomal dominant disease, classified as an ectodermal dysplasia, affecting the eyelids and skin as well as exhibiting dysmorphic facial features. BSS exhibits variable phenotypes. Molecular analysis of patients with BSS identified mutations in the TWIST2 gene, involving amino acid substitutions of Glutamate 75 to Glutamine (E75Q) or Alanine (E75A).TWIST2 is a transcription factor of the bHLH superfamily, belonging to the class II group. Glutamate 75 is a highly conserved amino acid in the basic region of bHLH proteins, which is responsible for nucleotide binding in both class I and II groups. Both the I and II bHLH classes are known for their specificity towards canonical E‐box (CANNTG) DNA response elements. The E75Q and E75A mutations have been suggested to alter the DNA‐binding activity of TWIST2, leading to both dominant‐negative and gain‐of‐function effects. In this study, we expressed His‐Tagged TWIST2, and the E75Q and E75A mutant proteins, in E. coli plysBL21 cells. Subsequently, proteins were purified by affinity chromatography and yields between 1.2 to 1.7 mg/L of pure recombinant protein were obtained from 2 liter cultures. Protein affinities towards known E‐boxes in the human TNFa, MCK1 and POSTN genes were measured using Biolayer Interferometry technology. TWIST2 wild type equilibrium constants were in the range of 0.003573 to 0.7929 uM. The equilibrium constants of the E75Q and E75A mutants were three to six order of magnitude higher when compared to the wild type protein. Therefore, the TWIST2 wild type protein showed higher binding affinities towards double‐stranded oligonucleotides containing E‐boxes, while the pathological variants found in BSS patients showed lower affinities to the same oligonucleotides. These results suggest that basic region variants of the TWIST2 glutamate 75 may cause gene dysregulation by their reduced capacity to bind their cognate DNA response elements.Support or Funding InformationThese studies were partially supported by grants U54MD007600 (NIMHD RCMI) and R24GM061838 (NIGMS RISE).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Small molecules bind human mTOR protein and inhibit mTORC1 specifically

Inhibition of mTOR activity (mechanistic target of rapamycin) is an anti-cancer therapeutic strategy. mTOR participates in two functional complexes, mTORC1 and mTORC2. Since mTORC1 is specifically activated in multiple tumors, novel molecules that inhibit mTORC1 could be therapeutically important. To identify potentially novel modulators of mTOR pathways, we screened 1600 small molecule human drugs for mTOR protein binding, using novel biolayer interferometry technology. We identified several small molecules that bound to mTOR protein in a dose-dependent manner, on multiple chemical scaffolds. As mTOR participates in two major complexes, mTORC1 and mTORC2, the functional specificities of the binders were measured by S6Kinase and Akt phosphorylation assays. Three novel ‘mTOR general’ binders were identified, carvedilol, testosterone propionate, and hydroxyprogesterone, which inhibited both mTORC1 and mTORC2. By contrast, the piperazine drug cinnarizine dose-dependently inhibited mTORC1 but not mTORC2, suggesting it as a novel mTORC1-specific inhibitor. Some of cinnarizine’s chemical analogs also inhibited mTORC1 specifically, whereas others did not. Thus we report the existence of a novel target for some related piperazines including cinnarizine and hydroxyzine, i.e. specific inhibition of mTORC1 activity. Since mTOR inhibition is a general anti-cancer strategy, and mTORC1 is specifically activated in some tumors, we suggest the piperazine scaffold, including cinnarizine and hydroxyzine, could be proposed for rational therapy in tumors in which mTORC1 is specifically activated. Related piperazines have shown toxicity to cancer cells in vitro as single agents and in combination chemotherapy. Thus piperazine-based mTOR inhibitors could become a novel chemotherapeutic strategy.

Universal label-free in-process quantification of influenza virus-like particles.

Virus-like particles (VLPs) are becoming established as vaccines, in particular for influenza pandemics, increasing the interest in the development of VLPs manufacturing bioprocess. However, for complex VLPs, the analytical tools used for quantification are not yet able to keep up with the bioprocess progress. Currently, quantification for Influenza relies on traditional methods: hemagglutination assay or Single Radial Immunodiffusion. These analytical technologies are time-consuming, cumbersome, and not supportive of efficient downstream process development and monitoring. Hereby we report a label-free tool that uses Biolayer interferometry (BLI) technology applied on an Octet platform to quantify Influenza VLPs at all stages of bioprocess. Human (α2,6-linked sialic acid) and avian (α2,3-linked sialic acid) biotinylated receptors associated with streptavidin biosensors were used, to quantify hemagglutinin content in several mono- and multivalent Influenza VLPs. The applied method was able to quantify hemagglutinin from crude samples up to final bioprocessing VLP product. BLI technology confirmed its value as a high throughput analytical tool with high sensitivity and improved detection limits compared to traditional methods. This simple and fast method allowed for real-time results, which are crucial for in-line monitoring of downstream processing, improving process development, control and optimization.

Expression, refolding and bio-structural analysis of a tetravalent recombinant dengue envelope domain III protein for serological diagnosis

Dengue is a mosquito-borne disease caused by four genetically and serologically related viruses that affect several millions of people. Envelope domain III (EDIII) of the viral envelope protein contains dengue virus (DENV) type-specific and DENV complex-reactive antigenic sites. Here, we describe the expression in Escherichia coli, the refolding and bio-structural analysis of envelope domain III of the four dengue serotypes as a tetravalent dengue protein (EDIIIT2), generating an attractive diagnostic candidate. In vitro refolding of denatured EDIIIT2 was performed by successive dialysis with decreasing concentrations of chaotropic reagent and in the presence of oxidized glutathione. The efficiency of refolding was demonstrated by protein mobility shifting and fluorescent visualization of labeled cysteine in non-reducing SDS-PAGE. The identity and the fully oxidized state of the protein were verified by mass spectrometry. Analysis of the structure by fluorescence, differential scanning calorimetry and circular dichroism showed a well-formed structural conformation mainly composed of β-strands. A label-free immunoassay based on biolayer interferometry technology was subsequently used to evaluate antigenic properties of folded EDIIIT2 protein using a panel of dengue IgM positive and negative human sera. Our data collectively support the use of an oxidatively refolded EDIIIT2 recombinant chimeric protein as a promising antigen in the serological diagnosis of dengue virus infections.

Discovery of a novel inhibitor of kinesin-like protein KIFC1.

Historically, drugs used in the treatment of cancers also tend to cause damage to healthy cells while affecting cancer cells. Therefore, the identification of novel agents that act specifically against cancer cells remains a high priority in the search for new therapies. In contrast with normal cells, most cancer cells contain multiple centrosomes which are associated with genome instability and tumorigenesis. Cancer cells can avoid multipolar mitosis, which can cause cell death, by clustering the extra centrosomes into two spindle poles, thereby enabling bipolar division. Kinesin-like protein KIFC1 plays a critical role in centrosome clustering in cancer cells, but is not essential for normal cells. Therefore, targeting KIFC1 may provide novel insight into selective killing of cancer cells. In the present study, we identified a small-molecule KIFC1 inhibitor, SR31527, which inhibited microtubule (MT)-stimulated KIFC1 ATPase activity with an IC50 value of 6.6 μM. By using bio layer interferometry technology, we further demonstrated that SR31527 bound directly to KIFC1 with high affinity (Kd=25.4 nM). Our results from computational modelling and saturation-transfer difference (STD)-NMR experiments suggest that SR31527 bound to a novel allosteric site of KIFC1 that appears suitable for developing selective inhibitors of KIFC1. Importantly, SR31527 prevented bipolar clustering of extra centrosomes in triple negative breast cancer (TNBC) cells and significantly reduced TNBC cell colony formation and viability, but was less toxic to normal fibroblasts. Therefore, SR31527 provides a valuable tool for studying the biological function of KIFC1 and serves as a potential lead for the development of novel therapeutic agents for breast cancer treatment.