Recombinant Fragments Research Articles

O-Fucose and Fringe-modified NOTCH1 extracellular domain fragments as decoys to release niche-lodged hematopoietic progenitor cells.

Successful hematopoietic progenitor cell (HPC) transplant therapy is improved by mobilizing HPCs from the bone marrow niche in donors. Notch receptor-ligand interactions are known to retain HPCs in the bone marrow, and neutralizing antibodies against Notch ligands, Jagged-1 or Delta-like ligand (DLL4), or NOTCH2 receptor potentiates HPC mobilization. Notch-ligand interactions are dependent on posttranslational modification of Notch receptors with O-fucose and are modulated by Fringe-mediated extension of O-fucose moieties. We previously reported that O-fucosylglycans on Notch are required for Notch receptor-ligand engagement controlling hematopoietic stem cell quiescence and retention in the marrow niche. Here, we generated recombinant fragments of NOTCH1 or NOTCH2 extracellular domain carrying the core ligand-binding regions (EGF11-13) either as unmodified forms or as O-fucosylglycan-modified forms. We found that the addition of O-fucose monosaccharide or the Fringe-extended forms of O-fucose to EGF11-13 showed substantial increases in binding to DLL4. Furthermore, the O-fucose and Fringe-extended NOTCH1 EGF11-13 protein displayed much stronger binding to DLL4 than the NOTCH2 counterpart. When assessed in an in vitro 3D osteoblastic niche model, we showed that the Fringe-extended NOTCH1 EGF11-13 fragment effectively released lodged HPC cells with a higher potency than the NOTCH2 blocking antibody. We concluded that O-fucose and Fringe-modified NOTCH1 EGF11-13 protein can be utilized as effective decoys for stem cell niche localized ligands to potentiate HPC egress and improve HPC collection for hematopoietic cell therapy.

Monoclonal antibody against Nile tilapia (Oreochromis niloticus) IgM heavy chain: A valuable tool for detection and quantification of IgM and IgM+ cells

Nile tilapia (Oreochromis niloticus) is a freshwater fish, which is extensively cultivated worldwide and constitutes one of the model species for the study of fish immunology. Monoclonal antibodies are very advantageous molecular tools for studying teleost immune system. Specifically, monoclonal antibodies that react with immunoglobulins are used successfully in the study of the humoral immune response of several fish species. In the present study, we produced and characterized a monoclonal antibody against tilapia IgM heavy chain using a peptide-based strategy. The peptide sequence was selected from the surface-exposed region between CH3–CH4 domains. The specificity of the polyclonal serum and the hybridoma culture supernatant obtained by immunization with the peptide conjugated to keyhole limpet hemocyanin were evaluated by western blotting, both showing reactivity against tilapia serum IgM. The purified mAb was able to recognize secreted IgM by western blotting and ELISA and membrane IgM by flow cytometry. We also demonstrated that the antibody doesn't cross-react with a recombinant IgT fragment. This tool allowed us to study for the first time the stimulation of mucosal immunity after Pituitary Adenylate Cyclase Activating Polypeptide administration. Overall, the results demonstrated the utility of this mAb to characterize humoral immune response in O. niloticus.

Optimized GAPDH-truncated immunogen of Streptococcus equi elicits an enhanced immune response and provides effective protection in a mouse model

Strangles is an acute and frequently diagnosed infectious disease caused by Streptococcus equi subsp. equi. Infection with this pathogen can cause grave losses to the equine industry. The present work investigates glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an important surface-localized virulence factor of S. equi, to determine whether it could be developed into an efficacious and suitable subunit vaccine against strangles. Two different recombinant fragments of S. equi GAPDH, namely, GAPDH-L and GAPDH-S, were constructed and expressed. Further, the antigenicity and immunogenicity of these two recombinant proteins were compared and evaluated in a mouse model. Our results revealed that immune responses were efficiently induced by the proteins in immunized mice. Remarkably, higher survival rates and significantly lower bacterial loads in the lung, liver, kidney, and spleen were observed in the GAPDH-S group compared with the GAPDH-L group after challenge with S. equi. High levels of specific antibodies, elevated antibody titers, and increased proportions of CD8 + T cells further indicated that GAPDH-S elicited better humoral and cellular immune responses than GAPDH-L. Furthermore, the induction of TCR, TLR-2, TLR-3, and TLR-4 significantly increased in the GAPDH-S group compared with those in the GAPDH-L and negative control groups. In summary, our results indicate that the optimized recombinant protein GAPDH-S is a promising candidate construct that may be further developed into a multivalent subunit vaccine for strangles.

The Intervening Removable Affinity Tag (iRAT) System for the Production of Recombinant Antibody Fragments.

Fv and Fab antibody fragments are versatile co-crystallization partners that aid in the structural determination of otherwise "uncrystallizable" proteins, including human/mammalian membrane proteins. Accessible methods for the rapid and reliable production of recombinant antibody fragments have been long sought. In this chapter, we describe the concept and protocols of the intervening removable affinity tag (iRAT) system for the efficient production of Fv and Fab fragments in milligram quantities, which are sufficient for structural studies. As an extension of the iRAT system, we also provide a new method for the creation of genetically encoded fluorescent Fab fragments, which are potentially useful as molecular devices in various basic biomedical and clinical procedures, such as immunofluorescence cytometry, bioimaging, and immunodiagnosis.

Fragments recombination, design, synthesis, safener activity and CoMFA model of novel substituted dichloroacetylphenyl sulfonamide derivatives.

Isoxaflutole (IXF), as a kind of 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor, has been widely used in many kinds of plants. IXF can cause injury in corn including leaf and stem bleaching, plant height reduction or stunting, and reduced crop stand. Safeners are co-applied with herbicides to protect crops without compromising weed control efficacy. With the ultimate goal of addressing Zea mays injury caused by IXF, a series of novel substituted dichloroacetylphenyl sulfonamide derivatives was designed on the basis of scaffold hopping and active substructure splicing. A total of 35 compounds were synthesized via acylation reactions. All the compounds were characterized by infrared (IR), proton and carbon-13 nuclear magnetic resonance (1 H-NMR and 13 C-NMR), and high-resolution mass spectrometry (HRMS). The configuration of compound II-1 was confirmed by single crystal X-ray diffraction. The bioassay results showed that all the title compounds displayed remarkable protection against IXF via improved content of carotenoid. Especially compound II-1 which possessed better glutathione transferases (GSTs) activity and carotenoid content than the contrast safener cyprosulfamide (CSA). All the satisfied parameters suggested that the Comparative Molecular Field Analysis (CoMFA) model was reliable and stable [with a cross-validated coefficient (q2 ) = 0.527, r2 = 0.995, r2 pred = 0.931]. The molecular docking simulation indicated that the compound II-1 and CSA could compete with diketonitrile (DKN) at the active site of HPPD, which is a hydrolyzed product of IXF in plants, causing the herbicide to be ineffective. The present work revealed that the compound II-1 deserves further attention as the candidate structure of safeners. © 2020 Society of Chemical Industry.

Preparation, characterization, and pharmacological study of a novel long-acting FGF21 with a potential therapeutic effect in obesity

FGF21 (Fibroblast Growth Factor 21), which is expressed in the liver, adipose tissue, and pancreas, has been widely known as a therapeutic candidate for metabolic diseases. Though FGF21 is crucial to glucose, lipid, and energy homeostasis, it is not straightforward to develop a new drug with FGF21 due to its short half-life in serum. Here, we derived a novel long-acting FGF21 (LAPS-FGF21), which is chemically conjugated to the human IgG4 Fc fragment for longer half-life in serum. The recombinant human IgG4 Fc fragment and FGF21 were prepared by the refolding of inclusion body and periplasmic expression in Escherichia coli overexpression systems, respectively. The efficacy study of LAPS-FGF21 in a Diet-Induced Obesity (DIO) mouse model revealed that LAPS-FGF21 reduced body weight effectively accompanied by improved glucose tolerance in a dose-dependent manner. The administration of LAPS-FGF21 also improved the blood profiles with a significant reduction in cholesterol and triglyceride levels. Additionally, the pharmacokinetic (PK) studies of LAPS-FGF21 using normal ICR mice demonstrated that the half-life of LAPS-FGF21 was approximately 64-fold longer than FGF21. Taken together, the LAPS-FGF21 could be a feasible drug candidate with excellent bodyweight loss efficacy and longer dosing interval by half-life increase in serum.

Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2.

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.

Identification of Functional Fragments in gMYL6 and the Mechanism of Improving NK Cell-Mediated Cytotoxicity.

In a previous study, the unrecognized role of gMYL6 in the up-regulation of human NK cells development and cytotoxicity was reported. To further elucidate the mechanism of action of small recombinant fragments of gMYL6 enhancing the NK cells activity. Mononuclear cells were isolated from umbilical cord blood (UCB) by density-gradient centrifugation and NK cells were propagated and cultured. The small peptides from the gMYL6, with the ability to enhance the cytotoxicity of NK cells were screened by CCK-8 method and one of the most powerful peptides was identified for the next study. Flow cytometry was used to assess the proliferation and apoptosis of K562 cells, as well as the cell cycle arrest. The apoptosis of target cells was observed by AO/EB fluorescence staining, and the degree of apoptosis was assessed by flow cytometry. Protein imprinting method was also used to explore the pathway of small peptides to enhance the NK cells' activity. On the other hand, Real-time Quantitative PCR Detecting System was used to verify the mechanism of K562 cells suppression. Small D peptide significantly increased NK cells cytotoxicity and induced both cell cycle arrest at G2/M and apoptosis of K562 cells. Small D peptide could be a novel promising peptide for cancer immunotherapy since it was shown to promote the cytotoxicity of cord blood-derived NK cells.

Assembly of Satsuma mandarin mitochondrial genome and identification of cytoplasmic male sterility–specific ORFs in a somatic cybrid of pummelo

Cytoplasmic male sterility (CMS) and seedlessness are highly desirable traits for citrus. However, a high-quality reference mitochondrial genome of citrus is unavailable, and the mechanisms of CMS remain to be elucidated in citrus. We previously generated a male sterile somatic cybrid of pummelo (G1+HBP) by fusing protoplasts from CMS ‘Guoqing No. 1’ Satsuma mandarin (G1) and fertile ‘Hirado Buntan’ pummelo (HBP). Here, we sequenced and assembled the mitochondrial genome of G1, G1+HBP, and HBP into circular molecules of 521.6 kb, 538.4 kb, and 518.3 kb, respectively. Mitochondrial genome of citrus encodes 35–36 proteins, three ribosomal RNAs, and 21 tRNAs. Comparative analysis indicated that nuclear genome and chloroplast genome of the cybrid are from HBP, whereas the mitochondrial genome sequences of the cybrid and G1 have 96.9% synteny, and that the cybrid has an extra recombinant mitochondrial DNA fragment of only 16.8 kb. Compared with the fertile HBP mitochondrial genome, 21 and 20 CMS-specific mitotype-specific sequences (MSSs), and 89 and 70 CMS-specific ORFs were identified in the cybrid and G1 mitochondrial genomes, respectively. Among them, 57 ORFs that co-transcribed with the same mitochondrial genes in the cybrid and G1 were identified as the CMS-specific ORFs. The assembly of mitochondrial genomes and the identification of the CMS-specific ORFs provide basis for determination of the CMS-associated gene and elucidation of CMS mechanisms in citrus.

The recombinant pseudorabies virus expressing African swine fever virus CD2v protein is safe and effective in mice

BackgroundAfrican swine fever (ASF) leads to high mortality in domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). Currently, no vaccine is commercially available for prevention, and the epidemic is still spreading. Here, we constructed a recombinant pseudorabies virus (PRV) (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) that expresses the CD2v protein of ASFV and evaluated its effectiveness and safety as a vaccine candidate in mice.MethodsA homologous recombination fragment containing ASFV CD2v was synthesized and co-transfected into HEK 293 T cells, a knockout vector targeting the PRV TK gene. The transfected cells were infected with PRV-ΔgE/ΔgI, and the recombinant strain (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) was obtained by plaque purification in Vero cells. The expression of ASFV CD2v in the recombinant virus was confirmed by sequencing, Western blotting, and immunofluorescence analysis, and the genetic stability was tested in Vero cells over 20 passages. The virulence, immunogenicity and protective ability of the recombinant virus were further tested in a mouse model.ResultsThe PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain is stable in Vero cells, and the processing of CD2v does not depend on ASFV infection. The vaccination of PRV-ΔgE/ΔgI/ΔTK-(CD2v) causes neither pruritus, not a systemic infection and inflammation (with the high expression of interleukin-6 (IL6)). Besides, the virus vaccination can produce anti-CD2v specific antibody and activate a specific cellular immune response, and 100% protect mice from the challenge of the virulent strain (PRV-Fa). The detoxification occurs much earlier upon the recombinant virus vaccination and the amount of detoxification is much lower as well.ConclusionsThe PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain has strong immunogenicity, is safe and effective, and maybe a potential vaccine candidate for the prevention of ASF and Pseudorabies.

Discovery of Pyrazine-Carboxamide-Diphenyl-Ethers as Novel Succinate Dehydrogenase Inhibitors via Fragment Recombination.

The discovery of novel succinate dehydrogenase inhibitors (SDHIs) has attracted great attention worldwide. Herein, a fragment recombination strategy was proposed to design new SDHIs by understanding the ligand-receptor interaction mechanism of SDHIs. Three fragments, pyrazine from pyraziflumid, diphenyl-ether from flubeneteram, and a prolonged amide linker from pydiflumetofen and fluopyram, were identified and recombined to produce a pyrazine-carboxamide-diphenyl-ether scaffold as a new SDHI. After substituent optimization, compound 6y was successfully identified with good inhibitory activity against porcine SDH, which was about 2-fold more potent than pyraziflumid. Furthermore, compound 6y exhibited 95% and 80% inhibitory rates against soybean gray mold and wheat powdery mildew at a dosage of 100 mg/L in vivo assay, respectively. The results of the present work showed that the pyrazine-carboxamide-diphenyl-ether scaffold could be used as a new starting point for the discovery of new SDHIs.

Development, Screening, and Validation of Camelid-Derived Nanobodies for Neuroscience Research.

Nanobodies (nAbs) are recombinant antigen-binding variable domain fragments obtained from heavy-chain-only immunoglobulins. Among mammals, these are unique to camelids (camels, llamas, alpacas, etc.). Nanobodies are of great use in biomedical research due to their efficient folding and stability under a variety of conditions, as well as their small size. The latter characteristic is particularly important for nAbs used as immunolabeling reagents, since this can improve penetration of cell and tissue samples compared to conventional antibodies, and also reduce the gap distance between signal and target, thereby improving imaging resolution. In addition, their recombinant nature allows for unambiguous definition and permanent archiving in the form of DNA sequence, enhanced distribution in the form of sequences or plasmids, and easy and inexpensive production using well-established bacterial expression systems, such as the IPTG induction method described here. This article will review the basic workflow and process for developing, screening, and validating novel nAbs against neuronal target proteins. The protocols described make use of the most common nAb development method, wherein an immune repertoire from an immunized llama is screened via phage display technology. Selected nAbs can then be taken through validation assays for use as immunolabels or as intrabodies in neurons. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Total RNA isolation from camelid leukocytes Basic Protocol 2: First-strand cDNA synthesis; VH H and VH repertoire PCR Basic Protocol 3: Preparation of the phage display library Basic Protocol 4: Panning of the phage display library Basic Protocol 5: Small-scale nAb expression Basic Protocol 6: Sequence analysis of selected nAb clones Basic Protocol 7: Nanobody validation as immunolabels Basic Protocol 8: Generation of nAb-pEGFP mammalian expression constructs Basic Protocol 9: Nanobody validation as intrabodies Support Protocol 1: ELISA for llama serum testing, phage titer, and screening of selected clones Support Protocol 2: Amplification of helper phage stock Support Protocol 3: nAb expression in amber suppressor E. coli bacterial strains.

Selection of an artificial paraquat-specific binding protein from a ribosome display library based on a lipocalin scaffold.

In this study, an anticalin that could specifically bind paraquat (PQ), a quaternary nitrogen herbicide, as a new set of engineered receptor protein with antibody-like properties was generated to detect PQ concentration. To this end, a native and random library was constructed and engineered to allow in vitro transcription and translation using an Escherichia coli lysate system. Meanwhile, a PQ derivative that carries an active aliphatic carboxylate group at the end of an aliphatic spacer arm was synthesized. Then, this compound was coupled covalently to the carrier protein bovine serum albumin/ovalbumin and amino-functionalized paramagnetic beads. Alternating selection in solution and immobilization in microtiter wells were used to pan mRNA-ribosome-antibody complexes. After several rounds of ribosome display, three variants were selected from a random library of the bilin-binding protein. The variants that could bind complex PQ with high affinity and exhibit IC50 values as low as 14.039±0.970ng/mL were identified. Moreover, the limits of detection reached 0.083±0.011ng/mL. Our data suggest that the generation of anticalins may provide a promising alternative to recombinant antibody fragments to create a stable receptor protein against hapten with bioanalytical relevance.

DDRE-20. GENERATION OF A HIGH-AFFINITY EGFR/EGFRvIII-TARGETED D2C7 CAR-T CELL

Abstract BACKGROUND The intent of this investigation is to validate a novel, high-affinity epidermal growth factor receptor (EGFR) chimeric antigen receptor (CAR) T-cell product for adoptive immunotherapy of EGFR-expressing malignancies of the central nervous system (CNS). Wild type EGFR is ubiquitously expressed on glial tumors in both children and adults, and the majority of solid tumors that metastasize to the CNS but is not expressed on healthy CNS tissues. EGFR and its isoform mutant, EGFRvIII, are hyperactivated or overexpressed receptor tyrosine kinases described in many human cancers. D2C7 is a recombinant monoclonal antibody short chain variable fragment (scFv) that binds to both wild type EGFR and EGFRvIII. We hypothesize that D2C7 adapted as a CAR on primary human T cells will generate potent, cytotoxic activity against EGFR-expressing tumors in vivo. METHODS In vitro interferon gamma release assays comparing the D2C7 CAR to the EGFRvIII CAR have proven that D2C7 CAR-T cells have high specificity and potent cytotoxicity against established murine glioblastoma (GBM) tumor cell lines, expressing both EGFR and EGFRvIII. Next steps include pre-clinical in vivo testing of the D2C7 CAR-T cells in murine models of CNS tumors expressing EGFR/EGFRvIII such as GBM, non-small cell lung carcinoma (NSCLC), breast carcinoma, and melanoma. CONCLUSIONS To improve and validate the effectiveness of CAR T-cell therapy for EGFR-expressing CNS tumors, our team has designed a high-affinity CAR-T cell that targets both EGFRvIII and wild-type EGFR, known as a D2C7 CAR-T cell. This investigation has established pre-clinical anti-tumor activity of D2C7-CAR T cells in vitro and we plan to present more mature data regarding efficacy in orthotopic murine models of GBM, NSCLC, breast carcinoma, and melanoma at the meeting this Fall.

Expression, purification and characterization of α-synuclein fibrillar specific scFv from inclusion bodies.

Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E. coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs.

KinFragLib: Exploring the Kinase Inhibitor Space Using Subpocket-Focused Fragmentation and Recombination.

Protein kinases play a crucial role in many cell signaling processes, making them one of the most important families of drug targets. In this context, fragment-based drug design strategies have been successfully applied to develop novel kinase inhibitors. These strategies usually follow a knowledge-driven approach to optimize a focused set of fragments to a potent kinase inhibitor. Alternatively, KinFragLib explores and extends the chemical space of kinase inhibitors using data-driven fragmentation and recombination. The method builds on available structural kinome data from the KLIFS database for over 2500 kinase DFG-in structures cocrystallized with noncovalent kinase ligands. The computational fragmentation method splits the ligands into fragments with respect to their 3D proximity to six predefined functionally relevant subpocket centers. The resulting fragment library consists of six subpocket pools with over 7000 fragments, available at https://github.com/volkamerlab/KinFragLib. KinFragLib offers two main applications: on the one hand, in-depth analyses of the chemical space of known kinase inhibitors, subpocket characteristics, and connections, and on the other hand, subpocket-informed recombination of fragments to generate potential novel inhibitors. The latter showed that recombining only a subset of 624 representative fragments generated 6.7 million molecules. This combinatorial library contains, besides some known kinase inhibitors, more than 99% novel chemical matter compared to ChEMBL and 63% molecules compliant with Lipinski's rule of five.

Binding of Gpibα to VWF A2 Domain Alters Mechanical Unraveling of the A2 Domain

Background: Von Willebrand factor (VWF), is a large, multimeric plasma glycoprotein that plays a critical role in hemostasis. VWF is synthesized and secreted as ultra large (UL) multimers that contain up to 100 protomers. If not processed by ADAMTS13, a plasma metalloprotease, ULVWF may initiate the spontaneous formation of life-threatening thrombi, as seen in thrombotic thrombocytopenic purpura (TTP). The cleavage site is buried under the central β-sheet within the VWF-A2 domain and tensile force is required to expose the cleavage site for ADAMTS13. Our prior studies demonstrate that several VWF-binding proteins, including coagulation factor VIII, apoB100/LDL, as well as the ectodomains of platelet glycoprotein Iba (GPIba), appear to function as cofactors that facilitate the proteolytic cleavage of VWF by ADAMTS13 under shear. However, the mechanism underlying GPIba enhancing effect on ADAMTS13-mediated VWF proteolysis is yet to be determined. Methods: Recombinant human GPIbα was purchased from Sino Biological. The recombinant VWF-A2 domain fragment with a SpyTag on the N-terminus and an AviTag-HisTag on the C-terminus was expressed in the HEK 293T cells and affinity-purified by the Ni-NTA affinity chromatography. Biotinylation was performed in vitro using a biotin-labeling kit. The binding between VWF-A2 and GPIbα was studied by a custom-built atomic force microscope (AFM) using our established single-molecule binding study protocol. MicroScale Thermophoresis was conducted by a Monolith device to detect the binding affinity between the VWF-A2 and GPIbα. A dual-beam mini-tweezers instrument was utilized to characterize the force-induced conformational changes of the A2 domain in the absence and presence of GPIbα with a pulling speed of 200 nm/s. Results: AFM results indicated that specific binding interactions occurred between GPIbα and VWF-A2. Monolith MST assay revealed a strong binding affinity (Kd of ~20 nM) between GPIbα and the VWF-A2 fragment. In the optical tweezer study, pulling on a single VWF-A2 resulted in an unfolding event at 10-30 pN with an extension ranging from 30 to 40 nm (Fig. 1A). Gaussian fits of the unfolding extension distributions revealed a most probable force-induced extension of 34.87 ± 2.2 nm (mean ± SEM) (Fig. 1B). Addition of 100 nM of GPIbα led to a noticeable decrease in both unfolding force and extension of VWF-A2 (Fig. 1A). The most probable unfolding extension reduced to 16.05 ± 0.2 nm in the presence of 100 nM of GPIbα (Fig. 1B), indicating the binding of GPIbα may influence mechanical unfolding of VWF-A2. Further, the unfolding results were analyzed by a worm-like chain model fit, which yielded a contour length for the initially folded structure of VWF-A2 at 58.83 ± 2.0 nm (mean ± SEM) and 24.53 ± 0.2 nm in the absence and presence of GPIbα, respectively (Fig. 1C), indicating that the specific interactions between GPIbα and A2 domain may partially unfold the A2 domain. Conclusions: These results demonstrate for the first time that binding of GPIbα to VWF-A2 may alter the force-induced conformational changes in the A2 domain. Under physiological conditions, the glycocalicin (or soluble GP1bα) may bind the VWF-A2 and cause A2 partial unfolding, which may result in excessive cleavage of VWF by ADAMTS13, thus regulating hemostasis. Figure legend: Fig.1. GPIbα influences the mechanical unfolding of the A2 domain of VWF. (A) Typical optical tweezer pulling traces of A2 domain in the absence (red) and presence (blue) of GPIbα (100 nM). The arrows point to the unfolding events. The pulling speed is 200 nm/s. (B) The histograms of the unfolding extension of pulling VWF-A2 in the absence (red) or presence (blue) of 100 nM of GPIbα at 200 nm/s. Solid lines are Gaussian fits to the distributions. (C) The relationship between unfolding force (pN) and unfolding extension (nm) of pulling the VWF-A2 in the absence (red) or presence (blue) of 100 nM of GPIbα. The data are fitted to the worm-like chain model (solid lines). Horizontal and vertical error bars are one standard deviation for force and half width of the half bin width for extension, respectively. Figure Disclosures Cao: Ivygen: Consultancy; Bayer: Research Funding. Zheng:Sanofi: Consultancy, Speakers Bureau; Clotsolution: Other: Co-Founder; Alexion: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau.

Acquired von Willebrand Syndrome in left Impella supported cardiogenic shock patients

Abstract Background Bleeding is a main cause of morbidity and mortality in critically ill cardiogenic shock patients, supported by short-term percutaneous mechanical circulatory support (pMCS) devices. Bleeding not only occurs because of obligatory heparin and antiplatelet therapy (both required in the prevention of pump and stent thrombosis) but possibly also results from device-related coagulopathy. Similar to long-term ventricular assist devices, mechanical shear-induced acquired von Willebrand syndrome (AVWS) might further increase the bleeding risk. Therefore, we aimed to investigate the effect of left Impella percutaneous continuous flow pumps on the development of AVWS due to shear-induced excessive cleavage of large vWF multimers by the metalloproteinase ADAMTS-13, resulting in loss of high-molecular-weight vWF multimers. Methods Between March 2019 and January 2020, all cardiogenic shock patients supported by a left Impella and referred to a single tertiary ICU were studied. Both vWF Antigen (vWF:Ag) and vWF:GPIbR (ristocetin-induced binding of vWF to a recombinant wildtype Glycoprotein Ib fragment) levels were measured by chemiluminescent immunoassays using an AcuStar (Werfen) assay to determine the vWF:GPIbR /vWF:Ag ratio (normal range ≥1.0). VWF multimer analysis was performed by electrophoresis. On-pump analyses were performed 12h after implantation and off-pump analyses 12h after Impella explantation. Patients who died on-pump were excluded because of lack of paired data after explantation. Results Eight left Impella patients (four Impella CP, four Impella 5.0) were analyzed for AVWS. The vWF:GPIbR /vWF:Ag ratio was <1.0 in all patients on-pump (mean±SD 0.68±0.1 versus 1.1±0.15 off-pump (panel A; p=0.0018)) and thus AVWS was detected in all Impella-supported patients. The presence of AVSW was also confirmed by loss of large vWF multimers on-pump (panel B). Four patients (50%) had mucosal bleeds (epistaxis or gastrointestinal), none of them requiring transfusion. The mean rise in ratio 12h after pump removal was 0.35 which was also reflected by recovery of large multimers by electrophoresis (panel B). Conclusions Our data highlight the rapid onset and reversal of AVWS in all studied cardiogenic shock patients, supported by a left Impella pump. The determination of the GPIbR /vWF:Ag ratio with the AcuStar appears a reliable and faster test to detect AVWS as compared to vWF multimers electrophoresis. Further research into innovative pharmacological interventions (e.g. ADAMTS-13 inhibitors) should target pMCS-induced AVWS in an effort to reduce hemostatic complications in this critically ill ICU population. AVWS in Impella supported patients Funding Acknowledgement Type of funding source: None

How Reliable are Commercially Available Glypican4 ELISA Kits?

Glypican4 is an interesting new adipokine, which seems to play an important role in developmental processes and is potentially associated with metabolic changes in obesity and type 2 diabetes mellitus. Currently, only a few studies examined glypican4 in human blood, mainly in adults. The aim of our study was to investigate glypican4 serum levels in lean, overweight, and obese children and adolescents, to unravel a possible association between glypican4 serum levels and parameters of obesity and insulin resistance. In order to determine a suitable method for investigating glypican4 serum levels, we validated two commercially available human glypican4 ELISA kits, using serum and plasma samples of an obese, insulin-resistant patient, and a healthy control subject, a human recombinant glypican4 protein fragment and glypican4-overexpressing cell lysate. Using ELISA kit #1 we were not able to detect values above background level, apart from standard curve values. ELISA kit #2 initially seemed suitable to measure glypican4, but further validation experiments showed non-linearity of serial dilutions, no recognition of a human recombinant glypican4 protein fragment and non-linearity in the recovery of glypican4-overexpressing cell lysate. In addition, there was a considerable decrease (approx. 68%) of measured values between two experiments, performed at different time points with aliquots of the same serum sample. Contrary to that, further experiments found sample stability not to be compromised. Extensive evaluation of the performance of two commercially available ELISA kits led to the conclusion that none of them is applicable for the measurement of glypican4 in human blood samples.