A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels.

Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca2+) uptake with a Ca2+-selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca2+ concentration ([Ca2+]cyt) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations.

Neutralizing antibodies (nAbs) hold promise as therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and tetravalent versions block entry with a potency exceeding bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show increased tolerance to potential virus escape mutants and an emerging variant of concern. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for enhancing antiviral therapies against COVID-19 and related viral threats, and our strategy can be applied to virtually any antibody drug.

Quantitative Profiling of Synuclein Species: Application to Transgenic Mouse Models of Parkinson's Disease.

INTRODUCTION: Improved analytical tools for detailed characterization of synucleins in pre-clinical models of Parkinson's disease (PD) and related synucleinopathies are needed. OBJECTIVE: Develop a multiple reaction monitoring (MRM) liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to quantify species-specific sequences and structural heterogeneity in soluble α- and ß-synucleins in brain tissue. METHODS: Using a proteolytic digestion workflow, the MRM LC-MS/MS method assayed six proteotypic peptides from the α-synuclein sequence; three unique to mouse or human α-synuclein and three conserved in α- and ß-synuclein. For quantification, we used labeled α-synuclein as the internal standard and an external calibration curve. As proof of concept, the synuclein LC-MS/MS method was applied to brain tissue specimens from M83 transgenic PD mice, which overexpresses human α-synuclein, relative to wild-type littermate controls. RESULTS: The synuclein MRM assay was linear over a wide concentration range (at least one order of magnitude). The assay had several advantages over ligand binding analytical methods, such as western blotting and enzyme-linked immunosorbent assays. These advantages included the ability to: quantify 1) total α-synuclein, 2) combined α- and ß-synucleins, 3) species-specific contributions to total α-synuclein (e.g., in mice expressing both mouse and human α-synuclein), and 4) identify peptide-specific profile differences that may reflect post-translational modifications, all within a single analysis. CONCLUSION: With improved and expanded analytical characteristics coupled with a streamlined sample preparation workflow, the quantitative synuclein profiling LC-MS/MS assay provides a versatile and efficient platform to characterize synuclein biology in pre-clinical models and the potential for application to human tissues and fluids.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations.

Neutralizing antibodies (nAbs) hold promise as effective therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and its tetravalent versions can block entry with a potency that exceeds the bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, observations consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show much increased tolerance to potential virus escape mutants. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for developing potent antiviral therapies against COVID-19 and related viral threats, and our strategy can be readily applied to any antibody drug currently in development.

In Vitro Conversion Assays Diagnostic for Neurodegenerative Proteinopathies.

BACKGROUND: In vitro conversion assays, including real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) techniques, were first developed to study the conversion process of the prion protein to its misfolded, disease-associated conformation. The intrinsic property of prion proteins to propagate their misfolded structure was later exploited to detect subfemtogram quantities of the misfolded protein present in tissues and fluids from humans and animals with transmissible spongiform encephalopathies. Currently, conversion assays are used clinically as sensitive and specific diagnostic tools for antemortem diagnosis of prion disease. CONTENT: In vitro conversion assays are now being applied to the development of diagnostics for related neurodegenerative diseases, including detection of misfolded α-synuclein in Parkinson disease, misfolded amyloid-ß in Alzheimer disease, and misfolded tau in Pick disease. Like the predicate prion protein in vitro conversion diagnostics, these assays exploit the ability of endogenously misfolded proteins to induce misfolding and aggregation of their natively folded counterpart in vitro. This property enables biomarker detection of the underlying protein pathology. Herein, we review RT-QuIC and PMCA for (a) prion-, (b) α-synuclein-, (c) amyloid-ß-, and (d) tau-opathies. SUMMARY: Although already in routine clinical use for the detection of transmissible spongiform encephalopathies, in vitro conversion assays for other neurodegenerative disorders require further development and evaluation of diagnostic performance before consideration for clinical implementation.

Cross-linked hemoglobin bis-tetramers from bioorthogonal coupling do not induce vasoconstriction in the circulation.

BACKGROUND: Hemoglobin-based oxygen carriers (HBOCs) are potential alternatives to red blood cells in transfusions. Clinical trials using early versions of HBOCs noted adverse effects that appeared to result from removal of the vasodilator nitric oxide (NO). Previous reports suggest that size-enlarged HBOCs may avoid NO-rich regions along the vasculature and therefore not cause vasoconstriction and hypertension. STUDY DESIGN AND METHODS: Hemoglobin (Hb) bis-tetramers (bis-tetramers of hemoglobin that are prepared using CuAAC chemistry [BT-Hb] and bis-tetramers of hemoglobin that are specifically acetylated and prepared using CuAAC chemistry [BT-acHb]) can be reliably produced by a bio-orthogonal cyclo-addition approach. We considered that an HBOC derived from chemical coupling of two Hbs would be sufficiently large to avoid NO scavenging and related side effects. The ability of intravenously infused BT-Hb and BT-acHb to remain in the circulation without causing hypertension were determined in wild-type (WT) and diabetic (db/db) mouse models. RESULTS: In WT mice, the coupled oxygen-carrying proteins retained their function over several hours after administration. No significant changes in systolic blood pressure from baseline were observed after intravenous infusion of BT-Hb or BT-acHb in awake WT and db/db mice. In contrast, infusion of native Hb or cross-linked Hb tetramers in both animal models induced systemic hypertension. CONCLUSION: The results of this study indicate that bis-tetrameric HBOCs derived from the bio-orthogonal cyclo-addition process are likely to overcome clinical issues that arise from NO scavenging by Hb derivatives.

Strain-promoted azide-alkyne cycloaddition for protein-protein coupling in the formation of a bis-hemoglobin as a copper-free oxygen carrier.

Conventional chemical approaches to protein-protein coupling present challenges due to the intrinsic competition between the desired interactions of reagents with groups of the protein as well as reactions with water. Biorthogonal Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC)-processes provide a basis to direct reactivity without functional group interference. However, the requirement for Cu(i) in CuAAC leads to complications that result from the metal ion's interactions with the protein. In principle, a similar but metal-free alternative approach to coupling could employ the reaction of an alkyne that is strained in combination with an azide (strain-promoted azide-alkyne cycloaddition, SPAAC). The method is exemplified by the combination of a cyclooctyne derivative of hemoglobin with an azide-modified hemoglobin. The bis-hemoglobin tetramer that is produced has properties consistent with those sought for use as a hemoglobin-based oxygen carrier (HBOC).

Self-Assembly of a Functional Triple Protein: Hemoglobin-Avidin-Hemoglobin via Biotin-Avidin Interactions.

Hypertension resulting from vasoconstriction in clinical trials of cross-linked tetrameric (α2ß2) human hemoglobins implicates the extravasation of the hemoglobins into endothelia where they scavenge nitric oxide (NO), which is the signal for relaxation of the surrounding smooth muscle. Thus, we sought an efficient route to create a larger species that avoids extravasation while maintaining the oxygenation function of hemoglobin. Selectively formed cysteine-linked biotin conjugates of hemoglobin undergo self-assembly with avidin into a stable triple protein, hemoglobin-avidin-hemoglobin (HbAvHb), which binds and releases oxygen with moderate affinity and cooperativity. The triple protein is likely to be stabilized by interactions of each constituent hemoglobin (pI 6.9) with the oppositely charged avidin (pI 10.5) as well as the strong association of the biotin moieties on hemoglobin with avidin.

Subunit-directed click coupling via doubly cross-linked hemoglobin efficiently produces readily purified functional bis-tetrameric oxygen carriers.

While cross-linked hemoglobin (Hb) tetramers can deliver oxygen as a supplement to red cells, they also cause unacceptable increases in blood pressure, presumably from their penetration of the linings of blood vessels (endothelia) where the internal hemes bind endogenous nitric oxide (NO). This penetration would lower the local concentration of NO that normally induces vasodilation. Enlarging the effective size of the oxygen-carrying protein by coupling two Hbs can prevent their extravasation. Efficient and selective protein-protein coupling to produce those species has been a significant challenge. Introduction of an azide within a protein provides a directionally-oriented reaction site for utilization of the Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) in the protein-protein-coupling process based on solubility-directed sequential addition to a bis-alkyne. However, it is known that Hb with an azide-containing cross-link between α-subunits is unreactive in CuAAC. To direct reaction away from the α-subunits of Hb, a specific fumaryl cross-link is installed exclusively between the most reactive sites on those subunits, thereby blocking the α-99 lysyl groups and preventing any further reaction. This modification allows installation of an azide-containing cross-link exclusively between lysine-82 ε-amino groups of the ß-subunits of Hb. The multiply interconnected sites establish a geometry that permits initial interfacial interaction of the cross-linked Hb-azide with Cu(i) and a bis-alkyne. After coupling, the protein-linked azide product undergoes CuAAC at the remaining alkyne with a second cross-linked Hb-azide, producing a fully functional cross-linked Hb bis-tetramer whose oxygenation and structural properties include cooperativity and oxygen affinity that should be suitable for testing as an alternative to red cells in transfusions.

Bioorthogonal phase-directed copper-catalyzed azide-alkyne cycloaddition (PDCuAAC) coupling of selectively cross-linked superoxide dismutase dimers produces a fully active bis-dimer.

Superoxide dismutase (SOD) is a 32 kDa dimeric enzyme that actively removes a toxic oxygen species within red cells. The acellular protein itself does not survive circulation as it is filtered through the kidney. Conjugating the protein to another SOD should increase the size of the dual protein above the threshold for filtration by the kidney, making the material a potential therapeutic in circulation. Site-selective chemical cross-linking of SOD introduces a bioorthogonal azide group on the cross-link so that two SODs react efficiently with a bis-alkyne through phase-directed copper-catalyzed azide-alkyne cycloaddition (PDCuAAC). The modification has a negligible effect on the catalytic activity of the constituent proteins. Consistent with the retained activity, circular dichroism (CD) spectroscopy indicates that the secondary structures of the proteins are similar to that of the native protein.

Attempts to prepare tethered bilayer lipid membranes using synthetic thioglycolipid anchors: synthesis of 6″-thiotrisaccharide glycolipid analogues and applications.

The synthesis of the three 6″-deoxy-6″-thio glycolipid analogues ß-d-Gal-(1→6)-ß-d-Gal-(1→4)-ß-d-Glu-(1→OCH2)-[1,2,3]-triazole-1-dodecane, ß-d-Gal-(1→4)-ß-d-Glu-(1→4)-ß-d-Glu-(1→OCH2)-[1,2,3]-triazole-1-dodecane and ß-d-Gal-(1→4)-ß-d-Glu-(1→4)-ß-d-Glu-(1→OCH2)-[1,2,3]-triazole-1-octadecane is presented. Glycosylation at position O-4' of a propargyl cellobioside glycosyl acceptor and position O-6' of a propargyl lactoside glycosyl acceptor with a 6-deoxy-6-thio galactosyl donor gave rise to two unique trisaccharides that in turn underwent copper-catalyzed azide-alkyne cycloadditions with either 1-azidododecane or 1-azidooctadecane. The potential for each of these analogues to function as tethers of lipid bilayers to Au(111) surface was assessed by differential capacitance experiments. A monolayer of the previously described monosaccharide 1-octadecane-4-(6-thio-ß-d-galacto-pyranosyloxymethyl)-[1,2,3]-triazole either self-assembled or prepared by Langmuir-Blodgett (LB) transfer was found to support an outer leaflet monolayer (DMPC/cholesterol, 70:30) deposited by Langmuir-Schaefer (LS) touch. The bilayers obtained with this monosaccharide analogue had minimum differential capacitances of 1.0 and 0.9µF/cm(2) when the inner monolayer was prepared by self-assembly and LS touch, respectively. Attempts to produce bilayers using the trisaccharides synthesized here were unsuccessful; we are attributing these unsuccessful results mostly to the high water solubility of trisaccharides combined with the relatively short length of the hydrocarbon chains used in this study.

Synthesis of 6-thio pseudo glycolipids and their orientation on a gold slide studied by IRRAS.

We have synthesized four 6-thio pseudo glycolipid analogues and assessed how two of them self-assembled on a gold surface. These structures were designed as candidate tethers molecules to anchor bilayer lipid membranes on gold. 6-Deoxy-6-thiogalactose was chosen to anchor the macromolecule to the gold and define an aqueous zone at the gold surface. A long alkane chain (C-12 or C-18) linked to the anomeric position of the sugar residue was chosen to anchor a bilayer lipid membrane. The linkage between the carbohydrate and the hydrophobic chains is either a glycosidic bond or a 1,4-disubstituted triazole formed by copper(I)-catalysed alkyne-azide cycloaddition (CuAAC) of the propargyl glycoside with azido-dodecane and azido-octadecane. We are expecting that the hydrocarbon chains will orient themselves perpendicular to the gold surface and be incorporated into the first leaflet of the bilayer membrane. We have studied self assembled monolayers of the C-12 aglycone analogues on gold using infrared reflection absorption spectroscopy (IRRAS). We compared the results given by the IRRAS experiments to the IR spectra recorded by attenuated total reflection (ATR) spectroscopy on films of the randomly oriented analogues. Our results demonstrate that the C-12 analogues did bind to gold and did orient themselves perpendicular to the gold slide.