Universal Ligand Research Articles

ASSESSMENT OF THE RELATIONSHIP BETWEEN THE CONCENTRATION OF PSGL-1, NO-SYNTHASES AND NITRIC OXIDE METABOLITES IN THE BLOOD SERUM OF PATIENTS WITH ACUTE ARTERIAL THROMBOSIS

Introduction. PSGL-1 is a complex protein, that provides contacts between blood cells and endothelial cells by connecting with cell-adhesion mole-cules selectins, and it is a universal participant in the processes of leukocyte adhesion, inflammation and immune response. The study of the possible regulation of its functioning is an urgent topic, since the process of its interactions with selectins can be a point of therapeutic application. Aim of the study. Evaluation of the relationship between the concentration of universal ligand selectins PSGL-1, inducible (iNOS) and endothelial (eNOS) NO synthases and final metabolites of nitric oxide (II) in the blood serum of patients with acute arterial thrombosis. Material and methods. The concentrations of PSGL-1, iNOS and eNOS in blood serum were determined, using enzyme immunoassay on an enzyme immunoassay analyzer Stat Fax 2100. The total concentration of the final stable metabolites of nitric oxide(II) (nitrates and nitrites) was determined by the spectrophotometric method modified by V.A. Metelskaya, by coloring in the reaction of diazotization with nitrite of sulfonamide present in the com-position of the Griss reagent. The results were processed, using nonparametric statistics methods of Microsoft Office Excel 2016 and IBM SPSS Statistics 26 (StatSoftInc., USA). Results. The study revealed a statistically significant increase in the level of endothelial nitric oxide synthase and the final stable metabolites of nitric oxide in the blood of patients with acute arterial thrombosis, compared with the control group. A direct correlation of moderate severity was also found between the level of eNOS and the concentration of nitrates and nitrites. A low positive correlation was found between the concentration of PSGL-1 and the level of eNOS, and a low negative correlation was found between PSGL-1 and iNOS. Conclusions. According to the results of the study, it can be concluded that nitric oxide, which generated by endothelial NO synthase in patients with arterial thrombosis, may have a regulatory effect on the expression or functioning of the universal glycoprotein ligand of selectins – PSGL-1, which re-quires more in-depth study.

A universal ligand for lead coordination and tailored crystal growth in perovskite solar cells.

Chemical environment and precursor-coordinating molecular interactions within a perovskite precursor solution can lead to important implications in structural defects and crystallization kinetics of a perovskite film. Thus, the opto-electronic quality of such films can be boosted by carefully fine-tuning the coordination chemistry of perovskite precursors via controllable introduction of additives, capable of forming intermediate complexes. In this work, we employed a new type of ligand, namely 1-phenylguanidine (PGua), which coordinates strongly with the PbI2 complexes in the perovskite precursor, forming new intermediate species. These strong interactions effectively retard the perovskite crystallization process and form homogeneous films with enlarged grain sizes and reduced density of defects. In combination with an interfacial treatment, the resulted champion devices exhibit a 24.6% efficiency with outstanding operational stability. Unprecedently, PGua can be applied in various PSCs with different perovskite compositions and even in both configurations: n-i-p and p-i-n, highlighting the universality of this ligand.

Role of Bis(phosphinimino)methanides as Universal Ligands in the Coordination Sphere of Metals across the Periodic Table.

The coordination chemistry of bis(phosphinimino)methanide ligands is widespread and accompanies a large number of metal ions in the periodic table ranging from lithium to neptunium. This unique class of ligand systems show copious coordination chemistry with the main-group, transition, rare-earth, and actinide metals and are considered to be among the most attractive ligand systems to researchers. The bis(phosphinimino)methanide metal complexes offer an extensive range of applications in various fields and have been demonstrated as one of the universal ligand systems to stabilize the metal ions in not only their usual but also their unusual oxidation states. The main-group and transition metal chemistry using bis(phosphinimino)methanides as ligands was last updated almost a decade ago. In this review, we provide a comprehensive overview of various state-of-the-art bis(phosphinimino)methanide-supported metal complexes by dealing with their synthesis, characterization, reactivity, and catalytic studies which were not included in the last critical reviews.

Tumor cell membrane remodeling with universal ligand for CAR-T cells to inhibit solid tumors

Tumor cell membrane remodeling with universal ligand for CAR-T cells to inhibit solid tumors

Repurposing Fc gamma receptor I (FcγRI, CD64) for site-oriented monoclonal antibody capture: A proof-of-concept study for real-time detection of tumor necrosis factor-alpha (TNF -α)

The controlled orientation of biomolecules on the sensor surface is crucial for achieving high sensitivity and accurate detection of target molecules in biosensing. FcγRI is an immune cell surface receptor for recognizing IgG-coated targets, such as opsonized pathogens or immune complexes. It plays a crucial role in T cell activation and internalization of the cargos, leading downstream signaling cascades. In this study, we repurposed the FcγRI as an analytical ligand molecule for site-oriented ADA capture, a monoclonal antibody-based biosimilar drug, on a plasmonic sensor surface and demonstrated the real-time detection of the corresponding analyte molecule, TNF-α. The study encompasses the analysis of comparative ligand behaviors on the surface, biosensor kinetics, concentration-dependent studies, and sensor specificity assays. The findings of this study suggest that FcγRI has a significant potential to serve as a universal ligand molecule for site-specific monoclonal antibody capture, and it can be used for biosensing studies, as it represents low nanomolar range affinity and excellent selectivity towards the target. However, there is still room for improvement in the surface stability and sensing response, and further studies are needed to reveal its performance on the monoclonal antibodies with various antigen binding sites and glycoforms.

Accelerated water activation and stabilized metal-organic framework via constructing triangular active-regions for ampere-level current density hydrogen production

Two-dimensional metal-organic frameworks (MOFs) have been explored as effective electrocatalysts for hydrogen evolution reaction (HER). However, the sluggish water activation kinetics and structural instability under ultrahigh-current density hinder their large-scale industrial applications. Herein, we develop a universal ligand regulation strategy to build well-aligned Ni-benzenedicarboxylic acid (BDC)-based MOF nanosheet arrays with S introducing (S-NiBDC). Benefiting from the closer p-band center to the Fermi level with strong electron transferability, S-NiBDC array exhibits a low overpotential of 310 mV to attain 1.0 A cm−2 with high stability in alkaline electrolyte. We speculate the newly-constructed triangular “Ni2-S1” motif as the improved HER active region based on detailed mechanism analysis and structural characterization, and the enhanced covalency of Ni-O bonds by S introducing stabilizes S-NiBDC structure. Experimental observations and theoretical calculations elucidate that such Ni sites in “Ni2-S1” center distinctly accelerate the water activation kinetics, while the S site readily captures the H atom as the optimal HER active site, boosting the whole HER activity.

Detection of Nipah and Hendra Viruses Using Recombinant Human Ephrin B2 Capture Virus in Immunoassays.

Nipah virus (NiV) and Hendra virus (HeV) are classified as high-consequence zoonotic viruses characterized by high pathogenicity and high mortality in animals and humans. Rapid diagnosis is essential to containing the outbreak. In this study, the henipavirus receptor ephrin B2 was examined to determine whether it could be used as a universal ligand for henipavirus detection in immunoassays. Enzyme-linked immunosorbent assays (ELISAs) were developed using recombinant ephrin B2 as the capture ligand and two monoclonal antibodies (mAbs) as detection reagents. Using mAb F27NiV-34, which cross-reacts with NiV and HeV, we were able to detect NiV and HeV, while mAb F20NiV-65 was used to detect NiV. Therefore, using these two ELISAs, we were able to differentiate between NiV and HeV. Furthermore, we developed a rapid lateral flow strip test for NiV detection using ephrin B2 as the capture ligand combined with mAb F20NiV-65 as the detector. Taken together, our results show that the combination of ephrin B2 and a specific mAb provides an excellent pairing for NiV and HeV detection.

The synthesis ofN‐containing heterocyclic compounds catalyzed by copper/L‐proline

N‐containing heterocyclic compounds are one of the most important motifs prevalent in various bioactive natural products and pharmaceuticals. Given the significance ofN‐containing heterocyclic compounds, various synthetic strategies have been developed. Copper/ligand (abbreviated as [CuL])‐catalyzed cascade reaction is a powerful and efficient tool for the construction ofN‐containing heterocyclic compounds.L‐proline is one universal ligand and has been utilized in the copper‐catalyzed reactions. In this review, copper/L‐proline‐catalyzed tandem reaction to synthesize the five‐ and six‐membered compounds is emphatically introduced. By comparing the yields in the presence and absence of the ligand, the importance ofL‐proline is recorded. Moreover, the function ofL‐proline is expounded on the basis of the reaction mechanism from original article.

Large Scale Synthesis of Transition Metal Single Atom Catalysts by a Universal Ligand Mediated Method

Series of transition metal single atom catalysts containing Cr, Mn, Fe, Co, Ni, Cu, Zn, Ru, Pt or combinations thereof were synthesized in a large scale by a universal ligand mediated method. The as-synthesized single atom catalysts exhibited excellent electrochemical CO2 reduction activity.

A universal ligand mediated method for large scale synthesis of transition metal single atom catalysts

There is interest in metal single atom catalysts due to their remarkable activity and stability. However, the synthesis of metal single atom catalysts remains somewhat ad hoc, with no universal strategy yet reported that allows their generic synthesis. Herein, we report a universal synthetic strategy that allows the synthesis of transition metal single atom catalysts containing Cr, Mn, Fe, Co, Ni, Cu, Zn, Ru, Pt or combinations thereof. Aberration-corrected high-angle annular dark-field scanning transmission electron microscopy and extended X-ray absorption fine structure spectroscopy confirm that the transition metal atoms are uniformly dispersed over a carbon black support. The introduced synthetic method allows the production of carbon-supported metal single atom catalysts in large quantities (>1 kg scale) with high metal loadings. A Ni single atom catalyst exhibits outstanding activity for electrochemical reduction of carbon dioxide to carbon monoxide, achieving a 98.9% Faradaic efficiency at −1.2 V.

Investigation of the Binding Affinity of a Broad Array of l-Fucosides with Six Fucose-Specific Lectins of Bacterial and Fungal Origin.

Series of multivalent α-l-fucoside containing glycoclusters and variously decorated l-fucosides were synthesized to find potential inhibitors of fucose-specific lectins and study the structure-binding affinity relationships. Tri- and tetravalent fucoclusters were built using copper-mediated azide-alkyne click chemistry. Series of fucoside monomers and dimers were synthesized using various methods, namely glycosylation, an azide-alkyne click reaction, photoinduced thiol-en addition, and sulfation. The interactions between compounds with six fucolectins of bacterial or fungal origin were tested using a hemagglutination inhibition assay. As a result, a tetravalent, α-l-fucose presenting glycocluster showed to be a ligand that was orders of magnitude better than a simple monosaccharide for tested lectins in most cases, which can nominate it as a universal ligand for studied lectins. This compound was also able to inhibit the adhesion of Pseudomonas aeruginosa cells to human epithelial bronchial cells. A trivalent fucocluster with a protected amine functional group also seems to be a promising candidate for designing glycoconjugates and chimeras.

An efficient protocol for the synthesis of thioethers via iron-catalyzed cross-coupling reaction and its mechanistic investigation

One of the most straightforward methods for the synthesis of diaryl sulfides is the transition metal catalyzed CS coupling reaction. Herein we report a study on the iron-catalyzed protocol for cross-coupling reaction of aryl halides with thiols. Structurally diverse diaryl sulfides were prepared efficiently by using a catalyst system involving cheap and environment-friendly FeCl3 and non-toxic universal ligand l-proline. The reaction mechanism for the iron-catalyzed CS coupling reaction was investigated by means of density functional theory (DFT) methods on a model system. The calculations were performed using hybrid PBE1PBE functional in conjugation with the LANL-2DZ basis set. The key step involved in the mechanism is the formation of a reactant complex in which both reactants are electrostatically bound to Fe(III) proline complex catalyst. The elimination of HI occurs with a much lower TS energy (20.0 kcal/mol) than the uncatalyzed reaction (44.7 kcal/mol).

Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: A potential carbamylation route involved in the formation of atherosclerotic plaques?

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

Recombinant FIX/X-Bp from the Japanese Habu Snake Is a Universal Ligand for Purification of Highly Carboxylated Factor IX Variants and Factor X

Background: The purification of vitamin K-dependent clotting factors typically involves multiple chromatographic steps, including an ion exchange-based pseudo-affinity step to enrich for species with sufficiently high gamma-carboxyglutamic acid (Gla) content to achieve maximal specific activity. Variants of these factors have been engineered to improve their pharmacokinetic properties by appending or inserting a variety of elements, including the Fc domain of IgG, unstructured hydrophilic peptides of defined amino acid composition (XTEN), albumin, and polyethylene glycol (PEG). In most cases, however, such modification alters both the hydrodynamic and electrostatic properties of the resulting molecule relative to those of the predicate molecule, thereby complicating their purification, particularly with regard to Gla enrichment by pseudo-affinity chromatography. Factor IX (FIX)- and factor X (FX)-binding protein (FIX/X-bp) isolated from the venom of the Japanese Habu snake (T. flavoviridis) has been shown to bind with high affinity and specificity to both FIX and FX, and structural studies have demonstrated that FIX/X-bp binds to the highly carboxylated calcium-bound forms of the Gla domains of these proteins. We therefore reasoned that FIX/X-bp could serve as a novel affinity ligand for rapid and simple purification of variants of FIX and FX with high specific activity.Aims: To generate and purify recombinant FIX/X-bp (rFIX/X-bp) and assess its utility for the purification of FIX, FIX-XTEN, FIX-albumin, and FX with high Gla content.Methods: A two-chained rFIX/X-bp molecule in which a polyhistidine tag was appended to one chain was generated by stable co-transfection of Chinese hamster ovary (CHO) cells. Culture medium was concentrated by tangential-flow filtration (TFF), and rFIX/X-bp was purified by one of two methods: 1) immobilized metal ion affinity chromatography (IMAC), followed by anion-exchange chromatography, or 2) affinity chromatography on immobilized FIX in calcium-containing buffer and subsequent elution in EDTA-containing buffer. The potent anticoagulant activity of rFIX/X-bp was verified by prothrombin time (PT) and activated partial thromboplastin time (APTT) assays, and its ability to bind to human FIX, FX, factor VII (FVII), protein S, and prothrombin was evaluated by biolayer interferometry. The affinity of rFIX/X-bp for FIX and FX was determined by surface plasmon resonance (SPR). An affinity column was then generated by chemical conjugation of rFIX/X-bp to NHS-activated Sepharose. Recombinant FIX, FIX-albumin, and FIX-XTEN were first affinity purified on IXSelect resin from the culture medium of transiently transfected HEK293 cells, and the resulting protein preparations, which were heterogeneous with regard to Gla content, were then applied to the rFIX/X-bp affinity column in calcium- or magnesium-containing buffer and eluted with EDTA-containing buffer. Activity was assessed by APTT assay, and Gla content was determined by mass spectrometric peptide mapping. Recombinant FX was purified from the culture medium of transiently transfected HEK293 cells by sequential barium citrate adsorption, anion exchange chromatography, and affinity chromatography on a rFIX/X-bp column.Results: In the presence of calcium or magnesium ions, rFIX/X-bp binds to FIX and FX with high affinity (KD≈ 10 pM), to a lesser extent to protein S and prothrombin, but not to FVII. FIX and FIX-albumin that had been affinity purified on a rFIX/X-bp column had specific activities that were consistent with published data and greater than 11 Gla residues per molecule. The Gla content of FX that had been affinity purified on a rFIX/X-bp column was 10 Gla residues per molecule (out of 11 possible).Conclusions: rFIX/X-bp is a universal ligand for the purification of highly carboxylated FX and FIX variants, including FIX-albumin and FIX-XTEN. DisclosuresMercury:Biogen: Employment. Liu:Biogen: Employment. Ismail:Biogen: Employment. Zhang:Biogen: Employment. Lu:Biogen: Employment. Cameron:Biogen: Employment. Goodman:Biogen: Employment. Culyba:Biogen: Employment. Ravindran Nair:Biogen: Employment. Holthaus:Biogen: Employment. Kulman:Biogen: Employment. Peters:Biogen: Employment.

A multifunctional peptide for targeted imaging and chemotherapy for nasopharyngeal and breast cancers

The L-peptide plays a role as a universal ligand binding specifically to nasopharyngeal carcinoma (NPC) and other cancers but not normal cells. It was used to link iron oxide nanoparticles, and injected intravenously to SCID mice bearing NPC and breast cancer xenografts for MR analysis, and showed significant change of MR signal intensity in the xenograft regions. Using this conjugate as a ligand to localize the L-peptide targeted protein in the cancer surgical specimens, a clear reaction product was identified in the tumor cells of both cancer types. If the L-peptide-linked-liposomal doxorubicin was used to treat the SCID mice bearing other NPC or breast cancer xenograft, a high efficacy of chemotherapy with minimal adverse effect was observed. In conclusion, the L-peptide has a considerable potential for clinical usage for targeted imaging, peptide histochemical localization of targeted protein, and targeted chemotherapy for different cancer types. From the Clinical EditorTargeted chemotherapy to cancer cells will enable maximum drug delivery but minimal systemic side effects. In this article, the authors identified a protein, L-peptide, on tumor cells. They also subsequently confirmed the specificity of this protein in animal experiments using iron oxide nanoparticles. The discovery of this marker could lead to future development of better chemotherapy.

Functionalization of Krebs-type polyoxometalates with N,O-chelating ligands: a systematic study.

The first organic derivatives of 3d-metal-disubstituted Krebs-type polyoxometalates have been synthesized under mild bench conditions via straightforward replacement of labile aqua ligands with N,O-chelating planar anions on either preformed or in situ-generated precursors. Nine hybrid clusters containing carboxylate derivatives of five- or six-membered aromatic N-heterocycles as antenna ligands have been obtained as pure crystalline phases and characterized by elemental and thermal analyses, infrared spectroscopy, and single-crystal X-ray diffraction. They all show the general formula [{M(II)L(H2O)}2(WO2)2(B-β-XW9O33)2](n-) and can be classified as follows: 1-SbM, where L = 1H-imidazole-4-carboxylate (imc), X = Sb(III), n = 12, and M(II) = Mn, Co, Ni, Zn; 1-TeM, where L = imc, X = Te(IV), n = 10, and M(II) = Mn, Co; 2-SbNi, where L = 1H-pyrazole-3-carboxylate (pzc), X = Sb(III), n = 12, and M(II) = Ni; and 3-SbM, where L = pyrazine-2-carboxylate (pyzc), X =Sb(III), n = 12, and M(II) = Co, Zn. The 3d-metal-disubstituted tungstotellurate(IV) skeleton of compounds 1-TeM is unprecedented in polyoxometalate chemistry. The stability of these hybrid Krebs-type species in aqueous solution has been confirmed by (1)H NMR spectroscopy performed on the diamagnetic 1-SbZn and 3-SbZn derivatives. Our systematic study of the reactivity of disubtituted Krebs-type polyoxotungstates toward diazole-, pyridine-, and diazinecarboxylates demonstrates that organic derivatization is strongly dependent on the nature of the ligand, as follows: imc displays a "universal ligand" character, as functionalization takes place regardless of the external 3d metal and heteroatom; pzc and pyzc show selectivity toward specific 3d metals; pyridazine-3-carboxylate and pyrimidine-4-carboxylate promote partial decomposition of specific precursors, leading to [M(II)L2(H2O)2] complexes; and picolinate is inert under all conditions tested.

Characterization of Molecular Interaction of Blood Coagulation Factor VIII and Its Clearance Receptor, Low-Density Lipoprotein Receptor.

AbstractAbstract 2206 Background.Clearance of blood coagulation factor VIII (FVIII) is mediated by several receptors (Saenko E. et al, 1999; Lenting P. et al, 1999 and Sarafanov A. et al, 2001) and one of them was proposed to be low-density lipoprotein receptor (LDLR) (Bovenschen N. et al, 2005). Besides FVIII, the major ligands of LDLR are plasma lipoproteins. The ligand-binding portion of LDLR is represented by a cluster of seven complement-type repeats. Each repeat forms a structurally autonomous domain, which are connected with flexible linkers. Two and five adjacent CRs were shown to form sites for the lipoproteins ApoE and ApoB, respectively, and the site for FVIII has not been determined. Objective.As a part of elucidation of mechanisms of FVIII clearance, our goal was to identify the FVIII-binding region on LDLR. Experimental Approach.Our experimental strategy was based on generation of recombinant CR-doublets that systematically overlap the CR cluster in LDLR (Fig. 1) and testing their interactions with FVIII. The proteins were expressed in insect cells using a baculovirus system and tested for structural integrity by circular dichroism. The functional properties of the fragments were assessed by their binding with alpha-2-macroglobulin receptor-associated protein (RAP), a universal ligand of a family of receptors that LDLR belongs to. The binding studies were performed using surface plasmon resonance technique. Using this assay, the LDLR fragments were further tested for interaction with different FVIII preparations, such as plasma-derived FVIII and recombinant FVIII: Advate (full-size FVIII) and Xyntha (B-domain deleted FVIII). The specificity of these interactions was tested in a competitive binding assay using an anti-FVIII single-chain variable antibody fragment KM33 known to inhibit binding FVIII to LDLR (Limburg V. et al, 2005). Also, the specificity of the interaction of LDLR fragments and FVIII was tested upon mutating the fragments by targeting conservative tryptophanes, one residue per selected CR. Upon expressing the mutants, their structural adequacy to the respective wild-type forms was confirmed by circular dichroism. Results.Three overlapping CR doublets were found active for binding with all FVIII preparations (Fig. 2). In presence of increased concentrations of KM33 and upon site-directed mutagenesis of the LDLR fragments, this binding was diminished confirming its specificity. Conclusions.The binding site of LDLR for FVIII is formed by a region spanned by the complement-type repeats from second to fifth. We proposed a tentative model of the interaction (Fig. 3), in which this region of the receptor contacts the A3 and C1 domains of FVIII. These portions of FVIII were previously shown to be involved in interaction with another FVIII clearance receptor known as LDLR-related protein (Bovenschen N. et al, 2003 and Meems H. et al, 2011). [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The molecular basis for high affinity of a universal ligand for human bombesin receptor (BnR) family members

There is increased interest in the Bn-receptor family because they are frequently over/ectopically expressed by tumors and thus useful as targets for imaging or receptor-targeted-cytotoxicity. The synthetic Bn-analog, [d-Tyr6, β-Ala11, Phe13, Nle14]Bn(6–14) [Univ.Lig] has the unique property of having high affinity for all three human BNRs (GRPR, NMBR, BRS-3), and thus could be especially useful for this approach. However, the molecular basis of this property is unclear and is the subject of this study. To accomplish this, site-directed mutagenesis was used after identifying potentially important amino acids using sequence homology analysis of all BnRs with high affinity for Univ.Lig compared to the Cholecystokinin-receptor (CCKAR), which has low affinity. Using various criteria 74 amino acids were identified and 101 mutations made in GRPR by changing each to those of CCKAR or to alanine. 22 GRPR mutations showed a significant decrease in affinity for Univ.Lig (>2-fold) with 2 in EC2[D97N, G112V], 1 in UTM6[Y284A], 2 in EC4[R287N, H300S] showing >10-fold decrease in Univ.Lig affinity. Additional mutations were made to explore the molecular basis for these changes. Our results show that high affinity for Univ.Lig by human Bn-receptors requires positively charged amino acids in extracellular (EC)-domain 4 and to a lesser extent EC2 and EC3 suggesting charge-charge interactions may be particularly important for determining the general high affinity of this ligand. Furthermore, transmembrane amino acids particularly in UTM6 are important contributing both charge-charge interactions as well as interaction with a tyrosine residue in close proximity suggesting possible receptor–peptide cation–π or H-bonding interactions are also important for determining its high affinity.

Orthogonal biofunctionalization of magnetic nanoparticles via “clickable” poly(ethylene glycol) silanes: a “universal ligand” strategy to design stealth and target-specific nanocarriers

The present work demonstrates a novel strategy to synthesize orthogonally bio-engineered magnetonanohybrids (MNPs) through the design of versatile, biocompatible linkers whose structure includes: (i) a robust anchor to bind with metal-oxide surfaces; (ii) tailored surface groups to act as spacers and (iii) a general method to implement orthogonal functionalizations of the substrate via “click chemistry”. Ligands that possess the synthetic generality of features (i)–(iii) are categorized as “universal ligands”. Herein, we report the synthesis of a novel, azido-terminated poly(ethylene glycol) (PEG) silane that can easily self-assemble on MNPs through hetero-condensation between surface hydroxyl groups and the silane end of the ligand, and simultaneously provide multiple clickable sites for high density, chemoselective bio-conjugation. To establish the universal-ligand-strategy, we clicked alkyl-functionalized folate onto the surface of PEGylated MNPs. By further integrating a near-infrared fluorescent (NIRF) marker (Alexa-Fluor 647) with MNPs, we demonstrated their folate-receptor mediated internalization inside cancer cells and subsequent translocation into lysosomes and mitochondria. Ex vivo NIRF imaging established that the azido-PEG-silane developed in course of the study can effectively reduce the sequestration of MNPs by macrophage organs (viz. liver and spleen). These folate–PEG-MNPs were not only stealth and noncytotoxic but their dual optical and magnetic properties aided in tracking their whereabouts through combined magnetic resonance and optical imaging. Together, these results provided a strong motivation for the future use of the “universal ligand” strategy towards development of “smart” nanohybrids for theragnostic applications.

High-Throughput Screen for the Chemical Inhibitors of Antiapoptotic Bcl-2 Family Proteins by Multiplex Flow Cytometry

The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.