High Ligand Concentrations Research Articles

Effects of oleylamine concentration on the synthesis of formamidinium lead halide perovskite nanocrystals and physical-optical properties

Perovskite nanocrystals (PNCs) has been extensively interested owing to their distinctive properties for applications in optoelectronics and energy harvesting. The properties of these nanocrystals, including optical and energy characteristics, can be tuned by adjusting the particle size using different synthesis techniques. Among these, the ligand-assisted reprecipitation (LARP) method has become popular for its simplicity and scalability. Nevertheless, it is vital to understand that the growth of PNCs is extremely sensitive to the conditions of synthesis, highlighting the importance of recognizing the factors that limit the formation and properties of PNCs. In this study, PNCs based on formamidinium lead bromide (FAPbBr3) were synthesized via the LARP method under room temperature and ambient atmospheric conditions. The structures and optical properties, including photoluminescence lifetime, of PNCs with varying amounts of organic ligands were investigated. Transmission electron microscopy showed that high concentrations of organic ligands lead to the formation of perovskite clusters. We also noted a slight red shift in the photoluminescence peak as the size of the PNCs increased. A peak photoluminescence quantum yield (PLQY) of 74% was achieved. This study provides crucial insights into the effects of ligand ratios and serves as a valuable resource for refining the synthesis parameters of PNCs.

Uptake of Cm (III) and Eu (III) by C–S–H phases under saline conditions in presence of EDTA: A batch sorption and TRLFS study

Eu (III) and Cm (III) uptake by calcium silicate hydrate phases (C–S–H) was investigated in presence of EDTA in NaCl and CaCl2 solutions. Different experimental parameters, i.e., ionic strength (0.1 m ≤ I'm ≤ 5.05 m), ligand concentration (10−5 m ≤ [EDTA] ≤ 10−2 m), calcium-to-silicon ratio (0.6 ≤ C/S ≤ 1.3) and sorption time (7 d ≤ t ≤ 365 d) were varied in the frame of batch sorption experiments and Time Resolved Laser Fluorescence spectroscopy (TRLFS) measurements. No effect of EDTA on the retention of Eu(III)/Cm(III) by C–S–H phases in NaCl or CaCl2 systems was observed at ligand concentrations ≤ 10−3 M. In NaCl solutions with [EDTA] = 10−2 M and C/S < 1.3, low retention (log Rd = 2–3, with Rd in L∙kg−1) of Eu(III) was detected after 7 d of sorption time, while strong retention (log Rd = 5–6) was observed after 50 d. This behaviour was explained by the initial stabilization of Eu(III)/Cm(III) in the aqueous phase due to the formation of two aqueous Eu(III)/Cm(III)-(OH)n-EDTA complexes, followed by the slow incorporation of Eu(III)/Cm(III) into the C–S–H structure. In CaCl2 solutions for all C/S ratios, as well as in NaCl solutions for C/S ∼ 1.3, the presence of [EDTA] = 10−2 M led to a significant decrease of the uptake (log Rd = 2–3) after 7 and 50 d of contact time. This effect was explained by the formation of stable aqueous Ca–Eu(III)/Cm(III)-EDTA complexes triggered by the presence of moderate to high Ca concentrations. No evident effect caused by increased ionic strength conditions could be confirmed in our sorption experiments.Results obtained in batch sorption experiments are underpinned by TRLFS data, with the observation of three main aqueous species, tentatively defined as Cm(OH)(EDTA)2-, Cm(OH)x (EDTA)-(x+1) and Ca–Cm(III)-EDTA, as well as a fourth species corresponding to Cm(III) incorporated in the CaO-layer of the C–S–H phases. In spite of the thermodynamic stability of the CaEDTA2− complex, which reduces the concentration of free EDTA, the formation of ternary Ca–Eu(III)/Cm(III)-EDTA complexes is expected to result in a significant impact of EDTA on the uptake of An (III)/Ln (III) by cement at high ligand concentrations. The assumption that Ca outcompetes actinides for the complexation with EDTA in cementitious systems may need to be revisited under these conditions.

Potassium Hexamethyldisilazide (KHMDS): Solvent-Dependent Solution Structures.

Solution structures of potassium hexamethyldisilazide [KHMDS] and labeled [15N]KHMDS were examined using a number of analytical methods including 29Si NMR spectroscopy and density functional theory computations. A combination of 15N-29Si couplings, 29Si chemical shifts, and the method of continuous variations reveals dimers, monomers, and ion pairs. Weakly coordinating monofunctional ligands such as toluene, N,N-dimethylethylamine, and Et3N afford exclusively dimers. 1,3-Dioxolane, THF, dimethoxyethane, hexamethylphosphoramide, and diglyme provide dimers at low ligand concentrations and monomers at high ligand concentrations. N,N,N',N'-Tetramethylethylenediamine and N,N,N',N'-tetramethylcyclohexanediamine provide exclusively dimers at all ligand concentrations at ambient temperatures and significant monomer at -80 °C. Studies of 12-crown-4 ran into technical problems. Equimolar 15-crown-5 forms a dimer, whereas excess 15-crown-5 affords a putative ion pair. Whereas equimolar 18-crown-6 also affords a dimer, an excess provides a monomer rather than a solvent-separated ion pair. [2.2.2]cryptand affords what is believed to be a contact-ion-paired cryptate. Solvation was probed using largely density functional theory (DFT) computations. Thermally corrected energies are consistent with lower aggregates and higher solvates at low temperatures, but the magnitudes of the computed temperature dependencies were substantially larger than the experimentally derived data.

Uranium(VI) hydrolysis up to 250 °C and its geological implications

The hydrolysis of uranium(VI) has been investigated by solubility experiments conducted on sodium uranate (Na2U2O7) over a pHT range of 4.5–8 at 200 and 250 °C. Equilibrium constants for the UO2OH+, UO2OH20, UO2OH3- and UO2OH42- complexes have all been derived at both temperatures. Alongside this investigation a comprehensive literature review has been conducted to document previous investigations into U(VI) hydrolysis at temperatures above 25 °C. Using the new data reported here alongside those available in the literature, modified Ryzhenko-Bryzgalin equation of state parameters have been generated for the UO2OH+, UO2OH20, UO2OH3- and UO2OH42- complexes along with the polynuclear UO22OH22+ and UO23OH5+ complexes. This permits future works to extrapolate thermodynamic properties for these complexes to pressure–temperature conditions beyond those for which experimental data are available. Results reported here highlight the potential inadequacies of using the Ryzhenko-Bryzgalin and revised Helgeson-Kirkham-Flowers equations of state to extrapolate thermodynamic properties solely from room temperature data, with discrepancies ranging over 2–6 orders of magnitude being observed between extrapolations and the equilibrium constants experimentally derived at 200/250 °C. Using the newly derived properties, we report the results of thermodynamic calculations of uranium solubility and aqueous speciation under hydrothermal conditions relevant to a range of geological systems. These calculations suggest that uranyl hydroxide complexes may be responsible for uranium transport in a wide range of crustal fluids potentially obviating the need for high ligand concentrations.

Extracellular domain mutations of the EGF receptor differentially modulate high-affinity and low-affinity responses to EGF receptor ligands

The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants--R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK, but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called ‘high’ affinity biological responses arise from activation by isolated receptor dimers, whereas ‘low’ affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.

Exploration of commercial cyclen-based chelators for mercury-197m/g incorporation into theranostic radiopharmaceuticals.

A comprehensive investigation of the Hg2+ coordination chemistry and 197m/gHg radiolabeling capabilities of cyclen-based commercial chelators, namely, DOTA and DOTAM (aka TCMC), along with their bifunctional counterparts, p-SCN-Bn-DOTA and p-SCN-Bn-TCMC, was conducted to assess the suitability of these frameworks as bifunctional chelators for the 197m/gHg2+ theranostic pair. Radiolabeling studies revealed that TCMC and DOTA exhibited low radiochemical yields (0%-6%), even when subjected to harsh conditions (80°C) and high ligand concentrations (10-4M). In contrast, p-SCN-Bn-TCMC and p-SCN-Bn-DOTA demonstrated significantly higher 197m/gHg radiochemical yields (100% ± 0.0% and 70.9% ± 1.1%, respectively) under the same conditions. The [197m/gHg]Hg-p-SCN-Bn-TCMC complex was kinetically inert when challenged against human serum and glutathione. To understand the differences in labeling between the commercial chelators and their bifunctional counterparts, non-radioactive natHg2+ complexes were assessed using NMR spectroscopy and DFT calculations. The NMR spectra of Hg-TCMC and Hg-p-SCN-Bn-TCMC suggested binding of the Hg2+ ion through the cyclen backbone framework. DFT studies indicated that binding of the Hg2+ ion within the backbone forms a thermodynamically stable product. However, competition can form between isothiocyanate binding and binding through the macrocycle, which was experimentally observed. The isothiocyanate bound coordination product was dominant at the radiochemical scale as, in comparison, the macrocycle bound product was seen at the NMR scale, agreeing with the DFT result. Furthermore, a bioconjugate of TCMC (TCMC-PSMA) targeting prostate-specific membrane antigen was synthesized and radiolabeled, resulting in an apparent molar activity of 0.089 MBq/nmol. However, the complex demonstrated significant degradation over 24h when exposed to human serum and glutathione. Subsequently, cell binding assays were conducted, revealing a Ki value ranging from 19.0 to 19.6nM. This research provides crucial insight into the effectiveness of current commercial chelators in the context of 197m/gHg2+ radiolabeling. It underscores the necessity for the development of specific and customized chelators to these unique "soft" radiometals to advance 197m/gHg2+ radiopharmaceuticals.

Growth and Dispersion Control of SnO2 Nanocrystals Employing an Amino Acid Ester Hydrochloride in Solution Synthesis: Microstructures and Photovoltaic Applications.

Tin oxide (SnO2) is a technologically important semiconductor with versatile applications. In particular, attention is being paid to nanostructured SnO2 materials for use as a part of the constituents in perovskite solar cells (PSCs), an emerging renewable energy technology. This is mainly because SnO2 has high electron mobility, making it favorable for use in the electron transport layer (ETL) in these devices, in which SnO2 thin films play a role in extracting electrons from the adjacent light-absorber, i.e., lead halide perovskite compounds. Investigation of SnO2 solution synthesis under diverse reaction conditions is crucial in order to lay the foundation for the cost-effective production of PSCs. This research focuses on the facile catalyst-free synthesis of single-nanometer-scale SnO2 nanocrystals employing an aromatic organic ligand (as the structure-directing agent) and Sn(IV) salt in an aqueous solution. Most notably, the use of an aromatic amino acid ester hydrochloride salt-i.e., phenylalanine methyl ester hydrochloride (denoted as L hereafter)-allowed us to obtain an aqueous precursor solution containing a higher concentration of ligand L, in addition to facilitating the growth of SnO2 nanoparticles as small as 3 nm with a narrow size distribution, which were analyzed by means of high-resolution transmission electron microscopy (HR-TEM). Moreover, the nanoparticles were proved to be crystallized and uniformly dispersed in the reaction mixture. The environmentally benign, ethanol-based SnO2 nanofluids stabilized with the capping agent L for the Sn(IV) ions were also successfully obtained and spin-coated to produce a SnO2 nanoparticle film to serve as an ETL for PSCs. Several SnO2 ETLs that were created by varying the temperature of nanoparticle synthesis were examined to gain insight into the performance of PSCs. It is thought that reaction conditions that utilize high concentrations of ligand L to control the growth and dispersion of SnO2 nanoparticles could serve as useful criteria for designing SnO2 ETLs, since hydrochloride salt L can offer significant potential as a functional compound by controlling the microstructures of individual SnO2 nanoparticles and the self-assembly process to form nanostructured SnO2 thin films.

The evolutionary divergence of receptor guanylyl cyclase C has implications for preclinical models for receptor-directed therapeutics

Mutations in receptor guanylyl cyclase C (GC-C) cause severe gastrointestinal disease, including meconium ileus, early onset acute diarrhea, and pediatric inflammatory bowel disease that continues into adulthood. Agonists of GC-C are FDA-approved drugs for the treatment of constipation and irritable bowel syndrome. Therapeutic strategies targeting GC-C are tested in preclinical mouse models assuming that murine GC-C mimics human GC-C in its biochemical properties and downstream signaling events. Here, we reveal important differences in ligand binding affinity and guanylyl cyclase activity between mouse GC-C and human GC-C. We generated a series of chimeric constructs of various domains of human and mouse GC-C to show that the extracellular domain of mouse GC-C contributed to log-orders lower affinity of mouse GC-C for ligands when compared to human GC-C. Further, the Vmax of the murine guanylyl cyclase domain was lower than that of human GC-C, and allosteric regulation of the receptor by ATP binding to the intracellular kinase-homology domain also differed. These altered properties are reflected in the high concentrations of ligands required to elicit signaling responses in the mouse gut in preclinical models and the specificity of a guanylyl cyclase inhibitor towards human GC-C. Therefore, our studies identify considerations in using the murine model to test molecules for therapeutic purposes that work as either agonists or antagonists of GC-C, and vaccines for the bacterial heat-stable enterotoxin that causes watery diarrhea in humans.

Strategies for Mitigating Commercial Sensor Chip Variability with Experimental Design Controls.

Surface plasmon resonance (SPR) is a popular real-time technique for the measurement of binding affinity and kinetics, and bench-top instruments combine affordability and ease of use with other benefits of the technique. Biomolecular ligands labeled with the 6xHis tag can be immobilized onto sensing surfaces presenting the Ni2+-nitrilotriacetic acid (NTA) functional group. While Ni-NTA immobilization offers many advantages, including the ability to regenerate and reuse the sensors, its use can lead to signal variability between experimental replicates. We report here a study of factors contributing to this variability using the Nicoya OpenSPR as a model system and suggest ways to control for those factors, increasing the reproducibility and rigor of the data. Our model ligand/analyte pairs were two ovarian cancer biomarker proteins (MUC16 and HE4) and their corresponding monoclonal antibodies. We observed a broad range of non-specific binding across multiple NTA chips. Experiments run on the same chips had more consistent results in ligand immobilization and analyte binding than experiments run on different chips. Further assessment showed that different chips demonstrated different maximum immobilizations for the same concentration of injected protein. We also show a variety of relationships between ligand immobilization level and analyte response, which we attribute to steric crowding at high ligand concentrations. Using this calibration to inform experimental design, researchers can choose protein concentrations for immobilization corresponding to the linear range of analyte response. We are the first to demonstrate calibration and normalization as a strategy to increase reproducibility and data quality of these chips. Our study assesses a variety of factors affecting chip variability, addressing a gap in knowledge about commercially available sensor chips. Controlling for these factors in the process of experimental design will minimize variability in analyte signal when using these important sensing platforms.

Liquid tumor microenvironment enhances WNT signaling pathway of peritoneal metastasis of gastric cancer

Gastric cancer remains one of the most prevalent tumors worldwide and peritoneal metastasis is responsible for approximately 60% of death in advanced gastric cancer patients. However, the underlying mechanism of peritoneal metastasis is poorly understood. We have established organoids derived from malignant ascites (MA) of gastric cancer patients and noticed that MA supernatant could strongly increase the colony formation of organoids. Thus, we realized the interaction between exfoliated cancer cells (ECCs) and liquid tumor microenvironment contributes to peritoneal metastasis. Further, we designed a medium component control test which proved that exosomes derived from MA could not enhance the growth of organoids. Using Immunofluorescence and confocal imaging as well as dual-luciferase reporter assay, our data showed WNT signaling pathway was upregulated by high concentrations of WNT ligands (wnt3a and wnt5a), which was verified by ELISA. Besides, suppressing WNT signaling pathway diminished the growth promoting function of MA supernatant. This result implicated WNT signaling pathway as a potential therapeutic target for peritoneal metastasis of gastric cancer.

Folding of Staphylococcal Nuclease Induced by Binding of Chemically Modified Substrate Analogues Sheds Light on Mechanisms of Coupled Folding/Binding Reactions.

Several proteins have been shown to undergo a shift in the mechanism of ligand binding-induced folding from conformational selection (CS; folding precedes binding) to induced fit (IF; binding precedes folding) with increasing ligand concentration. In previous studies of the coupled folding/binding reaction of staphylococcal nuclease (SNase) in the presence of a substrate analogue, adenosine-3',5'-diphosphate (prAp), we found that the two phosphate groups make important energetic contributions toward stabilizing its complex with the native protein as well as transient conformational states encountered at high ligand concentrations favoring IF. However, the structural contributions of each phosphate group during the reaction remain unclear. To address this question, we relied on fluorescence, nuclear magnetic resonance (NMR), absorption, and isothermal titration calorimetry to study the effects of deletion of the phosphate groups of prAp on the kinetics of ligand-induced folding, using a strategy analogous to mutational ϕ-value analysis to interpret the results. Kinetic measurements over a wide range of ligand concentrations, together with structural characterization of a transient protein-ligand encounter complex using 2D NMR, indicated that, at high ligand concentrations favoring IF, (i) the 5'-phosphate group interacts weakly with denatured SNase during early stages of the reaction, resulting in loose docking of the two domains of SNase, and (ii) the 3'-phosphate group engages in some specific contacts with the polypeptide in the transition state prior to formation of the native SNase-prAp complex.

Enhancing response of a protein conformational switch by using two disordered ligand binding domains.

Introduction: Protein conformational switches are often constructed by fusing an input domain, which recognizes a target ligand, to an output domain that establishes a biological response. Prior designs have employed binding-induced folding of the input domain to drive a conformational change in the output domain. Adding a second input domain can in principle harvest additional binding energy for performing useful work. It is not obvious, however, how to fuse two binding domains to a single output domain such that folding of both binding domains combine to effect conformational change in the output domain. Methods: Here, we converted the ribonuclease barnase (Bn) to a switchable enzyme by duplicating a C-terminal portion of its sequence and appending it to its N-terminus, thereby establishing a native fold (OFF state) and a circularly permuted fold (ON state) that competed for the shared core in a mutually exclusive fashion. Two copies of FK506 binding protein (FKBP), both made unstable by the V24A mutation and one that had been circularly permuted, were inserted into the engineered barnase at the junctions between the shared and duplicated sequences. Results: Rapamycin-induced folding of FK506 binding protein stretched and unfolded the native fold of barnase via the mutually exclusive folding effect, and rapamycin-induced folding of permuted FK506 binding protein stabilized the permuted fold of barnase by the loop-closure entropy principle. These folding events complemented each other to turn on RNase function. The cytotoxic switching mechanism was validated in yeast and human cells, and in vitro with purified protein. Discussion: Thermodynamic modeling and experimental results revealed that the dual action of loop-closure entropy and mutually exclusive folding is analogous to an engine transmission in which loop-closure entropy acts as the low gear, providing efficient switching at low ligand concentrations, and mutually exclusive folding acts as the high gear to allow the switch to reach its maximum response at high ligand concentrations.

Design, Synthesis, and Pharmacological Evaluation of Benzimidazolo-thiazoles as Potent CXCR3 Antagonists with Therapeutic Potential in Autoimmune Diseases: Discovery of ACT-672125.

The chemokine receptor CXCR3 allows the selective recruitment of innate and adaptive inflammatory immune cells into inflamed tissue. CXCR3 ligands are secreted after exposure to pro-inflammatory cytokines. Upon binding to CXCR3 ligands, CXCR3 expressing T-lymphocytes migrate toward sites of inflammation and can promote tissue damage. Therefore, antagonizing this receptor may provide clinical benefits for patients suffering from autoimmune diseases characterized by high concentrations of CXCR3 ligands. Herein, we report the second part of our CXCR3 discovery program where we explored the benzimidazolo-thiazole core scaffold. The optimization of potency and the mitigation of an hERG liability are described. Further pharmacokinetic considerations led to the identification of the potent CXCR3 antagonist ACT-672125 (29). The compound showed good physicochemical properties and safety profile. In a proof-of-mechanism model of lung inflammation, ACT-672125 inhibited the recruitment of CXCR3 expressing T cells into the inflamed lung in a dose-dependent manner.

Intermittent ERK oscillations downstream of FGF in mouse embryonic stem cells.

ABSTRACTSignal transduction networks generate characteristic dynamic activities to process extracellular signals and guide cell fate decisions such as to divide or differentiate. The differentiation of pluripotent cells is controlled by FGF/ERK signaling. However, only a few studies have addressed the dynamic activity of the FGF/ERK signaling network in pluripotent cells at high time resolution. Here, we use live cell sensors in wild-type and Fgf4-mutant mouse embryonic stem cells to measure dynamic ERK activity in single cells, for defined ligand concentrations and differentiation states. These sensors reveal pulses of ERK activity. Pulsing patterns are heterogeneous between individual cells. Consecutive pulse sequences occur more frequently than expected from simple stochastic models. Sequences become more prevalent with higher ligand concentration, but are rarer in more differentiated cells. Our results suggest that FGF/ERK signaling operates in the vicinity of a transition point between oscillatory and non-oscillatory dynamics in embryonic stem cells. The resulting heterogeneous dynamic signaling activities add a new dimension to cellular heterogeneity that may be linked to divergent fate decisions in stem cell cultures.

Abstract P201: Zenocutuzumab is an effective HER2/HER3 Biclonics® antibody in cancers with NRG1 fusions

Abstract Neuregulin 1 (NRG1) fusion proteins are oncogenic drivers in multiple cancer types, including non-small cell lung cancer, pancreas adenocarcinoma, and other solid tumors. NRG1 fusion proteins bind to HER3 and signal through HER2/HER3 heterodimers, leading to oncogenic transformation. Zenocutuzumab is an HER2/HER3 humanized IgG1 bispecific antibody that specifically and potently blocks NRG1 fusion-driven signaling of the heterodimeric complex. Zenocutuzumab binds via its anti-HER2 monovalent Fab arm to domain I of HER2. Docking of zenocutuzumab in this position increases the effective affinity of the anti-HER3 Fab arm, which binds to domain 3, a critical epitope that blocks binding of the HER3 ligand NRG1. Consequently, zenocutuzumab displaces NRG1 binding to HER3, even at high ligand concentrations, keeping HER3 in an inactive state and blocking HER2/HER3 heterodimerization and NRG1-mediated signaling. The clinical efficacy and safety of zenocutuzumab were recently demonstrated in patients with NRG1 fusion-driven cancers who were enrolled in the ongoing global multicenter eNRGy study and a global early access program NCT02912949 (J Clin Oncol 39;15_suppl; abstract 3003). Treatment with zenocutuzumab led to rapid and major radiologic tumor regression and biomarker responses in heavily pretreated patients with multiple NRG1 fusion-positive cancers with several different N-terminal fusion partners. Zenocutuzumab has been enhanced for antibody-dependent cell-mediated cytotoxicity (ADCC) to recruit and bolster immune effector cells to aid in tumor elimination. Here we explored the different Fc-mediated immune effector functions of zenocutuzumab. In several cancer cell lines, zenocutuzumab induced Fc-mediated dose-dependent ADCC and antibody-dependent cellular phagocytosis. Insertion of ATP1B1-NRG1 and SLC3A2-NRG1 into immortalized pancreatic ductal epithelial cells (H6c7) led to activation of the HER3 oncogenic signaling pathways including AKT. The H6c7-SLC3A2-NRG1 cells formed tumors when implanted in the subcutaneous flank of immune compromised mice (after about 10 weeks). Furthermore, a dependency of the transformed cell on this oncogene for growth and survival was also observed. Zenocutuzumab effectively inhibited proliferation of these NRG1 fusion-expressing, transformed cells. In addition, proteomic analysis showed that zenocutuzumab inhibited the HER3 and AKT-related downstream signaling pathways. In conclusion, these data provide strong mechanistic evidence supporting the clinical efficacy of zenocutuzumab in patients with NRG1 fusion-driven cancers. Citation Format: Jan Gerlach, Igor Odintsov, Ron Schackmann, Marc Ladanyi, Jeroen Lammerts van Bueren, Romel Somwar, Cecile Geuijen. Zenocutuzumab is an effective HER2/HER3 Biclonics® antibody in cancers with NRG1 fusions [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P201.

Fabrication of Flexible Co‐salen Integrated Polymers for Hydration of Epoxides and Alkynes via Cooperative Activation

AbstractMost cross‐linked polymers with integrated active sites in the network have poor flexibility, which inhibits their applications in catalysis involving a cooperative activation pathway. Herein, a flexible organic polymer with high concentration of salen ligand in the network (SOP) was successfully synthesized by the reaction of meso‐1,2‐diphenylethylenediamine and 1,3,5‐tris(3’‐tert‐butyl‐4’‐hydroxy‐5’‐formylphenyl)benzene. The post‐synthesis coordination with CoII followed by further oxidation results in the formation of SOP−Co−OTs. The large amounts uptake of benzene of SOP and variation of the BET surface area after coordination with different metal cations confirmed the network flexibility. Consequently, SOP−Co−OTs could efficiently catalyze the epoxides hydration to afford turnover frequency up to 2400 h−1 via cooperative activation. Furthermore, SOP−Co−OTs works cooperatively with Amberlyst‐15 in the hydration of phenylacetylene to afford higher yield than homogeneous Co‐salen. Our primary results suggest the potential applications of flexible polymer catalysts in reactions involving cooperative activation pathway.

Understanding Symmetry Breaking at the Single-Particle Level via the Growth of Tetrahedron-Shaped Nanocrystals from Higher-Symmetry Precursors.

The vast majority of single crystalline metal nanoparticles adopt shapes in the Oh point group as a consequence of the symmetry of the underlying face-centered cubic (FCC) crystal lattice. Tetrahedra are a notable exception to this rule, and although they have been observed in several syntheses, their growth mechanism, and the symmetry-reduction process that necessarily characterizes it, is poorly understood. Here, a symmetry breaking mechanism is revealed by in situ liquid flow cell transmission electron microscopy (TEM) observation of seeded growth in which tetrahedra nanoparticles are formed from higher symmetry seeds. Real-time observation of the growth demonstrates a kinetically driven pathway during which rhombic dodecahedra nanoparticles transition to tetrahedra through tristetrahedra intermediates, with an accompanying surface facet evolution from {110} to {111} via {hhl} (where h > l), respectively. On the basis of these data, we propose a mechanism that relies on a rapid loss of inversion symmetry in the initial stages of the reaction, followed by differential reactivity of tips vs faces under conditions of relatively high supersaturation and moderate ligand concentration. The application of these insights to ex situ synthesis conditions allowed for an improved yield of tetrahedra nanoparticles. This work sheds an important mechanistic light on the crystallographic underpinnings of nanoparticle shape and symmetry transformations and highlights the importance of single-particle characterization tools for monitoring nanoscale phenomena.

Bioaffinity Screening with a Rapid and Sample-Efficient Autosampler for Native Electrospray Ionization Mass Spectrometry.

Fast and efficient handling of ligands and biological targets are required in bioaffinity screening based on native electrospray ionization mass spectrometry (ESI-MS). We use a prototype microfluidic autosampler, called the "gap sampler", to sequentially mix and electrospray individual small molecule ligands together with a target protein and compare the screening results with data from thermal shift assay and surface plasmon resonance. In a first round, all three techniques were used for a screening of 110 ligands against bovine carbonic anhydrase II, which resulted in five mutual hits and some false positives with ESI-MS presumably due to the high ligand concentration or interferences from dimethyl sulfoxide. In a second round, 33 compounds were screened in lower concentrations and in a less complex matrix, resulting in only true positives with ESI-MS. Within a cycle time of 30 s, dissociation constants were determined within an order of magnitude accuracy consuming only 5 pmol of ligand and less than 15 pmol of protein per screened compound. In a third round, dissociation constants of five compounds were accurately determined in a titration experiment. Thus, the gap sampler can rapidly and efficiently be used for high-throughput screening.

Impact of the mouse model and molar amount of injected ligand on the tissue distribution profile of PSMA radioligands

PurposeVarious preclinical study designs are described in the literature for the evaluation of PSMA radioligands. In this study, [177Lu]Lu-Ibu-DAB-PSMA, an albumin-binding radioligand, and [177Lu]Lu-PSMA-617 were investigated and compared under variable experimental conditions.MethodsIn vitro cell uptake studies were performed with PC-3 PIP and LNCaP tumor cells using a range of molar concentrations (0.75–500 nM) of both radioligands. Biodistribution and SPECT/CT imaging studies were carried out with the respective tumor mouse models using 0.05 nmol and 1.0 nmol injected ligand per mouse.ResultsIn both tumor cell lines, the uptake of the radioligands was increased when using low molar concentrations of the respective ligand. The observed saturation effect at high ligand concentrations was more pronounced for LNCaP cells that express PSMA at lower levels than for PC-3 PIP cells. At all investigated timepoints, the in vivo uptake of both radioligands was higher in PC-3 PIP tumors than in LNCaP tumors. A low molar amount of injected ligand increased the PC-3 PIP tumor uptake mainly for [177Lu]Lu-Ibu-DAB-PSMA; however, the molar amount of ligand was relevant for both radioligands when using LNCaP tumors. Renal retention of both radioligands was, however, up to fourfold higher during the first hours after application of a low ligand amount compared to the high ligand amount.ConclusionThe results of this preclinical study underline the relevance of the tumor model and applied ligand amount for the characterization of PSMA radioligands. The application of equal preclinical study designs is crucial to allow the comparison of novel radioligands with existing ones and, thus, predict potential advantages of new radioligands in view of a clinical application.

Abstract 957: Zenocutuzumab: An antibody that can overcome HER3 mediated HRG signalling in tumor cells by docking on HER2

Abstract HER2-driven cancers require phosphatidylinositide-3 kinase (PI3K)/Akt signaling through HER3 to promote tumor growth and survival. The HER2/HER3 complex is most active in the presence of heregulin (HRG) and upregulation of HRG is one of the key resistance mechanisms after TKI treatment. Zenocutuzumab is an HER2/HER3 bispecific antibody that specifically and potently blocks HRG-driven signaling of the heterodimeric complex. Zenocutuzumab has demonstrated clinical activity in patients with advanced cancer where the HRG-driven HER2/HER3 pathway is active [Ref].Zenocutuzumab was discovered through a large unbiased screen with HER2×HER3 Biclonics® (bAbs). Zenocutuzumab specifically blocks HRG-induced signaling of HER2:HER3 but not of HER2:HER4, EGFR:HER2 or EGFR:HER3 heterodimers. Zenocutuzumab demonstrates an in vitro potency superior to other high affinity anti-HER2 and anti-HER3 antibodies in cells stimulated with high concentrations of HRG, thereby overcoming one of the resistance mechanisms of current HER2 therapies. The potent activity of Zenocutuzumab is based in-part on its binding to domain I of HER2. Docking of Zenocutuzumab in this position increases the effective affinity of the anti-HER3 Fab arm. As a result, Zenocutuzumab displaces HRG binding to HER3, even at high ligand concentrations, keeping HER3 in an inactive state and blocking HRG-mediated HER2/HER3 signaling. In binding experiments on cells expressing different levels of HER2 and HER3, HER2:HER3 ratios point to the anti-HER2 Fab binding arm being the main driver of Zenocutuzumab avidity. In an in vivo orthotopic breast cancer xenograft model that used Micro-PET imaging, the tumor penetration of a variant of Zenocutuzumab is HER2-dependent despite the high subnanomolar affinity of the HER3 Fab arm for its receptor. Gamma-counter quantification of radioactivity confirmed that antibody levels in the tumors with the HER2xHER3 bAb were 2.5-fold higher than for the parental HER3 mAb. Zenocutuzumab reduced tumor growth in an in vivo orthotopic pancreatic cancer xenograft model that expresses HRG. The brain is one of the organs with high HRG concentrations to which many tumors metastasize. Mice bearing intracranial HER2-amplified breast cancer cells presented at the end of the treatment period 100% survival when treated with MCLA-128, in contrast to 38% survival when treated with HER2 antibody drug conjugate T-DM1.Conclusion: Zenocutuzumab uses a Dock &amp; Block™ mechanism for potent and specific tumor targeting and simultaneous inhibition of HRG-driven proliferation of HER2-amplified tumors, even at supramaximal concentrations of HRG. Citation Format: Cecile Geuijen, David Maussang-Detaille, Tristan Gallenne, Linda Hendriks, Arjen Kramer, Jan Gerlach, Mark Throsby, John de Kruif. Zenocutuzumab: An antibody that can overcome HER3 mediated HRG signalling in tumor cells by docking on HER2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 957.