Modulation Of Affinity Research Articles

Modulation of the binding affinity of naproxen to bovine serum albumin by conversion of the drug into amino acid ester salts

Naproxen (NAP) is one of the most widely prescribed non-steroidal anti-inflammatory drugs. Novel formulations of NAP aiming at better water-solubility, dosage, and the onset of action or new routes of application in order to minimize or prevent side effects of NAP are in the current interest of the pharmaceutical industry. Here, we report the synthesis, chemical, spectral and physicochemical characterization of a series of salts containing cations amino acid alkyl esters (AAE) and NAP anion, which potentially can be used as novel drug formulation. The [l-AAE][NAP] were obtained in three steps: preparation of the AAE hydrochlorides, neutralization of the hydrochlorides to the corresponding AAE, and formation of the target organic salts. All NAP derivatives, tested at a concentration as high as 100 μM, exhibited no toxicity against murine macrophage cell line (RAW 264.7). The binding parameters and stoichiometry of [AAE][NAP]s to bovine serum albumin (BSA) are in the range of that estimated for the parent NAP. Only l-valine isopropyl ester naproxenate characterizes with about one order of magnitude lower binding affinity for BSA, which suggests a faster diffusion rate in the circulatory system than the parent NAP and other derivatives, and therefore faster reach to the target system. Using molecular modeling seven binding pockets of BSA were probed for their suitability to binds the cation and the anion and results are discussed in correlation with the obtained thermodynamic parameters for the binding of NAP derivatives to BSA.

Agent-Based Modeling of T Cell Receptor Cooperativity.

Immunological synapse (IS) formation is a key event during antigen recognition by T cells. Recent experimental evidence suggests that the affinity between T cell receptors (TCRs) and antigen is actively modulated during the early steps of TCR signaling. In this work, we used an agent-based model to study possible mechanisms for affinity modulation during IS formation. We show that, without any specific active mechanism, the observed affinity between receptors and ligands evolves over time and depends on the density of ligands of the antigen peptide presented by major histocompatibility complexes (pMHC) and TCR molecules. A comparison between the presence or absence of TCR–pMHC centrally directed flow due to F-actin coupling suggests that centripetal transport is a potential mechanism for affinity modulation. The model further suggests that the time point of affinity measurement during immune synapse formation is critical. Finally, a mathematical model of F-actin foci formation incorporated in the agent-based model shows that TCR affinity can potentially be actively modulated by positive/negative feedback of the F-actin foci on the TCR-pMHC association rate kon.

Application of the Gyrolab microfluidic platform to measure picomolar affinity of a PD-L1-binding Adnectin™ radioligand forpositron emission tomography.

Advances in in vitro display and protein engineering yield therapeutics with affinities in the picomolar range. The Gyrolab® microfluidics platform uses the kinetic exclusion assay principle to measure subnanomolar solution affinities. This work describes application of the Gyrolab solution affinity module and the new multi-curve analysis feature to determine affinity of the PD-L1 Adnectin™ positron emission tomography radioligand, which was measured as20pM for human PD-L1. We also report key parameters that affect assay signal-to-background ratio and data quality, such as detection reagent concentration. Gyrolab offers the necessary throughput for rapid assay development with low sample consumption, as demonstrated in this study, whichalso provides helpful tips for assay optimization for solution affinity measurement.

Modulation of neuronal cell affinity of composite scaffolds based on polyhydroxyalkanoates and bioactive glasses

The biocompatibility and neuron regenerating properties of various bioactive glass (BG)/polyhydroxyalkanoate (PHA) blend composites were assessed in order to study their suitability for peripheral nerve tissue applications, specifically as lumen structures for nerve guidance conduits. BG/PHA blend composites were fabricated using Bioactive glass® 45 S5 (BG1) and BG 1393 (BG2) with the 25:75 poly(3-hydroxyoctanoate/poly3-hydroxybutyrate), 25:75 P(3HO)/P(3HB) blend (PHA blend). Various concentrations of each BG (0.5 wt%, 1.0 wt% and 2.5 wt%) were used to determine the effect of BG on neuronal growth and differentiation, in single culture using NG108-15 neuronal cells and in a co-culture along with RN22 Schwann cells. NG108-15 cells exhibited good growth and differentiation on all the PHA blend composites showing that both BGs have good biocompatibility at 0.5 wt%, 1.0 wt% and 2.5 wt% within the PHA blend. The Young’s modulus values displayed by all the PHA blend/BG composites ranged from 385.6 MPa to 1792.6 MPa, which are able to provide the required support and protective effect for the regeneration of peripheral nerves. More specifically, the tensile strength obtained in the PHA blend/BG1 (1.0 wt%) (10.0 ± 0.6 MPa) was found to be similar to that of the rabbit peroneal nerve. This composite also exhibited the best biological performance in supporting growth and neuronal differentiation among all the substrates. The neurite extension on this composite was found to be remarkable with the neurites forming a complex connection network.

Modulation of Hb-O2 affinity to improve hypoxemia in COVID-19 patients.

This opinion paper aims at discussing the potential impact of modulating the Hb-O2 affinity by the nutritional supplement 5-HMF on patients affected by COVID-19. The paper describes the critical role of the oxygen affinity in hypoxemic COVID-19 patients and the potential positive effect of 5-HMF, a compound shown to increase the Hb-O2 affinity.

Effects of an allosteric hemoglobin affinity modulator on arterial blood gases and cardiopulmonary responses during normoxic and hypoxic low-intensity exercise.

Numerous pathophysiological conditions induce hypoxemia-related cardiopulmonary perturbations, decrements in exercise capacity, and debilitating symptoms. Accordingly, this study investigated the efficacy of an allosteric hemoglobin modulator (voxelotor) to enhance arterial oxygen saturation during low-intensity exercise in hypoxia. Eight normal healthy subjects (36 ± 7 yr; 73.8 ± 9.5 kg; 3 women) completed a submaximal cycling test (60 W) under normoxic ([Formula: see text]: 0.21; O2 partial pressure: 144 mmHg) and hypoxic ([Formula: see text]: 0.125; O2 partial pressure: 82 mmHg) conditions before (day 1) and after (day 15) 14 days of oral drug administration. While stationary on a cycle ergometer and during exercise, ratings of perceived exertion (RPE) and dyspnea, oxygen consumption (V̇o2), and cardiac output (Q) were measured noninvasively, while arterial blood pressure (MAP) and blood gases ([Formula: see text], [Formula: see text], and [Formula: see text]) were measured invasively. The 14-day drug administration left shifted the oxygen-hemoglobin dissociation curve (ODC; p50 measured at standard pH and Pco2; day 1: 28.0 ± 2.1 mmHg vs. day 15: 26.1 ± 1.8 mmHg, P < 0.05). RPE, dyspnea, V̇o2, Q, and MAP were not different between day 1 and day 15. [Formula: see text] was similar during normoxia on day 1 and day 15 while stationary but higher during exercise (day 1: 95.2 ± 0.4% vs. day 15: 96.6 ± 0.3%, P < 0.05). [Formula: see text] was higher during hypoxia on day 15 while stationary (day 1: 82.9 ± 3.4% vs. day 15: 90.9 ± 1.8%, P < 0.05) and during exercise (day 1: 73.6 ± 2.5% vs. day 15: 84.8 ± 2.7%, P < 0.01). [Formula: see text] and [Formula: see text]were systematically higher and lower, respectively, after drug (P < 0.01), while the alveolar-arterial oxygen difference was unchanged suggesting hyperventilation contributed to the rise in [Formula: see text]. Oral administration of voxelotor left shifted the ODC and stimulated a mild hyperventilation, leading to improved arterial oxygen saturation without altering V̇o2 and central hemodynamics during rest and low-intensity exercise. This effect was more pronounced during submaximal hypoxic exercise, when arterial desaturation was more evident. Additional studies are needed to determine the effects of voxelotor during maximal exercise and under chronic forms of hypoxia.NEW & NOTEWORTHY In humans, a novel allosteric hemoglobin-oxygen affinity modulator was administered to comprehensively examine the cardiopulmonary consequences of stabilizing a portion of the available hemoglobin in a high-oxygen affinity state during submaximal exercise in normoxia and hypoxia. Oral administration of voxelotor enhanced arterial oxygen saturation during submaximal exercise without altering oxygen consumption and central hemodynamics; however, the partial pressure of arterial carbon dioxide was reduced and the partial pressure of arterial oxygen was increased implying that hyperventilation also contributed to the increase in oxygen saturation. The preservation of arterial oxygen saturation and content was particularly evident during hypoxic submaximal exercise, when arterial desaturation typically occurs, but this did not influence arterial-venous oxygen difference.

CIAN: Cross-Image Affinity Net for Weakly Supervised Semantic Segmentation

Weakly supervised semantic segmentation with only image-level labels saves large human effort to annotate pixel-level labels. Cutting-edge approaches rely on various innovative constraints and heuristic rules to generate the masks for every single image. Although great progress has been achieved by these methods, they treat each image independently and do not take account of the relationships across different images. In this paper, however, we argue that the cross-image relationship is vital for weakly supervised segmentation. Because it connects related regions across images, where supplementary representations can be propagated to obtain more consistent and integral regions. To leverage this information, we propose an end-to-end cross-image affinity module, which exploits pixel-level cross-image relationships with only image-level labels. By means of this, our approach achieves 64.3% and 65.3% mIoU on Pascal VOC 2012 validation and test set respectively, which is a new state-of-the-art result by only using image-level labels for weakly supervised semantic segmentation, demonstrating the superiority of our approach.

Pharmacological characterization of a novel, orally available GluN2B antagonist JNJ‐63612445

IntroductionBlockade of GluN2B (NR2B)‐containing NMDA receptors has been proposed as a therapy for a number of neurological and psychiatric diseases including mood disorders. A number of GluN2B antagonists have been identified and developed over the last three decades. These compounds, however, have significant limitations in regard to their selectivity or drug‐like properties (e.g. oral bioavailability).MethodsThe pharmacology of JNJ‐63612445 was explored using in‐vitro assays (calcium mobilization, competitive radioligand binding, electrophysiology), ex‐vivo slice autoradiography and in‐vivo electrophysiology (synaptic transmission and plasticity) and behavior.ResultsJNJ‐63612445 is a high affinity (pKi=8.0), potent (pIC50=7.8) and selective negative allosteric modulator of GluN2B receptors. Upon oral dosing, the compound occupied GluN2B receptors in rat hippocampus in time‐ and concentration‐dependent manner, reaching 50% occupancy at the plasma concentration of 443 ng/ml. JNJ‐63612445 at 1 μM blocked 54% of NMDA receptor‐mediated EPSC in rat neonatal slices similar to other GluN2B antagonists. The compounds at the dose 10 mg/kg s.c. inhibited in vivo long‐term depression measured in the CA1 of anesthetized rats without effects on the basal synaptic transmission. Given orally at the dose 30 mg/kg in mice, the compound significantly reduces immobility time in a tail‐suspension test, while occupying approximately 89% of the GluN2B receptors in the hippocampus.ConclusionsJNJ‐63612445 represents an example of new generation of potent, selective, and orally available GluN2B antagonists.Support or Funding InformationFunding informationAuthors are full time employees of Janssen Research &amp; Development, LLC. The research was funded by the company.

Cytochrome c Oxidase Subunit 4 Isoform Exchange Results in Modulation of Oxygen Affinity.

Cytochrome c oxidase (COX) is regulated through tissue-, development- or environment-controlled expression of subunit isoforms. The COX4 subunit is thought to optimize respiratory chain function according to oxygen-controlled expression of its isoforms COX4i1 and COX4i2. However, biochemical mechanisms of regulation by the two variants are only partly understood. We created an HEK293-based knock-out cellular model devoid of both isoforms (COX4i1/2 KO). Subsequent knock-in of COX4i1 or COX4i2 generated cells with exclusive expression of respective isoform. Both isoforms complemented the respiratory defect of COX4i1/2 KO. The content, composition, and incorporation of COX into supercomplexes were comparable in COX4i1- and COX4i2-expressing cells. Also, COX activity, cytochrome c affinity, and respiratory rates were undistinguishable in cells expressing either isoform. Analysis of energy metabolism and the redox state in intact cells uncovered modestly increased preference for mitochondrial ATP production, consistent with the increased NADH pool oxidation and lower ROS in COX4i2-expressing cells in normoxia. Most remarkable changes were uncovered in COX oxygen kinetics. The p50 (partial pressure of oxygen at half-maximal respiration) was increased twofold in COX4i2 versus COX4i1 cells, indicating decreased oxygen affinity of the COX4i2-containing enzyme. Our finding supports the key role of the COX4i2-containing enzyme in hypoxia-sensing pathways of energy metabolism.

The Role of HCN Channel Helices D and E in the Modulation of Camp Affinity

Hyperpolarization-activated cyclic-nucleotide regulated (HCN) channels are non-selective cation channels controlling key physiological functions such as cardiac pacemaking and repetitive neuronal firing. They are activated by voltage and modulated by binding of cAMP to their cytoplasmic C-terminal region named cyclic nucleotide binding domain (CNBD). Altered cAMP responses caused either by single point mutations in the protein or by non-physiological levels of the cyclic nucleotide in the cell lead to pathological conditions both in the heart and in the nervous system. The recently obtained cryo-EM structure of human HCN1 in the bound and unbound forms, confirms previous NMR studies on cAMP-induced conformational changes and reveals the presence of two additional helices at the C-terminus of the CNBD which fold in the presence of cAMP. We have previously shown, by ITC and patch clamp, that deletion of these helices (D and E) decreases the affinity for cAMP in the most studied HCN isoforms (HCN1, HCN2 and HCN4). Here we introduced single point mutations in HCN4 isoform, designed either to delete the single E helix or to selectively perturb the folding of the D helix, with the aim to dissect the role of the single D and E helices in this regulatory mechanism. Moreover, different truncations of the D helix were analyzed in order to clarify its role in the folding of the preceding C helix, known to be a key element in the transition of the CNBD from the unbound to the bound state. The patch clamp experiments were performed in whole cell configuration using a new all-in-one integrated amplifier, the ePatch.

A Role of Cholesterol in Modulating the Binding of α-Synuclein to Synaptic-Like Vesicles.

α-Synuclein (αS) is a presynaptic protein whose aggregation is associated with Parkinson’s disease (PD). Although the physiological function of αS is still unclear, several lines of evidence indicate that this protein may play a role in the trafficking of synaptic vesicles (SVs) during neurotransmitter release, a task associated with its ability to bind SVs and promote their clustering. It is therefore crucial to identify the cellular factors that modulate this process. To address this question, using nuclear magnetic resonance (NMR) spectroscopy we have characterized the role of cholesterol, a major component of the membrane of SVs, in the binding of αS with synaptic-like vesicles. Our results indicate that cholesterol can act as a modulator of the overall affinity of αS for SVs by reducing the local affinity of the region spanning residues 65–97 in the non-amyloid-β component (NAC) of the protein. The increased population of bound states that expose the region 65–97 to the solvent was found to induce stronger vesicle-vesicle interactions by αS. These results provide evidence that cholesterol modulates the clustering of synaptic vesicles induced by (α)S, and supports the role of the disorder-to-order equilibrium of the NAC region in the modulation of the biological properties of the membrane-bound state of αS.

Antibody Structure and Function: The Basis for Engineering Therapeutics.

Antibodies and antibody-derived macromolecules have established themselves as the mainstay in protein-based therapeutic molecules (biologics). Our knowledge of the structure–function relationships of antibodies provides a platform for protein engineering that has been exploited to generate a wide range of biologics for a host of therapeutic indications. In this review, our basic understanding of the antibody structure is described along with how that knowledge has leveraged the engineering of antibody and antibody-related therapeutics having the appropriate antigen affinity, effector function, and biophysical properties. The platforms examined include the development of antibodies, antibody fragments, bispecific antibody, and antibody fusion products, whose efficacy and manufacturability can be improved via humanization, affinity modulation, and stability enhancement. We also review the design and selection of binding arms, and avidity modulation. Different strategies of preparing bispecific and multispecific molecules for an array of therapeutic applications are included.

The CC\u2032 loop of IgV domains of the immune checkpoint receptors, plays a key role in receptor:ligand affinity modulation

Antibodies targeting negative regulators of immune checkpoints have shown unprecedented and durable response against variety of malignancies. While the concept of blocking the negative regulators of the immune checkpoints using mAbs appears to be an outstanding approach, their limited effect and several drawbacks, calls for the rational design of next generation of therapeutics. Soluble isoforms of the negative regulators of immune checkpoint pathways are expressed naturally and regulate immune responses. This suggests, affinity-modified versions of these self-molecules could be effective lead molecules for immunotherapy. To obtain better insights on the hotspot regions for modification, we have analysed structures of 18 immune receptor:ligand complexes containing the IgV domain. Interestingly, this analysis reveals that the CC′ loop of IgV domain, a loop which is distinct from CDRs of antibodies, plays a pivotal role in affinity modulation, which was previously not highlighted. It is noteworthy that a ~5-residue long CC′ loop in a ~120 residue protein makes significant number of hydrophobic and polar interactions with its cognate ligand. The post-interaction movement of CC′ loop to accommodate the incoming ligands, seems to provide additional affinity to the interactions. In silico replacement of the CC′ loop of TIGIT with that of Nectin-2 and PVR followed by protein docking trials suggests a key role of the CC′ loop in affinity modulation in the TIGIT/Nectin pathway. The CC′ loop appears to be a hotspot for the affinity modification without affecting the specificity to their cognate receptors.

Differential Efficacy of Anti-Sickling and Anti-Inflammatory Mechanisms in a Fluorescent Intravital Microscopy Dorsal Skinfold Vaso-Occlusion Model in Sickle Cell Disease Townes Mice

Sickle cell disease (SCD) is characterized by acute and repetitive vaso-occlusive crises (VOC). These crises have been hypothesized to occur when blood flow is reduced following obstruction of sickle-shaped red blood cells in the vasculature. However, it is now well established that inflammation, oxidative stress, endothelial activation and pro-coagulation in sickle cell disease patients also contribute to the formation of heterocellular aggregates that can lead to VOC (Vercellotti and Belcher, 2014). Transgenic SCD mice recapitulate the pathology of human disease in response to stimuli such as heme injection and hypoxia/reoxygenation. SCD SS Townes mice, which express human α and sickle γAβS globins, AA Townes mice expressing normal human α and normal γAβA globins and heterozygous AS mice which express only one allele of the γAβS sickle gene were used. To characterize vaso-occlusion in these mice and evaluate the efficacy of different pharmacological mechanisms, we modified the skinfold vaso-occlusion model (Kalambur et al, 2004) using fluorescent intravital microscopy to visualize blood flow occlusion following hemin injection or hypoxia/reoxygenation challenge. Dorsal skinfold chambers were implanted and24 hours post-surgery mice were injected with FITC-dextran for visualization of flowing blood vessels. Skinfold bearing mice were then subjected to hemin treatment (16 μmoles/kg) or hypoxia (7%; 1 hour)/reoxygenation (1 hour) followed by the injection of Alexa fluor 647-labeled albumin to allow quantification of occluded vessels through dual fluorescent image analysis. Following hemin injection, SS mice showed significant ~30% vaso-occlusion in comparison to AA mice with ~8%, whereas the AS mice showed an intermediate phenotype with ~20% vaso-occlusion. Hypoxia/reoxygenation challenge also resulted in significant vaso-occlusion for SS mice (~25%) whereas only 5% was observed in AA mice. Interestingly, AS mice also showed a significant amount of vaso-occlusion (~25%) similar to SS mice when challenged with hypoxia/reoxygenation. Although no sickling can be observed in an ex vivo sickling assay using AS red blood cells, an intermediate amount of free Hemoglobin (Hb) can be detected in the plasma of these mice and rolling can be observed. This suggests that these vaso-occlusive models relate more on the inflammatory and endothelial activation state and are independent of the sickling potential of the red blood cell. We then used our model with hypoxia/reoxygenation challenge to evaluate the effects of dimethyl fumarate (DMF, 15 mpk BID), an anti-P-Selectin antibody (150ug/mouse) and the covalent hemoglobin oxygen affinity modulator GBT-440 (300 mpk). As anti-inflammatory agents, DMF and Anti-P-Selectin significantly reduced vaso-occlusion in SS mice by ~60% compared to the vehicle treated mice, but GBT-440 did not inhibit vaso-occlusion at a dose where a significant reduction in p50 was observed. In conclusion, our data have shown that obstruction of blood flow in the skinfold vaso-occlusion model in SCD Townes mice reflects the vascular inflammatory state of the disease and is independent of the ex vivo capacity of red blood cell to sickle. Disclosures Sturtevant: Sanofi: Employment. Macias-Garcia:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment. van der Flier:Sanofi: Employment. Hicks:Sanofi: Employment. Demers:Sanofi: Employment.

New role for an old molecule: The 2,3-diphosphoglycerate case

BackgroundAerobic organisms have to overcame the dangerous species derived from the unquestionable favorable effects due to the utilization of oxygen in the cellular respiration. 2,3-Diphosphoglycerate (DPG) could be one of the molecules able to perform different role inside the cells and (from the data obtained from our experimental work) may help cellular components, in particular hemoglobin, to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS). MethodsTherefore, we have investigated the kinetic and antioxidant properties of this molecule against the main biological reactive species and the protective role of this molecules on hemoglobin treated with strong oxidant. ResultsDPG, at the physiological concentration is able to scavenge hydroxyl radical, peroxyl radical, cation radicals and to chelate iron in the reduced state. Moreover it is able to avoid oxidation of iron inside the hemoglobin following treatment with nitrite and tert-butyl hydroperoxide (t-BOOH). On the other side, it is not able to protect membrane components from oxidative burning. This different behavior towards radical species is probably linked to the polarity of the molecule and also the high levels of charged groups present on the surface of DPG, that avoid the possibility to act in an environment almost completely hydrophobic, as inside the membrane, where reactive species produce the main damages during the reactions of peroxidation. ConclusionsThis is the first paper dealing with the potential role of DPG not only as a modulator of oxygen affinity in hemoglobin, but also as a scavenger of radicals.

Surface affinity modulation of MoS2 by hydrothermal synthesis and its intermediary function in interfacial chemistry

This paper reported a simple hydrothermal method to prepare MoS2 with different surface affinity. By adjusting the reaction time and Mo/S proportion of hydrothermal synthesis, MoS2 powder with a face-to-edge ratio of 1.19 can be obtained on the surface. Interestingly, for the first time, MoS2 with such ratio can function as an intermediary similar to surfactant. It tends to distribute on the interface between water and n-hexane, and further adsorb methylene blue from water to the interface. By this route, it has a great implication for the interaction of incompatible molecules and the interfacial process in future applications.

S147 RESULTS FROM THE RANDOMIZED PLACEBO‐CONTROLLED PHASE 3 HOPE TRIAL OF VOXELOTOR IN ADULTS AND ADOLESCENTS WITH SICKLE CELL DISEASE

Background:Sickle cell disease (SCD) is an inherited disorder caused by a single amino acid substitution in the β‐chain of hemoglobin (Hb) resulting in the production of sickle hemoglobin (HbS). When deoxygenated, HbS polymerizes, leading to red blood cell sickling and damage. This results in a triad of clinical features (anemia, hemolysis, and vaso‐occlusion), which contribute to the acute and chronic manifestations of SCD. These long‐term complications contribute to the decreased quality of life and reduced life expectancy observed in patients with SCD. Voxelotor is an oral, once‐daily hemoglobin‐oxygen affinity modulator designed to inhibit HbS polymerization, thus improving anemia and reducing hemolysis. The randomized phase 3 HOPE trial (NCT03036813) evaluates the efficacy and safety of voxelotor in patients with SCD aged 12 to 65 years.Aims:To present the results of the pre‐specified Part A analysis of the first approximately 150 randomized patients in the HOPE trial.Methods:Patients with SCD (HbSS, HbSC, HbSβ0 thalassemia, or other variants), Hb ≥5.5 and ≤10.5 g/dL, and between 1 and 10 vaso‐occlusive crises in the prior 12 months were eligible. Concurrent hydroxyurea was allowed if the dose had been stable for ≥90 days. Patients were randomly assigned to receive voxelotor 1500 mg/day, 900 mg/day, or placebo for at least 24 weeks. The primary endpoint was the proportion of patients with a &gt;1.0‐g/dL increase in Hb from baseline at week 24. Secondary endpoints included change from baseline to week 24 in measures of hemolysis (absolute and percent reticulocyte counts, indirect bilirubin levels, and lactate dehydrogenase levels) and safety.Results:154 patients were included in the preliminary Part A analysis; median age was 25 years (range, 12–59), and 42% were male. Most patients were HbSS/HbSβ0: 92% (1500 mg), 94% (900 mg), and 90% (placebo). Hydroxyurea use at study entry was 62% (1500 mg), 67% (900 mg), and 64% (placebo), and median baseline Hb was 8.6 g/dL (1500 mg; range, 5.9–10.8), 8.3 g/dL (900 mg; range, 6.3–10.8), and 8.5 g/dL (placebo; range, 6.1–10.4). At week 24, the proportion of patients with a &gt;1.0‐g/dL increase in Hb from baseline was significantly higher for both voxelotor 1500 mg (65%; P &lt; 0.0001) and 900 mg (33%; P = 0.0159) compared with placebo (10%) (Figure). The mean change in Hb from baseline to week 24 was 1.5 g/dL with 1500 mg, 0.6 g/dL with 900 mg, and 0 g/dL with placebo. Consistent with improvement in Hb, voxelotor also resulted in concordant improvements in measures of hemolysis (Table). Overall, the treatment‐emergent adverse events (TEAEs) were similar across all treatment arms except for diarrhea, which was higher with voxelotor (1500 mg, 21%; 900 mg, 19%) compared with placebo (10%). The majority of TEAEs were grade 1 or 2 in severity. The efficacy and safety data from the full patient population of the phase 3 HOPE trial (N = 274) will be presented.Summary/Conclusion:Voxelotor treatment demonstrated a dose‐dependent increase in Hb, with the majority of patients on voxelotor 1500 mg achieving a &gt;1.0‐g/dL improvement in Hb from baseline to week 24. In addition, there was a dose‐dependent decrease in measures of hemolysis with voxelotor. Furthermore, voxelotor was generally well tolerated. These results suggest that voxelotor has the potential to be disease‐modifying by improving anemia and reducing hemolysis and their associated morbidity and mortality.image

Imatinib-Induced Changes in Protein Expression and ATP-Binding Affinities of Kinases in Chronic Myelocytic Leukemia Cells.

Kinases are crucial components in numerous cell signaling pathways. Aberrant expression and activation of protein kinases are known to be accompanied by many types of cancer, and more than 30 small-molecule kinase inhibitors have been approved by the Food and Drug Administration (FDA) for cancer chemotherapy. Biological and clinical applications of small-molecule kinase inhibitors require comprehensive characterizations about how these inhibitors modulate the protein expression and activities of kinases at the entire proteome scale. In this study, we developed a parallel-reaction monitoring (PRM)-based targeted proteomic method to monitor the alterations in protein expression of kinases in K-562 chronic myelocytic leukemia (CML) cells elicited by treatment with imatinib, an ABL kinase inhibitor approved by the FDA for CML treatment. By employing isotope-coded ATP affinity probes together with liquid chromatography-multiple-reaction monitoring (LC-MRM) analysis, we also examined the modulation of the ATP-binding affinities of kinases induced by imatinib treatment. The results revealed profound increases in protein expression levels of a large number of kinases in K-562 cells upon treatment with imatinib, which is accompanied by substantial decreases in ATP-binding capacities of many kinases. Apart from ABL kinases, we identified a number of other kinases whose ATP-binding affinities are markedly diminished upon imatinib treatment, including CHK1, a checkpoint kinase involved in DNA damage response signaling. Together, our targeted quantitative proteomic methods enabled, for the first time, dual assessments of small-molecule kinase inhibitor-induced changes in protein expression and ATP-binding affinities of kinases in live cells.

Toward reducing biomaterial antigenic potential: a miniaturized Fc-binding domain for local deposition of antibodies.

A peptide derived from staphylococcal protein A (SpA) was developed as an affinity module for antibody delivery applications. The miniaturized protein consists of the first helix of the engineered SpA Z domain fused with the self-assembling peptide (SAP) AEAEAKAKAEAEAKAK, or EAK. The resulting peptide, named Z15_EAK, was shown to possess fibrillization properties and an Fc-binding function. The peptide induced a red shift in the Congo red absorbance characteristic of peptide fibrils, also evidenced in transmission electron microscopy images. The one-site binding affinity (Kd) of a gel-like coacervate generated by admixing Z15_EAK with EAK for IgG was determined to be 1.27 ± 0.14 μM based on a microplate-based titration assay. The coacervate was found to localize IgG subcutaneously in mouse footpads for 8 to 28 days. A set of in vivo data was fit to a one-compartment model for simulating the relative fractions of IgG dissociated from the materials in the depot. The model predicted that close to 27% of the antibodies injected were available unbound for the duration of the experiment. Z15_EAK did not appear to induce innate immune responses; injecting Z15_EAK into mouse footpads elicited neither interleukin-6 (IL-6) nor tumor necrosis factor-alpha (TNF-α) from splenocytes isolated from the animals one day, seven days, or eleven days afterward. The antigenic potential of Z15 was analyzed using a bioinformatic approach in predicting sequences in SpA and Z15 dually presented by class I and class II human MHC alleles covering the majority of the population. A peptide in SpA identified as a potential T cell epitope cross reacting with a known epitope in a microbial antigen was eliminated by miniaturization. These results demonstrate that Z15_EAK is a potential platform for generating antibody depots by which the impacts of Fc-based biotherapeutics can be enhanced through spatiotemporal control.

Differential Recognition of Various Anions Utilizing Aromatic CH Hydrogen Bonding

Dihydrogen phosphate selective anion receptors 1, 2, and 3 that utilize amide NH, and CH for anion recognition have been designed and synthesized. For 1 and 2, aromatic CH hydrogen bonding element is present on naphthyl rings (H a). In addition, for receptor 1, this aromatic hydrogen bonding element is further polarized by the nitro groups, which would enhance the hydrogen bonding ability. Partial charges derived from Natural Population Analysis also supported the existence of this polarization. That is, positive partial charge of H a in receptor 1 was more positive than that in receptor 2. As expected from the partial charge values, receptor 1 consistently showed highest affinity among three receptors, followed by receptor 2 regardless of the anions tested in this study. Therefore, we have shown that affinity modulation of a receptor was possible by changing the nature of the aromatic CH hydrogen. In addition, the receptors showed selectivity toward dihydrogen phosphate among various anions including acetate and chloride.