A Pharmacological perspective on the temporal properties of sweeteners.

Several non-caloric sweeteners exhibit a delay in sweetness onset and a sweetness linger after sampling. These temporal properties are thought to be the result of non-specific interactions with cell membranes and proteins in the oral cavity. Data and analysis presented in this report also support the potential involvement of receptor affinity and binding kinetics to this phenomenon. In general, affected sweeteners exhibit distinctly higher binding affinity compared to carbohydrate sweeteners, which do not have temporal issues. In addition, binding kinetic simulations illustrate much slower receptor binding association and dissociation kinetics for a set of non-caloric sweeteners presenting temporal issues, in comparison to carbohydrate sweeteners. So, the higher affinity of some non-caloric sweeteners, dictating lower use levels, and affecting binding kinetics, could contribute to their delay and linger in sweetness perception. Simple pharmacology principles could explain, at least in part, some of the temporal issues of sweeteners.

Safety and efficacy of a novel anti-CD19 chimeric antigen receptor T cell product targeting a membrane-proximal domain of CD19 with fast on- and off-rates against non-Hodgkin lymphoma: a first-in-human study.

BACKGROUND: Commercial anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are efficacious against advanced B-cell non-Hodgkin lymphoma (NHL); however, most patients ultimately relapse. Several mechanisms contribute to this failure, including CD19-negative escape and CAR T dysfunction. All four commercial CART19 products utilize the FMC63 single-chain variable fragment (scFv) specific to a CD19 membrane-distal epitope and characterized by slow association (on) and dissociation (off) rates. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 and that is characterized by faster on- and off-rates could mitigate CART19 failure and improve clinical efficacy. METHODS: We developed an autologous CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218). This antibody is specific to a membrane-proximal CD19 epitope and harbors faster on/off rates compared to FMC63. We tested h1218-CART19 in vitro and in vivo using FMC63-CART19-resistant models. We conducted a first-in-human multi-center phase I clinical trial to test AT101 (clinical-grade h1218-CART19) in patients with relapsed or refractory (r/r) NHL. RESULTS: Preclinically, h1218- but not FMC63-CART19 were able to effectively eradicate lymphomas expressing CD19 point mutations (L174V and R163L) or co-expressing FMC63-CAR19 as found in patients relapsing after FMC63-CART19. Furthermore, h1218-CART19 exhibited enhanced killing of B-cell malignancies in vitro and in vivo compared with FMC63-CART19. Mechanistically, we found that h1218-CART19 had reduced activation-induced cell death (AICD) and enhanced expansion compared to FMC63-CART19 owing to faster on- and off-rates. Based on these preclinical results, we performed a phase I dose-escalation trial, testing three dose levels (DL) of AT101 (the GMP version of h1218) using a 3 + 3 design. In 12 treated patients (7 DLBCL, 3 FL, 1 MCL, and 1 MZL), AT101 showed a promising safety profile with 8.3% grade 3 CRS (n = 1) and 8.3% grade 4 ICANS (n = 1). In the whole cohort, the overall response rate was 91.7%, with a complete response rate of 75.0%, which improved to 100% in DL-2 and -3. AT101 expansion correlates with CR and B-cell aplasia. CONCLUSIONS: We developed a novel, safe, and potent CART19 product that recognizes a membrane-proximal domain of CD19 with fast on- and off-rates and showed significant efficacy and promising safety in patients with relapsed B-cell NHL. TRIAL REGISTRATION: NCT05338931; Date: 2022-04-01.

Tuning ensemble-averaged cargo run length via fractional change in mean kinesin number.

The number of motors carrying cargos in biological cells is not well-defined, instead varying from cargo to cargo about a statistical mean. Predictive understanding of motility in cells therefore requires quantitative insights into mixed ensembles of cargos. Toward this goal, here we employed Monte Carlo simulations to investigate statistical ensembles of cargos carried by a Poisson-distributed number of motors. Focusing on the key microtubule-based motor kinesin-1, our simulations utilized experimentally determined single-kinesin characteristics and alterations in kinesin's on- and off-rates caused by cellular factors and/or physical load. We found that a fractional increase in mean kinesin number enhances the ensemble-averaged cargo run length and amplifies run-length sensitivity to changes in single-kinesin on-rate and off-rate. These tuning effects can be further enhanced as solution viscosity increases over the range reported for cells. Together, our data indicate that the physiological range of kinesin number sensitively tunes the motility of mixed cargo populations. These effects have rich implications for quantitative and predictive understanding of cellular motility and its regulation.

A Novel Method for Designing General Window Functions with Flexible Spectral Characteristics.

In the field of sensor signal processing, windows are time-/frequency-domain weighting functions that are widely applied to reduce the well-known Gibbs oscillations. Conventional methods generally control the spectral characteristics of windows by adjusting several of the parameters of closed-form expressions. Designers must make trade-offs among the mainlobe width (MW), the peak sidelobe level (PSL), and sometimes the sidelobe fall-off rate (SLFOR) of windows by carefully adjusting these parameters. Generally, not all sidelobes need to be suppressed in specified applications. In this paper, a novel method, i.e., the inverse of the shaped output using the cyclic algorithm (ISO-CA), for designing window functions with flexible spectral characteristics is proposed. Simulations are conducted to test the effectiveness, flexibility and versatility of the method. Some experiments based on real measured data are also presented to demonstrate the practicability. The results show that the window functions generated using the cyclic algorithm (CA) yield better performance overall than the windows of conventional methods, achieving a narrower MW, a lower PSL, and a controllable SLFOR. In addition, steerable sidelobes over specified regions can be acquired both easily and flexibly while maintaining the original properties of the initial window as much as possible.

Inter-domain Synergism Is Required for Efficient Feeding of Cellulose Chain into Active Site of Cellobiohydrolase Cel7A.

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase.

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases.

Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (P(Intr)) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a (14)C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site -3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils.

Building homogeneous time-resolved fluorescence resonance energy transfer assays for characterization of bivalent inhibitors of an inhibitor of apoptosis protein target.

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is a central apoptosis regulator that blocks cell death by inhibiting caspase-3, caspase-7, and caspase-9 via binding interactions with the XIAP BIR2 and BIR3 domains (where BIR is baculovirus IAP repeat). Smac protein, in its dimeric form, effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. Here we describe the development of highly sensitive homogeneous time-resolved fluorescence resonance energy transfer (HTRF) assays to measure binding affinities of potent bivalent peptidomimetic inhibitors of XIAP. Our results indicate that these assays can differentiate Smac-mimetic inhibitors with a wide range of binding affinities down to the picomolar range. Furthermore, we demonstrate the utility of these fluorescent tools for characterization of inhibitor off-rates, which as a crucial determinant of target engagement and cellular potency is another important parameter to guide optimization in a structure-based drug discovery effort. Our study also explores how increased inhibitor valency can lead to enhanced potency at multimeric proteins such as IAP.

Forces and dynamics of glucose and inhibitor binding to sodium glucose co-transporter SGLT1 studied by single molecule force spectroscopy.

Single molecule force spectroscopy was employed to investigate the dynamics of the sodium glucose co-transporter (SGLT1) upon substrate and inhibitor binding on the single molecule level. CHO cells stably expressing rbSGLT1 were probed by using atomic force microscopy tips carrying either thioglucose, 2'-aminoethyl ß-d-glucopyranoside, or aminophlorizin. Poly(ethylene glycol) (PEG) chains of different length and varying end groups were used as tether. Experiments were performed at 10, 25 and 37 °C to address different conformational states of SGLT1. Unbinding forces between ligands and SGLT1 were recorded at different loading rates by changing the retraction velocity, yielding binding probability, width of energy barrier of the binding pocket, and the kinetic off rate constant of the binding reaction. With increasing temperature, width of energy barrier and average life time increased for the interaction of SGLT1 with thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations.

Residence time and kinetic efficiency analysis of extracellular signal-regulated kinase 2 inhibitors.

The RAS/RAF/MEK/ERK signal transduction cascade plays an important role in the regulation of critical cellular processes such as cell proliferation, migration, and differentiation. The up-regulation of this pathway can negatively affect cell homeostasis and is responsible for the development of various forms of cancer and inflammation processes. Therefore, there is a strong interest in pursuing drug programs targeting some of the enzymes involved in this pathway. In addition to the determination of Ki, Kd, IC50, and/or EC50, a more thorough kinetic analysis can provide useful information for the selection of the best lead series during the early stage of the drug discovery process. This study describes a medium-throughput fluorescent probe displacement assay for the rapid determination of the k(off) constant, residence time, and kinetic efficiency for ERK (extracellular signal-regulated kinase) inhibitors. Using this method, we have identified several inhibitors that we have subjected to further kinetic analysis by comparing k(off) constants determined for these time-dependent inhibitors using either the active or inactive form of ERK2.

Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step.

Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the "affinity maturation" step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold.

Exploring monocyclic core: Discovery of pyrrol-2-one derivatives as a new series of potent MCHR1 antagonists with in vivo efficacy.

With an objective to improve the profiles of the 1st generation non-basic MCHR1 antagonists, a lean design approach of replacing the bicyclic thienopyrimidine core with a monocyclic pyrrol-2-one chemotype was examined in the context of reducing aromatic ring count, while also contemplating enhanced flexibility as a means of decreasing flat character. The new compounds exhibited potent antagonism up to the sub-nanomolar range, thereby implying that the monocyclic ring could effectively serve as an effective bioisostere of the bicyclic system. The prototype compound 2m offered benefits like improved potency, reduced half-life, and enhanced solubility, while also demonstrating >5% reduction in weight gain in rats, thereby providing proof-of-concept for this new class of compounds as anti-obesity agents.

HDL-Associated Proteins in Subjects with Polycystic Ovary Syndrome: A Proteomic Study.

INTRODUCTION: Serum lipoproteins, with the exception of high-density lipoprotein cholesterol (HDL-C), are increased in polycystic ovary syndrome (PCOS) and their levels may reflect the associated obesity and insulin resistance, but the nature of this association is not fully explained. Therefore, proteomic analysis of key proteins in lipoprotein metabolism was performed. METHODS: In this cohort study, plasma was collected from 234 women (137 with PCOS and 97 controls without PCOS). Somalogic proteomic analysis was undertaken for the following 19 proteins involved in lipoprotein, and particularly HDL, metabolism: alpha-1-antichymotrypsin; alpha-1-antitrypsin; apolipoproteins A-1, B, D, E, E2, E3, E4, L1, and M; clusterin; complement C3; hemopexin; heparin cofactor II; kininogen-1; serum amyloid A-1; amyloid beta A-4; and paraoxonase-1. RESULTS: The levels of apolipoprotein E were higher in PCOS (p = 0.012). However, the other isoforms of ApoE, ApoE2, E3, and E4, did not differ when compared with controls. ApoM was lower in PCOS (p = 0.000002). Complement C3 was higher in PCOS (p = 0.037), as was heparin cofactor II (HCFII) (p = 0.0004). The levels of the other proteins associated with lipoprotein metabolism did not differ between PCOS and controls. CONCLUSIONS: These data contribute to the concern of the deleterious dyslipidemia found in PCOS, with the novel combination reported here of higher levels of ApoE, C3 and HCFII together with lower ApoM. The dysregulation of these proteins could circumvent the protective effect of HDL-C and contribute to a more atherogenic profile that may increase cardiovascular risk.

Antibody Affinity and Stability Maturation by Error-Prone PCR.

Human antibodies are the most important class of biologicals, and antibodies - human and nonhuman - are indispensable as research agents and for diagnostic assays. When generating antibodies, they sometimes show the desired specificity profile but lack sufficient affinity for the desired application. In this article, a phage display-based method and protocol to increase the affinity of recombinant antibody fragments is given.The given protocol starts with the construction of a mutated antibody gene library by error-prone PCR. Subsequently, the selection of high-affinity variants is performed by panning on immobilized antigen with washing conditions optimized for off-rate-dependent selection. A screening ELISA protocol to identify antibodies with improved affinity and an additional protocol to select antibodies with improved thermal stability is described.

Two-dimensional measurements of receptor-ligand interactions.

Gaining insight into the two-dimensional receptor-ligand interactions, which play a significant role in various pivotal biological processes such as immune response and cancer metastasis, will deepen our understanding of numerous physiological and pathological mechanisms and contribute to biomedical applications and drug design. A central issue involved is how to measure the in situ receptor-ligand binding kinetics. Here, we review several representative mechanical-based and fluorescence-based methods, and briefly discuss the strengths and weaknesses for each method. In addition, we emphasize the great importance of the combination of experimental and computational methods in studying the receptor-ligand interactions, and further studies should focus on the synergistic development of experimental and computational methods.

Aerogel Technology for Thermal Insulation of Cryogenic Tanks-Numerical Analysis for Comparison with Traditional Insulating Materials.

The traditional choice of insulation material for liquefied natural gas (LNG) transportation with cryogenic tankers is the back-filled perlite-based system. However, aiming to further cut down the insulation cost, spare additional arrangement space, and provide safety in installation and maintenance, the requirement of looking for alternative materials still exists. Fiber-reinforced aerogel blankets (FRABs) could represent good candidates in designing insulation layers for LNG cryogenic storage because of their ability to ensure adequate thermal performance without the need to create deep vacuum conditions in the annular space of the tank. In this work, a finite element method (FEM) model was developed to study the thermal insulation performance of a commercial FRAB (Cryogel ® Z) for application in cryogenic storage/transport LNG tanks, comparing it with the performance of traditional perlite-based systems. Within the reliability limits of the computational model, the analysis proved that FRAB insulation technology gave encouraging results and might be potentially scalable for transporting cryogenic liquid. In addition to demonstrating superior performance in terms of thermal insulating efficiency and boil-off rate over the perlite-based system, as far as a perspective of cost savings and space gain, FRAB technology allows for higher levels of insulation without vacuum and with lower thickness of the outer shell, which is therefore beneficial for storing more material and lightening the weight of the LNG transportation semitrailer.

Simultaneous affinity maturation and developability enhancement using natural liability-free CDRs.

ABBREVIATIONS: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; ka: association rate; kd: dissociation rate; KD: dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.

The Role of Ligand Rebinding and Facilitated Dissociation on the Characterization of Dissociation Rates by Surface Plasmon Resonance (SPR) and Benchmarking Performance Metrics.

Surface plasmon resonance (SPR) is a real-time kinetic measurement principle that can probe the kinetic interactions between ligands and their binding sites, and lies at the backbone of pharmaceutical, biosensing, and biomolecular research. The extraction of dissociation rates from SPR-response signals often relies on several commonly adopted assumptions, one of which is the exponential decay of the dissociation part of the response signal. However, certain conditions, such as high density of binding sites or high concentration fluctuations near the surface as compared to the bulk, can lead to non-exponential decays via ligand rebinding or facilitated dissociation. Consequently, fitting the data with an exponential function can underestimate or overestimate the measured dissociation rates. Here, we describe a set of alternative fit functions that can take such effects into consideration along with plasmonic sensor design principles with key performance metrics, thereby suggesting methods for error-free high-precision extraction of the dissociation rates.

Presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection.

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 119 CoViD-19 patients. The SPRi assay measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19 and this new method can furthermore be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 in vaccination programs.

High throughput surface plasmon resonance imaging method for clinical detection of presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection.

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 102 CoViD-19 and non-CoViD-19 patients. The SPRi assay simultaneously measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19. This new high throughput method can be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 and its mutants in vaccination programs.•Surface Plasmon Resonance imaging is an unprecedented technology for high throughput screening of antibody profiling of CoViD19 patients.•Fingerprinting of isotypes IgM, IgG and IgA can be performed for 384 patients in one run.•An affinity maturation effect was shown for patients recovering from CoViD19.

A semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes.

A tRNA interacts with numerous proteins throughout its biogenesis and during translation, and a significant portion of these interacting proteins are involved in post-transcriptional modifications. While some of the modifying enzymes use relatively simple recognition elements for substrate recognition, many enzymes selectively modify a specific subset of tRNA species without obvious recognition rules. In this chapter we describe a semi-quantitative pull-down assay to study tRNA substrate specificity of modification enzymes, by using the yeast Saccharomyces cerevisiae m3C32 methyltransferase Trm140 as an example. We also discuss some overall considerations for a successful pull-down experiment, with a focus on practical applications of the dissociation constant KD between the protein and the tRNA and the off-rate.