Dissociation Rate Constants Research Articles

Inhibition of E. coli RecQ Helicase Activity by Structurally Distinct DNA Lesions: Structure—Function Relationships

DNA helicase unwinding activity can be inhibited by small molecules and by covalently bound DNA lesions. Little is known about the relationships between the structural features of DNA lesions and their impact on unwinding rates and processivities. Employing E.coli RecQ helicase as a model system, and various conformationally defined DNA lesions, the unwinding rate constants kobs = kU + kD, and processivities P = (kU/(kU + kD) were determined (kU, unwinding rate constant; kD, helicase-DNA dissociation rate constant). The highest kobs values were observed in the case of intercalated benzo[a]pyrene (BP)-derived adenine adducts, while kobs values of guanine adducts with minor groove or base-displaced intercalated adduct conformations were ~10-20 times smaller. Full unwinding was observed in each case with the processivity P = 1.0 (100% unwinding). The kobs values of the non-bulky lesions T(6-4)T, CPD cyclobutane thymine dimers, and a guanine oxidation product, spiroiminodihydantoin (Sp), are up to 20 times greater than some of the bulky adduct values; their unwinding efficiencies are strongly inhibited with processivities P = 0.11 (CPD), 0.062 (T(6-4)T), and 0.63 (Sp). These latter observations can be accounted for by correlated decreases in unwinding rate constants and enhancements in the helicase DNA complex dissociation rate constants.

Assay Development and Small Molecule Screens for Inhibitors of an RNA Binding Protein Implicated in Hematologic Malignancies

RNA-binding proteins (RBPs) play a critical role in regulating RNA splicing, polyadenylation, and translation. RBPs typically contain discrete RNA-binding domains (RBDs) that bind to specific nucleotide motifs. Given their important role in regulating gene expression, RBPs are often mutated or aberrantly expressed in a variety of malignancies, particularly hematological cancers. As a result, RBPs appear to be excellent therapeutic targets. However, given that most RBPs lack enzymatic activity, it has been challenging to develop targeted therapies. Over the past several years we and others have used genetic and molecular biology approaches to understand the consequence of aberrant expression of the poly-C RBP, heterogeneous ribonucleoprotein K (hnRNP K), and determine the mechanisms by which it drives hematologic malignancies (Cancer Cell, 2015; NEJM, 2013, JNCI, 2020; and BioRxiv, 2021). To develop a pharmacologic strategy to target hnRNP K's RNA binding activities, we developed a robust fluorescent anisotropy (FA) assay to evaluate hnRNP K's ability to bind target transcripts that are critical for tumorigenesis; such as, BRD4, MYC, RUNX1, and 7SK. We next performed small molecule screens using ~80,000 compounds and identified 34 compounds that inhibited hnRNP K's ability to bind RNA targets. To gain a deeper understanding of whether these compounds directly interact with hnRNP K to block its RNA binding functions, we performed SYPRO-orange thermal shift assays. Here we identified compounds that bound hnRNP K directly and altered hnRNP K thermostability. To evaluate association rate, dissociation rate, and KD constants, we next performed SPR assays. These experiments identified compounds bound hnRNP K with affinities between (350nM and 20uM). We next performed cell-based studies, such as CETSA. Here we identified compounds that crossed the plasma membrane, directly engaged hnRNP K in vivo, and significantly stabilized hnRNP K. Molecular analyses revealed that cells treated with these lead compounds have reduced c-Myc expression, a direct mRNA target of hnRNP K. To develop a granular understanding of how these hnRNP K-small molecule interactions impact growth and cell survival, we performed high-throughput cell-based assays. These studies demonstrated that cells with inducible hnRNP K overexpression were sensitive to these small molecules. To begin medicinal chemistry approaches and to understand each compound's chemical requirements for binding to hnRNP K, we scanned molecules for reactive moieties. Here, we identified two compounds possessing amide groups within two carbons of either a furan or pyrrole moiety. These compounds, #20 and #23, respectively, were then chemically modified and FA assays were repeated in the presence of target mRNAs. Here, we observed that both the furan and isoxazole ring of compound #20 and pyrrole rings of compound #23 are critical for hnRNP K interactions, while the cyclopropane moiety on compound #23 is dispensable. Further analyses revealed additional structural analogs with either increased or decreased affinities and these results have been critical to our understanding of the importance of each moiety. Currently, we are using cryo-EM to evaluate positional interactions between hnRNP K (hnRNP K's RBDs) and these small molecules. These results will be used to further our medicinal chemistry approaches to generate more potent analogs. Collectively, our results demonstrate that hnRNP K functions can be directly inhibited by small molecules that block its RNA binding functions and demonstrates that the development of effective therapies for RBPs is possible. 1. hnRNP K Is a Haploinsufficient Tumor Suppressor that Regulates Proliferation and Differentiation Programs in Hematologic Malignancies. Cancer Cell, 2015. 28(4): p. 486-499. 2. Cancer Genome Atlas Research Network, Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013 368(22):2059-74. 3. Uncovering the Role of RNA-Binding Protein hnRNP K in B-Cell Lymphomas. JNCI: Journal of the National Cancer Institute, 2020. 4. Heterogeneous nuclear ribonucleoprotein K is overexpressed in acute myeloid leukemia and causes myeloproliferative disease in mice via altered Runx1 splicing. BioRxiv. doi: https://doi.org/10.11.02.05.429385, 2021 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Quantitative assessment of the in-vitro binding kinetics of antisickling aromatic aldehydes with hemoglobin A: A universal HPLC-UV/Vis method to quantitate Schiff-base adduct formation

Aromatic aldehydes act as allosteric effectors of hemoglobin (AEH), forming Schiff-base adducts with the protein to increase its oxygen (O2) affinity; a desirable property in sickle cell disease (SCD) treatment, as the high-O2 affinity hemoglobin (Hb) does not polymerize and subsequently prevents erythrocytes sickling. This study reports the development, validation, and application of a weak cation-exchange HPLC assay – quantifying the appearance of Hb-AEH adduct – as a “universal” method, allowing for the prioritization of AEH candidates through an understanding of their Hb binding affinity and kinetics. Concentration- and time-dependent Hb binding profiles of ten AEHs were determined with HPLC, followed by the appropriate non-linear modeling to characterize their steady-state binding affinity (KDss), and binding kinetics second-order association (kon) and first-order dissociation (koff) rate constants. Vanillin-derived AEHs exhibited enhanced binding affinity to Hb, primarily due to their faster kon. Across AEH, kon and koff values are strongly correlated (r = 0.993, n = 7), suggesting that modifications of the AEH scaffold enhanced their interactions with Hb as intended, but inadvertently increased their Hb-AEH adduct dissociation. To our knowledge, the present study is the first to provide valuable insight into Hb binding kinetics of antisickling aromatic aldehydes, and the assay will be a useful platform in screening/prioritizing drug candidates for SCD treatment.

Supramolecular association studied by Fluorescence correlation spectroscopy.

A comprehensive description of a supramolecular system involves a full understanding of its thermodynamic and dynamic properties, as well as detailed knowledge of its structure. Fluorescence Correlation Spectroscopy (FCS) constitutes a powerful technique to acquire this information. Fluorescence correlation curves show a characteristic diffusion term that is related to the binding equilibrium constant or other thermodynamic properties of the supramolecular system. The association and dissociation rate constants of the binding process can be determined in FCS when the relaxation time of the binding is faster than the observation time-a regime called fast-exchange dynamics - in opposition to the slow-exchange regime. In all cases, structural information can be inferred from the diffusional properties of the supramolecular complexes. A short overview of the use of FCS for the study of supramolecular systems is given with examples which belong to the fast and slow regime.

Probing the Synergistic Effects of Mg2+ on CO2 Reduction Reaction on CoPc by In Situ Electrochemical Scanning Tunneling Microscopy.

We report herein the in situ electrochemical scanning tunneling microscopy (ECSTM) study on the synergistic effect of Mg2+ in CO2 reduction reaction (CO2RR) catalyzed by cobalt phthalocyanine (CoPc). ECSTM measurement molecularly resolves the self-assembled CoPc monolayer on the Au(111) substrate. In the CO2 environment, high-contrast species are observed in the adlayer and assigned to the CO2 adsorption on CoPc. Furthermore, the contrast of the CO2-bound complex is higher in Mg2+-containing electrolytes than in Mg2+-free electrolytes, indicating the formation of the CoPc-CO2-Mg2+ complex. The surface coverage of adsorbed CO2 is positively correlated with the Mg2+ concentration as the additive in electrolytes up to a plateau of 30.8 ± 2.7% when c(Mg2+) > 30 mM. The potential step experiment indicates the higher CO2 adsorption dynamics in Mg2+-containing electrolytes than without Mg2+. The rate constants of CO2 adsorption and dissociation in different electrolytes are extracted from the data fitting of statistical results from in situ ECSTM experiments.

Measurement of Protein Kinetics Using a Liquid Phase-Based Biosensing Platform.

Macromolecular association is crucial to many fields in biomedical sciences, including drug development, gene editing, and diagnostics. In particular, protein-protein association and dissociation rate constants are typically determined using surface plasmon resonance systems, which require costly instrumentation and cumbersome procedures (e.g., blocking, washing, and separation). Herein, we demonstrate that protein-binding constants can be readily determined using a real-time biosensing platform facilitated by graphene oxide-modified microwell plates and fluorophore-labeled proteins, where the fluorescent probes remain highly fluorescent during protein association, whereas fluorescent bioprobes that are not associated with their counterparts are quenched by graphene oxide. Binding data of three pairs of proteins were systematically determined employing this single-step platform and compared with those data reported by the suppliers or the literature, suggesting that this approach is comparable and consistent with the existing ones. Such pairs include (i) human immunoglobulin G (H-IgG)-fluorophore-labeled anti-H-IgG, (ii) prostate-specific antigen (PSA)-quantum dot-labeled anti-PSA, and (iii) anti-RBD-fluorophore-labeled SARS-CoV-2 spike receptor-binding domain recombinant protein. We also offer an open-source software that automatically determines the binding kinetics constants of proteins. This Technical Note introduces a simple, yet effective, platform to determine relevant information on protein kinetics, which can be performed using a microwell plate reader and economical materials like graphene oxide. We foresee a new generation of diagnostics based on our affordable protein kinetics analysis.

FcγRIIIA affinity chromatography complements conventional functional characterization of rituximab.

Analytical and functional characterization of batches of biologics/biosimilar products are imperative towards qualifying them for pre-clinical and clinical investigations. Several orthogonal strategies are employed to characterize the functional attributes of these drugs. However, the use of conventional techniques for online monitoring of functional attributes is not feasible. Liquid chromatography is one of the crucial unit operations during the downstream processing of biopharmaceuticals. In this work, we have demonstrated the utility of FcγRIIIA affinity chromatography as an independent quantitative functional characterization tool. FcγRIIIA affinity chromatography aided in sequential elution of Rituximab glycoform mixtures, based on varying levels of galactosylation, and thereby the affinity for the receptor protein. The predominant glycans present in the three Rituximab glycoform mixture peaks were G0F, G1F, and G2F, respectively. Dissociation rate constants were derived from the chromatographic elution profiles by the peak profiling method, for the control and glucose stress conditions. The glucose stress conditions did not result in unfavorable binding kinetics of Rituximab and FcγRIIIA. The dissociation rate constants of the glycoform mixture 2, predominantly consisting of G1F, were similar to the dissociation rate constants obtained by surface plasmon resonance. Moreover, the glycosylation profiles obtained from chromatographic estimation can be corroborated with the ADCC activity. However, the ex vivo ADCC reporter assay indicated that there was an increase in the effector activity with increasing glucose stress. Thus, FcγRIIIA affinity chromatography permitted three independent assessments via a single analysis. Such approaches can be utilized as potential process analytical technology (PAT) tools in the biosimilar development process.

Structural and Mechanistic Bases of Viral Resistance to HIV-1 Capsid Inhibitor Lenacapavir.

ABSTRACTLenacapavir (LEN) is a long-acting, highly potent HIV-1 capsid (CA) inhibitor. The evolution of viral variants under the genetic pressure of LEN identified Q67H, N74D, and Q67H/N74D CA substitutions as the main resistance associated mutations (RAMs). Here, we determined high-resolution structures of CA hexamers containing these RAMs in the absence and presence of LEN. Our findings reveal that the Q67H change induces a conformational switch, which adversely affects the inhibitor binding. In the unliganded protein, the His67 side chain adopts the closed conformation by projecting into the inhibitor binding pocket and thereby creating steric hindrance with respect to LEN. Upon the inhibitor binding, the His67 side chain repositions to the open conformation that closely resembles the Gln67 side chain in the WT protein. We propose that the switch from the closed conformation to the open conformation, which is needed to accommodate LEN, accounts for the reduced inhibitor potency with respect to the Q67H CA variant. The N74D CA change results in the loss of a direct hydrogen bond and in induced electrostatic repulsions between CA and LEN. The double Q67H/N74D substitutions exhibited cumulative effects of respective single amino acid changes. An examination of LEN binding kinetics to CA hexamers revealed that Q67H and N74D CA changes adversely influenced the inhibitor binding affinity (KD) by primarily affecting the dissociation rate constant (koff). We used these structural and mechanistic findings to rationally modify LEN. The resulting analog exhibited increased potency against the Q67H/N74D viral variant. Thus, our studies provide a means for the development of second-generation inhibitors with enhanced barriers to resistance.

Re-evaluation of rate constants for the reaction N2H4 (+ M) ⇄ NH2 + NH2 (+ M)

Rate constants for the dissociation/recombination reaction N2H4 (+ M) ⇄ NH2 + NH2 (+ M) are determined by a combination of quantum-chemical calculations and statistical unimolecular rate theory. Between 1100 and 2500 K, limiting low-pressure rate constants for hydrazine dissociation in the bath gas Ar of k0 = [Ar] 6.1 × 1020 (T/1000 K)−7.3 exp (-34490 K/T) cm3 mol−1 s−1, limiting high-pressure rate constants of k∞ = 7.6 × 1016 (T/1000 K)−1.0 exp(-33600 K/T) s − 1, and center broadening factors of the falloff curves (between 1100 and 1600 K in Ar) of Fcent = 0.71 exp(- T/1460 K) + 0.29 exp(- T/21 K) + exp(-13400 K/T) were calculated. Using equilibrium constants KC = 1.7 × 103 (T/1000 K)−1.5 exp(-33,460 K/T) mol cm−3, between 300 and 600 K limiting low-pressure rate constants for the reverse recombination of NH2 radicals in the bath gas N2 of krec,0 = [N2] 4.4 × 1020(T/400 K)−6.9 exp (-1630 K/T) cm6 mol−2 s−1, limiting high-pressure rate constants of krec,∞ = 4.4 × 1013 (T/1000 K)0.44 exp(-140 K/T) cm3 mol-1 s−1, and center broadening factors (between 300 and 600 K in N2) of Fcent = exp(- T/1130 K) + exp (-10340 K/T) were obtained. A comparison with experimental results for hydrazine dissociation from the literature suggests incomplete falloff extrapolations toward k∞ and experimental problems in the determination of k0 at temperatures above about 1600 K. Implications of the present re-evaluated rate constants for the modeling of high temperature ammonia oxidation kinetics are discussed, showing an only small influence of their precise values on the overall properties of the process.

Computationally Elucidating the Binding Kinetics for Different AChE Inhibitors to Access the Rationale for Improving the Drug Efficacy.

Traditional drug discovery is based on a binding affinity (thermodynamics)-driven paradigm. Numerous examples, however, demonstrated that drug efficacy does not always depend only on binding affinity but positively correlates with binding kinetics, that is, the dissociation rate constant (koff). Binding free energy landscape (BFEL) constructor is a computational binding kinetics prediction method, previously developed by us, that estimates the binding kinetics for ligand-protein based on their constructed binding free energy landscape, but it also reveals the detailed molecular mechanism of the binding event, hence, providing the position of transition states at the molecular level to modify/improve the binding kinetics. Acetylcholinesterase (AChE) is a well-known Alzheimer's disease (AD) target for which there is still not an ideal drug on the market. Therefore, to improve the drug design strategy for AD, the binding kinetics and binding molecular mechanisms of the four inhibitors of AChE, that is, E2020 (Aricept), HupA, Rivastigmine, and Galantamine, were studied. Also, the differentiation of the binding kinetics between mAChE and TcAChE was studied to evaluate the sensitiveness of BFEL constructor. The flexibility of molecules has a noticeable effect on the nature of BFEL. To the same target, flexible molecules (i.e., E2020 and Rivastigmine) which contain more rotatable bonds tend to have more complicated BFELs reflecting more complicated molecular action mechanisms than the rigid ones (i.e., HupA and Galantamine), which therefore could be more challenging to be optimized. The binding kinetics is highly dependent on the structure of the molecules, such as the length and the functional groups. Therefore, E2020 presents better binding kinetic and thermodynamic properties with either TcAChE or mAChE. Therefore, it is the most promising lead drug for binding kinetics-based drug design. In addition, the binding kinetics of a drug may present different values in the proteins of different organisms because the residue compositions of the binding gorges of the targets are variant, that is, E2020 shows lower binding affinity and association energy barrier in binding with mAChE than TcAChE. However, HupA presents a better binding property with TcAChE than mAChE.

Flash pyrolysis mechanism of trimethylchlorosilane

The thermal decomposition mechanism of trimethylchlorosilane at temperatures up to 1400 K was investigated using flash pyrolysis microreactor coupled with vacuum ultraviolet (118.2 nm) photoionization time-of-flight mass spectrometry. The main initiation reaction of the parent molecule was identified to be HCl molecular elimination producing Me2Si=CH2, and its onset temperature was estimated to be around 1210 K. The methyl loss channel was also observed at an onset temperature of ∼1280 K. Other possible initiation pathways such as chlorine atom loss and methane molecular elimination were considered to be insignificant. Quantum chemistry calculations at the UCCSD(T)/cc-pVTZ//UM05–2X/aug-cc-pVDZ level of theory were performed to study the energetics of the possible initiation pathways. The theoretical calculations revealed that the HCl elimination channel via a van der Waals intermediate was the most energetically favored pathway among all the initiation channels, in agreement with the experimental observations. Transition state theory (TST) and variational transition state theory (VTST) calculations were employed to calculate unimolecular dissociation rate constants of the initiation reactions at elevated temperatures. The results supported the experimental observations and reaction energetics calculations that the HCl molecular elimination reaction was the most prominent initiation reaction and the CH3 loss channel was a significant initiation reaction channel. Secondary reactions of the initial products were identified, and the possible mechanisms were proposed.

Identification of Plant-Derived Bioactive Compounds Using Affinity Mass Spectrometry and Molecular Networking.

Affinity selection-mass spectrometry (AS-MS) is a label-free binding assay system that uses UHPLC-MS size-based separation methods to separate target-compound complexes from unbound compounds, identify bound compounds, classify compound binding sites, quantify the dissociation rate constant of compounds, and characterize affinity-extracted ligands. This label-free binding assay, in contrast to conventional biochemical (i.e., high-throughput screening (HTS)) approaches, is applicable to any drug target, and is also concise, accurate, and adaptable. Although AS-MS is an innovative approach for identifying lead compounds, the possibilities of finding bioactive compounds are limited by competitive binding, which occurs during the equilibration of extracts with the target protein(s). Here, we discuss the potential for metabolite profiling complemented with molecular networking to be used alongside AS-MS to improve the identification of bioactive compounds in plant extracts. AS-MS has gained significant prominence in HTS labs and shows potential to emerge as the driving force behind novel drug development in the future.

Human eukaryotic initiation factor 4E (eIF4E) and the nucleotide-bound state of eIF4A regulate eIF4F binding to RNA

During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate that eIF4E binding to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. In addition, we show that the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus the absence of ATP. Monitoring uncapped RNA dissociation in real time reveals that ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined that the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A-binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.

Transient Inhibition of Trastuzumab-Tumor Binding to Overcome the "Binding-Site Barrier" and Improve the Efficacy of a Trastuzumab-Gelonin Immunotoxin.

We have recently shown that coadministration of mAbs with anti-idiotypic distribution enhancers (AIDE) that inhibit mAb binding to tumor antigens enabled increased intratumoral mAb distribution and increased efficacy of an antibody-drug conjugate (trastuzumab emtansine, T-DM1). In this article, a pharmacokinetic/pharmacodynamic (PK/PD) model was applied to predict the impact of this optimization strategy on the within-tumor distribution and antitumor efficacy of trastuzumab-gelonin, where the released payload (gelonin) is expected to exhibit negligible bystander activity. Immunofluorescence histology was used to investigate trastuzumab-gelonin distribution in solid tumors following dosing with or without coadministration of anti-trastuzumab AIDEs. Antitumor efficacy of trastuzumab-gelonin, with or without coadministration of AIDEs, was also evaluated in tumor-bearing mice. Trastuzumab-gelonin efficiently induced cytotoxicity when applied to NCI-N87 cells in culture (IC50: 0.224 ± 0.079 nmol/L). PK/PD simulations predicted that anti-idiotypic single-domain antibodies AIDEs with dissociation rate constants between 0.03 and 0.2 per hour would provide optimal enhancement of trastuzumab-gelonin efficacy. LE8 and 1HE, anti-trastuzumab AIDEs, were selected for evaluation in vivo. Coadministration of trastuzumab-gelonin with the inhibitors increased the portion of tumor area that stained positive for trastuzumab-gelonin by 58% (P = 0.0059). In addition, LE8 or 1HE coadministration improved trastuzumab-gelonin efficacy in NCI-N87 xenograft-bearing mice by increasing the percent increase in life span (%ILS) from 27.8% (for trastuzumab-gelonin administered alone) to 62.5% when administered with LE8 (P = 0.0007) or 83.3% (P = 0.0007) when administered with 1HE. These findings support the hypothesis that transient, competitive inhibition of mAb-tumor binding can improve the intratumoral distribution and efficacy of immunotoxins when applied for treatment of solid tumors.

Insight into the mechanism of DNA synthesis by human terminal deoxynucleotidyltransferase.

Terminal deoxynucleotidyltransferase (TdT) is a member of the DNA polymerase X family that is responsible for random addition of nucleotides to single-stranded DNA. We present investigation into the role of metal ions and specific interactions of dNTP with active-site amino acid residues in the mechanisms underlying the recognition of nucleoside triphosphates by human TdT under pre-steady-state conditions. In the elongation mode, the ratios of translocation and dissociation rate constants, as well as the catalytic rate constant were dependent on the nature of the nucleobase. Preferences of TdT in dNTP incorporation were researched by molecular dynamics simulations of complexes of TdT with a primer and dNTP or with the elongated primer. Purine nucleotides lost the "summarised" H-bonding network after the attachment of the nucleotide to the primer, whereas pyrimidine nucleotides increased the number and relative lifetime of H-bonds in the post-catalytic complex. The effect of divalent metal ions on the primer elongation revealed that Me<sup>2+</sup> cofactor can significantly change parameters of the primer elongation by strongly affecting the rate of nucleotide attachment and the polymerisation mode.

Easy and Rapid Approach to Obtaining the Binding Affinity of Biomolecular Interactions Based on the Deep Learning Boost.

Recently, the deep learning (DL) dimension of artificial intelligence has received much attention from biochemical researchers and thus has gradually become the key approach adopted in the area of biosensing applications. Studies have shown that the use of DL techniques for sensing can not only shorten the time of data analysis but also significantly increase the accuracy of data analysis and prediction, resulting in the performance improvement of biosensing systems in comparison to conventional methods. However, obtaining reliable equilibrium and rate constants of biomolecular interactions during the detection process remains difficult and time-consuming to date. In this study, we propose a transformed model based on the deep transfer learning and sequence-to-sequence autoencoder that can successfully transfer the SPR sensorgram to the protein-binding constants, that is, the association rate constant (ka) and dissociation rate constant (kd), which provide crucial information to understand the mechanisms of drug action and the functional structures of biomolecules. Experimentally, we first trained and tested the pre-trained model using the Langmuir model which generated ideal SPR sensorgrams and then we fine-tuned the pre-trained model through the augmented SPR sensorgrams which were synthesized by using the synthesized minority oversampling technique (SMOTE) through the moderate-scale experiment. Next, the fine-tuned model was inputted with a short experimental SPR sensorgram that only needs 110 s, and the sensorgram was directly transformed into a reconstructed ideal sensorgram. Finally, the binding kinetic constants, that is, ka and kd, as outputs, were obtained through fitting the reconstructed ideal sensorgram. The results showed that the prediction errors of ka and kd obtained by our model were less than 12 and 24%, respectively. Based on the convenience, accuracy, and reliability of the proposed DL approach, we believe our strategy significantly boosts the feasibility to monitor the binding affinity of antibodies online during production.

Characterization of acetonitrile ice irradiated by X-rays employing the procoda code – I. Effective rate constants and abundances at chemical equilibrium

ABSTRACT In this work, the chemical evolution of pure acetonitrile ice at 13 K irradiated with broad-band soft X-rays (from 6 eV to 2 keV) is determined by using a computational methodology (procoda code) to best fit the experimental data. To simulate the chemical evolution of the acetonitrile ice under an astrophysical analogous situation, the code employs 273 reaction rates involving 33 molecular species (5 species observed in the experiment and 28 non-observed or unknown). The considered reaction network describes 240 chemical reactions (including dissociation, bimolecular, and termolecular rates) and 33 individual desorption rates. The summed desorption yield was determined to be 0.23 molecules per photon, in agreement with previous estimates. Average values for dissociation, bimolecular, and termolecular effective rate constants were determined as 2.3 × 10−3 s−1, 9.7 × 10−26 cm3 molecule−1 s−1, and 3.2 × 10−47 cm6 molecule−2 s−1, respectively. Some branching ratios within reaction groups were also determined. Molecular abundances at chemical equilibrium were obtained, such as CH3CN (67.5 per cent), H (10.6 per cent), CN (6.7 per cent), CH2 (6.4 per cent), CH (2.5 per cent), CH3 (1.2 per cent), CH4 (1.1 per cent), C2N2 (0.8 per cent), HCN (0.8 per cent), and CH3NC (0.6 per cent). The results of this work can be employed in future astrochemical models to map chemical evolution embedded species in astrophysical regions in the presence of an ionizing radiation field.

Strategies for selection and identification of rabbit single-chain Fv antibodies as ligand in affinity chromatography

We developed affinity chromatographic resins that immobilized rabbit single-chain Fv antibodies (scFvs). By biopanning using antigen-coupled multilamellar vesicles (Ag-MLVs), 152 types of original scFv clones that specifically bind to human IgG were isolated and identified. Apparent dissociation rate constants, appkoff, of six different candidates were less than 10-3 s-1 and their dissociation constants, KDs, were ranged from 5.56× 10-10 to 4.04× 10-8M. Consequently, the clones, R1-27, R2-18, and R3-26 were further investigated for use in affinity purification of human IgG. Both the clones, R1-27 and R3-26 maintained more than 40% of antigen-binding activities on the surface of affinity resins. Especially, R3-26 had a relatively high alkaline resistance. The direct separation of human IgG from 10% FBS-D-MEM by use of the column with R1-27 achieved 97.2% purity, while the column with R3-26 showed almost 100% recovery. The affinity resins at the densities between 4.32 and 15.19mg-scFv/cm3 exhibited maximum binding amount of human IgG, while the highest ligand utilization was achieved by use of the resin at approximately 9mg-scFv/cm3. The resin exhibited 7.69mg/cm3 of equilibrium binding capacity (EBC) in affinity chromatography. It was expected that the EBC of affinity resins was strongly dependent on the specific surface area as well as the pore volume of the base resin. Therefore, the strategies to develop affinity ligands will be beneficial for development of on-demand affinity columns with higher affinity/selectivity, chemical resistance, while optimization of pore size and pore volume for scFv-coupled resins will further improve the EBC.

Screening the bioactive compound from Coptis chinensis inflorescence by immobilized peroxisome proliferator-activated receptor-gamma.

Coptis chinensis inflorescence is a by-product of Coptis chinensis Franch and riches in alkaloids. We screened the bioactive compounds in the by-product through an immobilized peroxisome proliferator-activated receptor gamma. The receptor was covalently immobilized on the macroporous silica gel through amino groups to generate the affinity stationary phase and was applied for screening. Berberine, palmatine, and jatrorrhizine were identified as the retained components of the herb on the affinity column. We evaluated the binding of the three bioactive compounds with the receptor by nonlinear chromatography and molecular docking. The affinities of the compounds to the receptor were (1.42 ± 0.10) ×108 /M, (4.88 ± 0.38) ×107 /M, and (1.65 ± 0.13) ×107 /M for berberine, palmatine, and jatrorrhizine, with dissociation rate constants of (17.70 ± 0.03) ×10-3 /S, (5.18 ± 0.25) ×10-2 /S, and (15.7 ± 0.05) ×10-2 /S, respectively. Cys285, Arg288, Ile326, Leu330, and His449 in the agonist binding pocket of the receptor participated in the formation of bioactive compound-receptor conjugates. These data indicated that the immobilized receptor is a reliable alternative for screening the bioactive compounds. In addition, Coptis chinensis inflorescence has the potential to be a source for drug discovery.

A Full Target-Mediated Drug Disposition (TMDD) Model to Explain the Changes in Recombinant Human Erythropoietin (rhEpo) Pharmacokinetics in Patients with Different Bone Marrow Integrity Following Hematopoietic Transplantation

Our aim was to build a mechanistic full target-mediated drug disposition (TMDD) model for rhEpo to better understand rhEpo disposition, Epo receptor (EpoR) synthesis, and degradation in hematopoietic transplant patients with four distinct bone marrow conditions. All PK data were analyzed simultaneously using the nonlinear mixed effect modeling approach with NONMEM. The final model was a two-compartmental full TMDD model, which adequately characterizes rhEpo PK in patients and provides insight into the dynamics of free EpoR, rhEpo-EpoR, and total EpoR. The model predicted association rate constant (kon), dissociation rate constant (koff), and internalization rate constant (kint) were 0.0276 pM−1h−1, 0.647 h−1, and 0.255h−1, respectively, which were supported by experimental data. Also, the EpoR degradation rate constant (kdeg) was estimated to be 0.461 h−1. EpoR production rate was estimated to be 37.5 pM/h for adults at pre-ablation baseline and 5.91 pM/h, and 4.19 pM/h in the early post-transplant post-engraftment, and late post-transplant full engraftment. Our model provides extensive information on the dynamics of free EpoR, total EpoR and rhEpo-EpoR, and proven to be more robust and can provide more physiologically relevant binding parameters than previous models.