The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody-antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.

HIV-1 integration into the human genome is dependent on 3\'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3\'-processed DNA remained resistant. Here, we describe the mechanism by which the 3\'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3\'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3\'-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼2-fold) for the 3\'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3\'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3\'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3\'-processed HIV-1 DNA.

Pharmaceutical pollution has received considerable attention because of the harmful effects of pharmaceutical compounds on human health, even in trace amounts. Amoxicillin is one of the frequently used antibiotics that was included in the list of emerging water pollutants. Therefore, a highly selective and rapid technique for amoxicillin detection is required. In this work, a new aptamer was selected for amoxicillin and utilized for the development of a label-free electrochemical aptasensor. Aptamer selection was performed using the systematic evolution of ligands by exponential enrichment. The selected aptamer showed good specificity against other antibiotics, including the structurally related antibiotics: ampicillin and ciprofloxacin. Among the selected aptamers, Amx3 exhibited the lowest dissociation constant value of 112.9 nM. An aptasensor was developed by immobilization of thiolated Amx3 aptamer onto gold screen-printed electrodes via self-assembly, which was characterized using cyclic voltammetry and electrochemical impedance spectroscopy. The detection was realized by monitoring the change in the differential pulse voltammetry peak current in the ferro/ferricyanide redox couple upon binding of the aptasensor to amoxicillin. The aptasensor showed very good sensitivity with an ultralow limit of detection of 0.097 nM. When the aptasensor was tested using actual spiked milk samples, excellent recovery percentages were observed. The label-free electrochemical aptasensor developed herein is a promising tool for the selective and sensitive detection of amoxicillin in environmental samples.

UNASSIGNED: Modern drug discovery revolves around designing ligands that target the chosen biomolecule, typically proteins. For this, the evaluation of affinities of putative ligands is crucial. This has given rise to a multitude of dedicated computational and experimental methods that are constantly being developed and improved.
UNASSIGNED: In this review, the authors reassess both the industry mainstays and the newest trends among the methods for protein - small-molecule affinity determination. They discuss both computational affinity predictions and experimental techniques, describing their basic principles, main limitations, and advantages. Together, this serves as initial guide to the currently most popular and cutting-edge ligand-binding assays employed in rational drug design.
UNASSIGNED: The affinity determination methods continue to develop toward miniaturization, high-throughput, and in-cell application. Moreover, the availability of data analysis tools has been constantly increasing. Nevertheless, cross-verification of data using at least two different techniques and careful result interpretation remain of utmost importance.

The octanol/water partition coefficient P (logP) is a hydrophobicity index and is one of the determining factors for the pharmacokinetics of orally administered substances because it influences membrane permeability. To illustrate the wide-ranging variety of compounds in the chemical space, a two-dimensional data plot consisting of 25 blocks was previously proposed based on a substance\'s in silico chemical descriptors. The logP values of approximately 200 diverse chemicals (test plus reference compounds covering all 25 blocks of the chemical space) were estimated experimentally using retention times in reverse-phase liquid chromatography; these values were compared with those of authentic reference compounds with established logP values (available for 17 of 60 reference substances in the Organization for Economic Co-operation and Development Test Guideline 117). The logP values of 140 of 165 chemicals successfully estimated using four different mobile phase conditions (pH 2, 4, 7, and 10 for molecular forms) correlated significantly with those calculated using the in silico packages ChemDraw and ACD/Percepta (r > 0.72). Although substances that neighbored authentic compounds in the chemical space had precisely correlated logP values estimated experimentally and in silico, some compounds that were more distant from authentic substances showed lower logP values than those estimated in silico. These results indicate that additional authentic reference materials with wider ranging chemical diversity and their logP values from reverse-phase liquid chromatography should be included in the international test guidance to promote simple and reliable estimation of octanol/water partition coefficients, which are important determinant factors for the pharmacokinetics of general chemicals.

Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein\'s active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the \'dormant complex\' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.

Trichome patterning in Arabidopsis is regulated by R2R3MYB, bHLH and WDR (MBW) genes. These are considered to form a trimeric MBW protein complex that promotes trichome formation. The MBW proteins are engaged in a regulatory network to select trichome cells among epidermal cells through R3MYB proteins that can move between cells and repress the MBW complex by competitive binding with the R2R3MYB to the bHLHL protein. We use quantitative pull-down assays to determine the relative dissociation constants for the protein-protein interactions of the involved genes. We find similar binding strength between the trichome promoting genes and weaker binding of the R3MYB inhibitors. We used the dissociation constants to calculate the relative percentage of all possible complex combinations and found surprisingly low fractions of those complexes that are typically considered to be relevant for the regulation events. Finally, we predict an increased robustness in patterning as a consequence of higher ordered complexes mediated by GL3 dimerization.

Clostridioides difficile, a gram-positive anaerobic bacterium, is one of the most frequent causes of nosocomial infections. C. difficile infection (CDI) results in almost a half a million infections and approximately 30,000 deaths in the U.S. each year. Broad-spectrum antibacterial use is a strong risk factor for development of recurring CDI. There is a critical need for narrow-spectrum antibacterials with activity limited to C. difficile. The C. difficile enoyl-acyl carrier protein (ACP) reductase II enzyme (CdFabK), an essential and rate-limiting enzyme in the organism\'s fatty acid biosynthesis pathway (FAS-2), is an attractive target for narrow-spectrum CDI therapeutics as it is not present in many of the non-pathogenic gut organisms. We have previously characterized inhibitors of the CdFabK enzyme with narrow-spectrum anti-difficile activity and favorable in vivo efficacy, ADME, and low dysbiosis. To expand our knowledge of the structural requirements for CdFabK inhibition, we seek to identify new inhibitors with novel chemical scaffolds. Herein we present the optimization of a thermo-FMN biophysical assay based on the principles of differential scanning fluorimetry, or thermal shift, which leverages the fluorescence signal of the FabK enzyme\'s FMN prosthetic group. The optimized assay was validated by pilot testing a 10K diversity-based chemical library and novel scaffold hit compounds were identified and biochemically characterized. Additionally, we show that the thermo-FMN assay can be used to determine the thermodynamic dissociation constant, Kd, of CdFabK inhibitors.

Predicting the potency of inhibitors is key to in silico screening of promising synthetic or natural compounds. Here we describe a predictive workflow that provides calculated inhibitory values, which concord well with empirical data. Calculations of the free interaction energy ΔG with the YASARA plugin FoldX were used to derive inhibition constants Ki from PDB coordinates of protease-inhibitor complexes. At the same time, corresponding KD values were obtained from the PRODIGY server. These results correlated well with the experimental values, particularly for serine proteases. In addition, analyses were performed for inhibitory complexes of cysteine and aspartic proteases, as well as of metalloproteases, whereby the PRODIGY data appeared to be more consistent. Based on our analyses, we calculated theoretical Ki values for trypsin with sunflower trypsin inhibitor (SFTI-1) variants, which yielded the more rigid Pro14 variant, with probably higher potency than the wild-type inhibitor. Moreover, a hirudin variant with an Arg1 and Trp3 is a promising basis for novel thrombin inhibitors with high potency. Further examples from antibody interaction and a cancer-related effector-receptor system demonstrate that our approach is applicable to protein interaction studies beyond the protease field.