The ability to accurately measure drug-target interaction is critical for the discovery of new therapeutics. Classical pharmacological bioassays such as radioligand or fluorescent ligand binding assays can define the affinity or Kd of a ligand for a receptor with the lower the Kd, the stronger the binding and the higher the affinity. However, in many drug discovery laboratories today, the target of interest if often artificially upregulated by means of transfection to modify the host cell\'s genetic makeup. This then potentially invalidates the assumptions of classical pharmacology affinity calculations as the receptor of interest is no longer at normal physiological densities. The CXCR4 receptor is expressed on many different cancer cell types and is associated with metastasis and poor prognosis. Therefore, the CXCR4 receptor is a desirable target for novel therapeutics. In this study, we explore the applicability of the newly developed fluorescently tagged CXCR4 antagonists, IS4-FAM as an investigative tool to study CXCR4 affinity and competitive antagonism in native, non-transfected cancer cells using confocal microscopy and flow cytometry. IS4-FAM directly labels CXCR4 in several cell lines including high CXCR4 expressing SK-MEL-28 (malignant melanoma) and PC3 (metastatic prostate cancer) and lower CXCR4 expressing THP-1 (acute monocytic leukemia) and was competitive with the established CXCR4 antagonist, AMD3100. This highlights the potential of IS4-FAM as a pharmacological tool for drug discovery in native cells lines and tissues.

Circular RNAs (circRNAs) are involved in various physiological and pathological processes in both vertebrates and invertebrates. However, most studies on circRNAs have focused on their roles as endogenous competitive RNAs. Here, we report a novel function of circRNA derived from the Fibrinogen-like protein 1 gene (circ-FGL1) that inhibits coelomocyte apoptosis via competing with the deubiquitinase AjOTUB1 to bind AjMyc in Apostichopus japonicus during Vibrio splendidus infection. The results showed that circ-FGL1 is significantly downregulated in coelomocytes of V. splendidus-induced A. japonicus and negatively regulates coelomocyte apoptosis through the AjBax-AjCyt c pathway. Mechanistically, the deubiquitinase AjOTUB1 and circ-FGL1 could interact with the transcription factor protein AjMyc in the same region with circ-FGL1/AjMyc having greater affinity. Under normal conditions, high levels of circ-FGL1 bind directly to AjMyc, inhibiting the deubiquitylation of AjMyc by AjOTUB1 and leading to the degradation of AjMyc. After V. splendidus infection, AjMyc disassociates from the depressed expression of circ-FGL1, promoting its deubiquitylation by binding to the induced deubiquitinase AjOTUB1 to inhibit its degradation. AjMyc is then transferred to the nucleus and promotes the transcription of AjCyt c and AjBax to induce coelomocyte apoptosis. The new finding will expand our present outstanding on the functional role of circRNAs and suggest new therapeutic targets for the treatment of echinoderms during bacterial invasion.

Microtubule organization in cells relies on targeting mechanisms. Cytoplasmic linker proteins (CLIPs) and CLIP-associated proteins (CLASPs) are key regulators of microtubule organization, yet the underlying mechanisms remain elusive. Here, we reveal that the C-terminal domain of CLASP2 interacts with a common motif found in several CLASP-binding proteins. This interaction drives the dynamic localization of CLASP2 to distinct cellular compartments, where CLASP2 accumulates in protein condensates at the cell cortex or the microtubule plus end. These condensates physically contact each other via CLASP2-mediated competitive binding, determining cortical microtubule targeting. The phosphorylation of CLASP2 modulates the dynamics of the condensate-condensate interaction and spatiotemporally navigates microtubule growth. Moreover, we identify additional CLASP-interacting proteins that are involved in condensate contacts in a CLASP2-dependent manner, uncovering a general mechanism governing microtubule targeting. Our findings not only unveil a tunable multiphase system regulating microtubule organization, but also offer general mechanistic insights into intricate protein-protein interactions at the mesoscale level.

Fluorescent ligands have proved to be powerful tools in the study of G protein-coupled receptors in living cells. Here we have characterized a new fluorescent ligand PSB603-BY630 that has high selectivity for the human adenosine A2B receptor (A2BR). The A2BR appears to play an important role in regulating immune responses in the tumor microenvironment. Here we have used PSB603-BY630 to monitor specific binding to A2BRs in M1- and M2-like macrophages derived from CD14+ human monocytes. PSB603-BY630 bound with high affinity (18.3 nM) to nanoluciferase-tagged A2BRs stably expressed in HEK293G cells. The ligand exhibited very high selectivity for the A2BR with negligible specific-binding detected at NLuc-A2AR, NLuc-A1R, or NLuc-A3R receptors at concentrations up to 500 nM. Competition binding studies showed the expected pharmacology at A2BR with the A2BR-selective ligands PSB603 and MRS-1706 demonstrating potent inhibition of the specific binding of 50 nM PSB603-BY630 to A2BR. Functional studies in HEK293G cells using Glosensor to monitor Gs-coupled cyclic AMP responses indicated that PSB603-BY630 acted as a negative allosteric regular of the agonist responses to BAY 60-6583. Furthermore, flow cytometry analysis confirmed that PSB603-BY630 could be used to selectively label endogenous A2BRs expressed on human macrophages. This ligand should be an important addition to the library of fluorescent ligands which are selective for the different adenosine receptor subtypes, and will enable study of the role of A2BRs on immune cells in the tumor microenvironment.

We derive approximate expressions for pre- and post-steady state regimes of the velocity-substrate-inhibitor spaces of the Michaelis-Menten enzyme kinetic scheme with fully and partial competitive inhibition. Our refinement over the currently available standard quasi steady state approximation (sQSSA) seems to be valid over wide range of enzyme to substrate and enzyme to inhibitor concentration ratios. Further, we show that the enzyme-inhibitor-substrate system can exhibit temporally well-separated two different steady states with respect to both enzyme-substrate and enzyme-inhibitor complexes under certain conditions. We define the ratios fS = vmax/(KMS + e0) and fI = umax/(KMI + e0) as the acceleration factors with respect to the catalytic conversion of substrate and inhibitor into their respective products. Here KMS and KMI are the Michaelis-Menten parameters associated respectively with the binding of substrate and inhibitor with the enzyme, vmax and umax are the respective maximum reaction velocities and e0, s0, and i0 are total enzyme, substrate and inhibitor levels. When (fS/fI) < 1, then enzyme-substrate complex will show multiple steady states and it reaches the full-fledged steady state only after the depletion of enzyme-inhibitor complex. When (fS/fI) > 1, then the enzyme-inhibitor complex will show multiple steady states and it reaches the full-fledged steady state only after the depletion of enzyme-substrate complex. This multi steady-state behavior especially when (fS/fI) ≠ 1 is the root cause of large amount of error in the estimation of various kinetic parameters of fully and partial competitive inhibition schemes using sQSSA. Remarkably, we show that our refined expressions for the reaction velocities over enzyme-substrate-inhibitor space can control this error more significantly than the currently available sQSSA expressions.

Venezuelan equine encephalitis virus (VEEV) is a highly virulent pathogen whose nuclear localization signal (NLS) sequence from capsid protein binds to the host importin-α transport protein and blocks nuclear import. We studied the molecular mechanisms by which two small ligands, termed I1 and I2, interfere with the binding of VEEV\'s NLS peptide to importin-α protein. To this end, we performed all-atom replica exchange molecular dynamics simulations probing the competitive binding of the VEEV coreNLS peptide and I1 or I2 ligand to the importin-α major NLS binding site. As a reference, we used our previous simulations, which examined noncompetitive binding of the coreNLS peptide or the inhibitors to importin-α. We found that both inhibitors completely abrogate the native binding of the coreNLS peptide, forcing it to adopt a manifold of nonnative loosely bound poses within the importin-α major NLS binding site. Both inhibitors primarily destabilize the native coreNLS binding by masking its amino acids rather than competing with it for binding to importin-α. Because I2, in contrast to I1, binds off-site localizing on the edge of the major NLS binding site, it inhibits fewer coreNLS native binding interactions than I1. Structural analysis is supported by computations of the free energies of the coreNLS peptide binding to importin-α with or without competition from the inhibitors. Specifically, both inhibitors reduce the free energy gain from coreNLS binding, with I1 causing significantly larger loss than I2. To test our simulations, we performed AlphaScreen experiments measuring IC50 values for both inhibitors. Consistent with in silico results, the IC50 value for I1 was found to be lower than that for I2. We hypothesize that the inhibitory action of I1 and I2 ligands might be specific to the NLS from VEEV\'s capsid protein.

ASCT2 (alanine serine cysteine transporter 2), a member of the solute carrier 1 family, mediates Na+-dependent exchange of small neutral amino acids across cell membranes. ASCT2 was shown to be highly expressed in tumor cells, making it a promising target for anticancer therapies. In this study, we explored the binding mechanism of the high-affinity competitive inhibitor L-cis hydroxyproline biphenyl ester (Lc-BPE) with ASCT2, using electrophysiological and rapid kinetic methods. Our investigations reveal that Lc-BPE binding requires one or two Na+ ions initially bound to the apo-transporter with high affinity, with Na1 site occupancy being more critical for inhibitor binding. In contrast to the amino acid substrate bound form, the final, third Na+ ion cannot bind, due to distortion of its binding site (Na2), thus preventing the formation of a translocation-competent complex. Based on the rapid kinetic analysis, the application of Lc-BPE generated outward transient currents, indicating that despite its net neutral nature, the binding of Lc-BPE in ASCT2 is weakly electrogenic, most likely because of asymmetric charge distribution within the amino acid moiety of the inhibitor. The preincubation with Lc-BPE also led to a decrease of the turnover rate of substrate exchange and a delay in the activation of substrate-induced anion current, indicating relatively slow Lc-BPE dissociation kinetics. Overall, our results provide new insight into the mechanism of binding of a prototypical competitive inhibitor to the ASCT transporters.

Understanding the thermodynamics and kinetics of the protein-ligand interaction is essential for biologists and pharmacologists. To visualize the equilibrium and kinetics of the binding reaction with 1:1 stoichiometry and no cooperativity, we obtained the exact relationship of the concentration of the protein-ligand complex and the time in the second-order binding process and numerically simulated the process of competitive binding. First, two common concerns in measuring protein-ligand interactions were focused on how to avoid the titration regime and how to establish the appropriate incubation time. Then, we gave examples of how the commonly used experimental conditions of [L]0 ≫ [P]0 and [I]0 ≫ [P]0 affected the estimation of the kinetic and thermodynamic properties. Theoretical inhibition curves were calculated, and the apparent IC50 and IC50 were estimated accordingly under predefined conditions. Using the estimated apparent IC50, we compared the apparent Ki and Ki calculated by using the Cheng-Prusoff equation, Lin-Riggs equation, and Wang\'s group equation. We also applied our tools to simulate high-throughput screening and compare the results of real experiments. The visualization tool for simulating the saturation experiment, kinetic experiments of binding and competitive binding, and inhibition curve, \"Binding Curve Viewer,\" is available at www.eplatton.net/binding-curve-viewer.

More than 3000 proteins are now known to bind to glycosaminoglycans (GAGs). Yet, GAG-protein systems are rather poorly understood in terms of selectivity of recognition, molecular mechanism of action, and translational promise. High-throughput screening (HTS) technologies are critically needed for studying GAG biology and developing GAG-based therapeutics. Microarrays, developed within the past two decades, have now improved to the point of being the preferred tool in the HTS of biomolecules. GAG microarrays, in which GAG sequences are immobilized on slides, while similar to other microarrays, have their own sets of challenges and considerations. GAG microarrays are rapidly becoming the first choice in studying GAG-protein systems. Here, we review different modalities and applications of GAG microarrays presented to date. We discuss advantages and disadvantages of this technology, explain covalent and non-covalent immobilization strategies using different chemically reactive groups, and present various assay formats for qualitative and quantitative interpretations, including selectivity screening, binding affinity studies, competitive binding studies etc. We also highlight recent advances in implementing this technology, cataloging of data, and project its future promise. Overall, the technology of GAG microarray exhibits enormous potential of evolving into more than a mere screening tool for studying GAG - protein systems.

Within a cell, synthetic and native genes compete for expression machinery, influencing cellular process dynamics through resource couplings. Models that simplify competitive resource binding kinetics can guide the design of strategies for countering these couplings. However, in bacteria resource availability and cell growth rate are interlinked, which complicates resource-aware biocircuit design. Capturing this interdependence requires coarse-grained bacterial cell models that balance accurate representation of metabolic regulation against simplicity and interpretability. We propose a coarse-grained E. coli cell model that combines the ease of simplified resource coupling analysis with appreciation of bacterial growth regulation mechanisms and the processes relevant for biocircuit design. Reliably capturing known growth phenomena, it provides a unifying explanation to disparate empirical relations between growth and synthetic gene expression. Considering a biomolecular controller that makes cell-wide ribosome availability robust to perturbations, we showcase our model\'s usefulness in numerically prototyping biocircuits and deriving analytical relations for design guidance.