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  • Mechanism of Gating and Isoform-Specific Inhibition in Renal CLC Chloride Channels. bioRxiv : the preprint server for biology Chien, C. T., Sobecks, B. L., Powers, A. S., Kreiter, J., Das, A., Barry, C. N., Chen, M., Hinman, A., Petrakian, C. F., Trifkovic, N., Williams, B., Wood, C. A., Xu, M., Dror, R. O., Chiu, W., Maduke, M. 2026

    Abstract

    Hyponatremia is a prevalent disorder marked by excess water retention and substantial morbidity, motivating interest in the CLC-Ka chloride channel as a therapeutic target. Selectively inhibiting CLC-Ka without affecting the closely related CLC-Kb is essential for preventing serious side effects. However, developing isoform-selective inhibitors has been challenging because most small molecules do not distinguish between CLC-Ka and CLC-Kb, and the basis for selectivity in the few known exceptions remains unclear. The small molecule BIM1 preferentially inhibits CLC-Ka over CLC-Kb, providing an opportunity to dissect isoform-specific pharmacology. To investigate this mechanism, we determined cryo-EM structures of BIM1 and BIM15, a related nonselective analog, bound to a CLC-K variant engineered to match the human CLC-Ka binding pocket. Structural and computational analyses reveal that inhibition and isoform selectivity are anchored by interactions with a conserved lysine, with surrounding binding-site residues subtly tuning the local electrostatic environment to promote or disfavor these contacts. These analyses further identify a dynamic extracellular loop that intermittently occludes the access pathway, indicating its role as a gate for ions and inhibitors. BIM15 engages this gating loop more extensively than BIM1, suggesting that differential loop engagement contributes to inhibitor selectivity. To probe how gating reshapes this region, we solved the structure in the presence of Ca2+, which favors channel opening, and found the gating loop ordered and withdrawn from the pathway. Together, these findings elucidate how CLC-K channels gate and how subtle binding-site differences and loop dynamics shape isoform-specific drug binding, providing a foundation for designing next-generation CLC-Ka inhibitors.

    View details for DOI 10.64898/2026.02.17.706469

    View details for PubMedID 41756880

    View details for PubMedCentralID PMC12934935

  • High throughput mutational characterization of the GPCR ligand C5a using yeast display and deep sequencing. Structure (London, England : 1993) Xu, Y., Thakkar, K., Guan, L., Miao, Y., Mehibel, M., Lee, R. B., Marciano, D., Viswanathan, V., Wang, Z., Wang, J., Ji, L., Cao, H., Petrakian, C. F., Valenzuela, J., LaGory, E., Jia, X., Moon, E. J., Martinez, R., Wu, F., Frock, R. L., Moding, E. J., Le, Q. T., Rankin, E. B., Zhang, C., Huang, P., Olcina, M. M., Giaccia, A. J., Graves, E. E. 2025

    Abstract

    High-throughput mutagenesis approaches are widely employed to systematically characterize protein functions and play a critical role in therapeutic developments. As the largest class of membrane receptors, G protein-coupled receptors (GPCRs) are a primary focus of these studies. However, while significant progress has been made in understanding GPCRs themselves, mutagenesis studies on their ligands have lagged behind, because of the difficulties in solubilizing the target receptor. In this study, we present a novel approach that employs lipid vesicles to embed and stabilize target membrane receptors, allowing direct ligand screening. We applied this platform to investigate the anaphylatoxin complement 5a (C5a) and examined how mutations affect binding to its two native GPCRs: complement 5a receptor 1 (C5aR1) and complement 5a receptor 2 (C5aR2). The screening revealed new insights into the molecular basis of the interaction and led to the discovery of novel ligands that selectively activate C5aR2, but not C5aR1.

    View details for DOI 10.1016/j.str.2025.10.002

    View details for PubMedID 41151574

  • A Generalizable Fluorescence Sensor Platform for Sample Preparation-Free Protein Detection. Advanced materials (Deerfield Beach, Fla.) Wu, H. D., Trinh, T., Li, T., Thapa, S., Lee, R. B., Nguyen, T. T., Petrakian, C. F., Eisenstein, M., Schneebeli, S. T., Li, J., Tom Soh, H. 2025: e19662

    Abstract

    Modern molecular detection assays such as enzyme-linked immunosorbent assays (ELISAs) offer excellent sensitivity and specificity, but typically require multiple reagents and extensive sample preparation, limiting their usefulness as rapid diagnostics. A generalizable biosensor platform is introduced that enables single-step, sample preparation-free detection of protein analytes with high sensitivity in complex samples. The NanoFluor system employs Janelia Fluor dyes coupled to a nanobody via HaloTag conjugation with a flexible glycine-serine linker, where the dye undergoes a switch from a non-fluorescent to a fluorescent state when the coupled nanobody binds to its target. It is demonstrated that the NanoFluor design achieves detection limits as low as picomolar concentrations across diverse protein targets. Molecular dynamics simulations, coupled with quantum mechanics/molecular mechanics computational models, reveal the mechanistic basis for the fluorescence change, and demonstrate the feasibility of multiplexed detection in complex samples including undiluted serum. This versatile, simple biosensor design can prove valuable for point-of-care diagnostics and other molecular detection applications.

    View details for DOI 10.1002/adma.202419662

    View details for PubMedID 40847924

  • An engineered interleukin-11 decoy cytokine inhibits receptor signaling and proliferation in lung adenocarcinoma. Bioengineering & translational medicine McIntosh, B. J., Hartmann, G. G., Yamada-Hunter, S. A., Liu, P., Williams, C. F., Sage, J., Cochran, J. R. 2023; 8 (6): e10573

    Abstract

    The cytokine interleukin (IL)-11 has been shown to play a role in promoting fibrosis and cancer, including lung adenocarcinoma, garnering interest as an attractive target for therapeutic intervention. We used combinatorial methods to engineer an IL-11 variant that binds with higher affinity to the IL-11 receptor and stimulates enhanced receptor-mediated cell signaling. Introduction of two additional point mutations ablates IL-11 ligand/receptor association with the gp130 coreceptor signaling complex, resulting in a high-affinity receptor antagonist. Unlike wild-type IL-11, this engineered variant potently blocks IL-11-mediated cell signaling and slows tumor growth in a mouse model of lung cancer. Our approach highlights a strategy where native ligands can be engineered and exploited to create potent receptor antagonists.

    View details for DOI 10.1002/btm2.10573

    View details for PubMedID 38023717

    View details for PubMedCentralID PMC10658506

  • An engineered interleukin-1 decoy cytokine inhibits receptor signaling and proliferation in lung adenocarcinoma BIOENGINEERING & TRANSLATIONAL MEDICINE McIntosh, B. J., Hartmann, G. G., Yamada-Hunter, S. A., Liu, P., Williams, C. F., Sage, J., Cochran, J. R. 2023

    View details for DOI 10.1002/btm2.10573

    View details for Web of Science ID 001031916000001