Lukasz Skalniak

NIF3L1 (also NIF3) is a highly conserved protein belonging to the DUF34 protein family with unknown molecular function, present in bacteria, archaea, and eukaryotes. Here, we present the first crystal structures of human NIF3, revealing a hexameric toroidal assembly with a central cavity gated by PII‐like insertion domains. Each subunit contains a mononuclear zinc‐binding site, marking a clear departure from the dinuclear metal centers of bacterial homologs and potential functional divergence of the scaffold. We further capture a half‐capped state with asymmetric disorder of the insertion domains and local rearrangements near the metal site, suggesting a gating mechanism that regulates access to the internal chamber. These findings uncover an unexpected level of structural plasticity in human NIF3 and provide a framework for future studies investigating whether the different lid conformations observed in our structures have functional relevance in vivo .

Testing of new small molecules for oncotherapy often requires discrimination between the actual, target-specific bioactivity and non-specific toxicity. We present a mathematical model for T-cell reactivation under the action of therapeutic compounds targeting the PD-1/PD-L1 interaction in a co-culture in vitro setup. The model enables the estimation of the maximum safe concentration and the EC ₅₀ value for T-cell activation from experimental data representing non-monotonic dose responses. The model describes the dose-dependent change in the strength of the luminescence signal using a system of ordinary differential equations. The estimates for the model parameters are based on experimental measurements of the signal for different concentrations of the compound. They are then used to calculate two parameters: C _{max_resp} – the concentration of the tested molecule at which maximal T-cell activation is achieved (illustrating a maximal safe concentration), and EC ₅₀ (reflecting the potency of the molecule) via a Monte Carlo method. By incorporating mechanistic assumptions regarding compound-induced T-cell activation and concentration-dependent toxicity, the model provides a coherent explanation of the observed luminescence trajectories across the entire dose range. The model serves as a platform supporting rational drug candidate optimization strategies within the domain of immune checkpoint modulation.

Antibodies targeting PD-1 and PD-L1 have achieved considerable success against various cancers, however their effectiveness is limited as both therapeutic and diagnostic tools. Meanwhile, small organic molecules and macrocyclic peptides are currently at various stages of development as modulators of the PD-1/PD-L1 axis. Here, we report an array of low molecular weight anti-PD-L1 macrocyclic peptides, among which two─FM2 and FM213─emerged as promising bifunctional suppressors of PD-1/PD-L1 binding. Through a compound-centric proteomic approach, we demonstrated that FM213 robustly binds to PD-L1 in NSCLC cells and exosomes. Furthermore, at its highest non-cytotoxic concentration (2 μM), FM213 (i) significantly enhances recognition and destruction of NSCLC cells by human PBMCs, and (ii) promotes the internalization of cell-surface PD-L1 into the cytosol, leading to its degradation via a lysosome-dependent pathway. Finally, coinhibition of TIGIT and PD-L1 by ^DTBP-3, a TIGIT inhibitor, and FM213, respectively, enhances the antitumor immunity of anti-PD-L1 ligands.

Infrared spectroscopy of biological samples in water environments remains challenging due to water’s strong IR absorption in the mid-infrared region and limitations in spatial resolution. Recently, a new optical photothermal infrared (O-PTIR) spectroscopy, which utilizes a laser working in the visible spectrum to probe the sample for IR absorption, has shown great capabilities in the measurements of biological samples, like cells, at sub-micron resolutions. Here, we report the first implementation of a water-dipping objective in a commercially available O-PTIR system. The measurements were carried out in a counter-propagating configuration, with IR illumination being done from below the sample through a calcium fluorite substrate, and visible light being directed through the 60x water-dipping objective from the top. Such a configuration allowed the direct acquisition of IR spectra from fixed cells in water. We present a detailed protocol for system calibration, background acquisition, and spectral measurement, further verified by measurements of U-2 OS cells and avian erythrocytes. Spectra acquired in water exhibit high signal-to-noise ratios and preserve biochemical specificity, with good differentiation between nuclear and cytoplasmic regions of cells. The spectra obtained in the water match the results of experiments done in the air in other configurations of the O-PTIR microscope. Compared to the usually used “sandwich” configurations, the water dipping objective offers much higher image quality and easier sample handling. The approach facilitates routine, high-resolution, label-free chemical imaging of cells in a more physiologically environment, with potential applications in experiments with live cells.

Programmed cell death protein 1 (PD-1) is crucial in inhibiting immune responses by modulating the activity of T cells. We present an in vitro assay that is based on a co-culture of primary immune cells represented by human peripheral blood mononuclear cells (PBMCs), isolated from healthy donors with Chinese hamster ovary-derived cell line (CHO-K1) overexpressing human PD-L1 protein ( h PD-L1) and an artificial TCR-activator construct (TCRAct). CHO-K1/TCRAct/ h PD-L1 cells mimic antigen-presenting cells by activating T cells via the T-cell receptor (TCR) and providing a ligand for the negative immune checkpoint (PD-L1 protein). The two components, PBMCs and CHO-K1/TCRAct/ h PD-L1 cells, when in co-culture, provide a T-cell activation (TCA) assay, which may be used to test the potency of molecules targeting the PD-1/PD-L1 immune checkpoint. This method relies on monitoring the activation of helper CD4+ and cytotoxic CD8+ T cells using flow cytometry by analyzing the expression levels of early (CD69), intermediate (CD25 and HLA-DR), and late (PD-1) activation/exhaustion markers. This is the well-established in vitro co-culture assay in which primary T-cell activation via the TCR is diminished by the concurrent presence of PD-1/PD-L1 immune checkpoint, which can be blocked resulting in increased expression of T-cell surface markers.

Cancer immunotherapy aims to employ the immune system to target cancer cells. The PD-1/PD-L1 axis is a critical immune checkpoint that tumors exploit to evade immune surveillance. In this study, we characterized three small-molecule PD-L1 inhibitors, Evixapodlin, MAX-10181, and INCB086550, currently undergoing clinical trials for cancers such as non-small cell lung cancer, renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, and melanoma. Using the homogeneous time resolved fluorescence assay, we confirmed that each compound potently disrupts human PD-1/PD-L1 binding with IC₅₀ values in the nanomolar range. PD-L1 oligomerization upon inhibitor binding was demonstrated through NMR analysis and confirmed by X-ray crystallography, which finally elucidated the binding interactions that stabilize these inhibitors at the PD-L1 interface. Cellular assays revealed dose-dependent T-cell activation, demonstrating the immunomodulatory potential of each compound and its cytotoxicity profiles. These findings underscore the promise of small-molecule PD-L1 inhibitors as viable alternatives to antibody-based therapies in cancer immunotherapy.

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