Analysis tools for single-monomer measurements of self-assembly processes

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin

Exploring the Surface of the Ectodomain of the PD-L1 Immune Checkpoint with Small-Molecule Fragments

Development of small molecules targeting the PD-L1/PD-1 interface is advancing both in industry and academia, but only a few have reached early-stage clinical trials. Here, we take a closer look at the general druggability of PD-L1 using in silico hot spot mapping and nuclear magnetic resonance (NMR)-based characterization. We found that the conformational elasticity of the PD-L1 surface strongly influences the formation of hot spots. We deconstructed several generations of known inhibitors into fragments and examined their binding properties using differential scanning fluorimetry (DSF) and protein-based nuclear magnetic resonance (NMR). These biophysical analyses showed that not all fragments bind to the PD-L1 ectodomain despite having the biphenyl scaffold. Although most of the binding fragments induced PD-L1 oligomerization, two compounds, TAH35 and TAH36, retain the monomeric state of proteins upon binding. Additionally, the presence of the entire ectodomain did not affect the binding of the hit compounds and dimerization of PD-L1. The data demonstrated here provide important information on the PD-L1 druggability and the structure-activity relationship of the biphenyl core moiety and therefore may aid in the design of novel inhibitors and focused fragment libraries for PD-L1.This research has been supported by Grants Maestro 2017/26/A/ST5/00572 (to T.A.H.) , Sonata UMO-2020/39/D/ST4/01344 (to E.S.) , Preludium UMO-2021/41/N/ST4/03485 (to M.Z.) , and Preludium UMO-2020/37/N/ST4/02691 (to D.M.) from the National Science Centre, Poland. X.d.C. thanks the Basque Country Government for the predoctoral and EGONLABUR grants

Nutlin-3a-aa: Improving the Bioactivity of a p53/MDM2 Interaction Inhibitor by Introducing a Solvent-Exposed Methylene Group

Nutlin-3a is a reversible inhibitor of the p53/MDM2 interaction. We have synthesized the derivative Nutlin-3a-aa bearing an additional exocyclic methylene group in the piperazinone moiety. Nutlin-3a-aa is more active than Nutlin-3a against purified wild-type MDM2, and is more effective at increasing p53 levels and releasing transcription of p53 target genes from MDM2-induced repression. X-ray analysis of wild-type MDM2-bound Nutlin-3a-aa indicated that the orientation of its modified piperazinone ring was altered in comparison to the piperazinone ring of MDM2-bound Nutlin-3a, with the exocyclic methylene group of Nutlin-3a-aa pointing away from the protein surface. Our data point to the introduction of exocyclic methylene groups as a useful approach by which to tailor the conformation of bioactive molecules for improved biological activity.This work was generously supported by the Deutsche Forschungsgemeinschaft (BE 4572/3-1 to T.B.). We extend our thanks to Barbara Klüver, Katrin Eckhardt, Nadiya Brovchenko, and Domenique Herbstritt for experimental support. Parts of the data described in this manuscript have been published in the dissertation of Florian Nietzold (Leipzig University, 2019).31 In addition, this work was financially supported by the National Science Centre, Poland (NCN) under Grant Symphony 2014/12/W/NZ1/00457 (to T.A.H). We thank HZB for the allocation of synchrotron radiation beamtime. We acknowledge the MCB Structural Biology Core Facility (supported by the TEAM TECH CORE FACILITY/2017-4/6 grant from the Foundation for Polish Science) for valuable support. Open Access funding enabled and organized by Projekt DEAL

Effect of Selected Silyl Groups on the Anticancer Activity of 3,4-Dibromo-5-Hydroxy-Furan-2(5H)-One Derivatives

The pharmacological effects of carbon to silicon bioisosteric replacements have been widely explored in drug design and medicinal chemistry. Here, we present a systematic investigation of the impact of different silyl groups on the anticancer activity of mucobromic acid (MBA) bearing furan-2(5H)-one core. We describe a comprehensive characterization of obtained compounds with respect to their anticancer potency and selectivity towards cancer cells. All four novel compounds exert stronger antiproliferative activity than MBA. Moreover, 3b induce apoptosis in colon cancer cell lines. A detailed investigation of the mechanism of action revealed that 3b activity stems from the down-regulation of survivin and the activation of caspase-3. Furthermore, compound 3b attenuates the clonogenic potential of HCT-116 cells. Interestingly, we also found that depending on the type of the silyl group, compound selectivity towards cancer cells could be precisely controlled. Collectively, we demonstrated the utility of silyl groups for adjusting both the potency and selectivity of silicon-containing compounds. These data reveal a link between the types of silyl group and compound potency, which could have bearings for the design of novel silicon-based anticancer drugs

Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1

SummaryTargeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has recently provided breakthrough progress in the treatment of melanoma, non-small cell lung cancer, and other types of cancer. Small-molecule drugs interfering with this pathway are highly awaited, but their development is hindered by insufficient structural information. This study reveals the molecular details of the human PD-1/PD-L1 interaction based on an X-ray structure of the complex. First, it is shown that the ligand binding to human PD-1 is associated with significant plasticity within the receptor. Second, a detailed molecular map of the interaction surface is provided, allowing definition of the regions within both interacting partners that may likely be targeted by small molecules