Competition NMR for detection of hit/lead inhibitors of protein–protein interactions

Screening for small-molecule fragments that can lead to potent inhibitors of protein-protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low mu M-mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak mu M-mM ligand-protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex's protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers.(This article belongs to the Special Issue NMR in the Drug Design

Computer- and NMR-aided design of small-molecule inhibitors of the Hub1 protein

By binding to the spliceosomal protein Snu66, the human ubiquitin-like protein Hub1 is a modulator of the spliceosome performance and facilitates alternative splicing. Small molecules that bind to Hub1 would be of interest to study the protein-protein interaction of Hub1/Snu66, which is linked to several human pathologies, such as hypercholesterolemia, premature aging, neurodegenerative diseases, and cancer. To identify small molecule ligands for Hub1, we used the interface analysis, peptide modeling of the Hub1/Snu66 interaction and the fragment-based NMR screening. Fragment-based NMR screening has not proven sufficient to unambiguously search for fragments that bind to the Hub1 protein. This was because the Snu66 binding pocket of Hub1 is occupied by pH-sensitive residues, making it difficult to distinguish between pH-induced NMR shifts and actual binding events. The NMR analyses were therefore verified experimentally by microscale thermophoresis and by NMR pH titration experiments. Our study found two small peptides that showed binding to Hub1. These peptides are the first small-molecule ligands reported to interact with the Hub1 protein

CA-170 : a potent small-molecule PD-L1 inhibitor or not?

CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins – important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 – a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57

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

Design, Synthesis, and Biological Evaluation of 2-Hydroxy-4-phenylthiophene-3-carbonitrile as PD-L1 Antagonist and Its Comparison to Available Small Molecular PD-L1 Inhibitors

In search of a potent small molecular PD-L1 inhibitor, we designed and synthesized a compound based on a 2-hydroxy-4-phenylthiophene-3-carbonitrile moiety. Ligand's performance was tested in vitro and compared side-by-side with a known PD-L1 antagonist with a proven bioactivity BMS1166. Subsequently, we modified both compounds to allow 18F labeling that could be used for PET imaging. Radiolabeling, which is used in drug development and diagnosis, was applied to investigate the properties of those ligands and test them against tissue sections with diverse expression levels of PD-L1. We confirmed biological activity toward hPD-L1 for this inhibitor, comparable with BMS1166, while holding enhanced pharmacological properties. </p

Synthesis of hybrid compound 64Cu-ATMS/Ru-bpy as bimodal imaging agents for PET and optical imaging

W ostatnich latach w terapii nowotworów szczególny nacisk kładzie się na diagnozę chorej tkanki. Precyzyjne obrazowanie nowotworu umożliwia jego pełniejszą charakterystykę i pozwala zastosować odpowiedni dla rodzaju guza sposób leczenia. Wśród najnowszych metod do obrazowania nowotworów pojawiają się rozwiązania łączące dwie lub więcej technik diagnostycznych do tzw. obrazowania jednoczesnego. Zastosowanie takiej metody jest ściśle powiązane z koniecznością syntezy specjalnego znacznika, który umożliwi obserwowanie chorej tkanki wieloma technikami w tym samym czasie. W pracy przedstawiono ścieżkę syntezy związku, który łączy cechy markerów dla obrazowania metodą PET i obrazowania optycznego (OI). Opisano także wstępne badania, służące ocenie przydatności uzyskanej substancji jako nowego znacznika nowotworów.Otrzymana struktura wydaje się być obiecująca dla celów przyszłego obrazowania tkanek nowotworowych metodą PET-OI. Posiada ona dobre właściwości fizykochemiczne takie jak: duże wartości molowych współczynników absorbcji, oraz duże przesunięcie Stokesa. Testy in vitro pokazują z kolei świetne możliwości akumulacji związku w komórkach żywych, oraz jego stosunkowo niską cytotoksyczność.Presently, diagnosis of cancer tissue play an important role in cancer treatment. The detailed imaging could provide a more accurate description of the disease and allow for more successful treatment.Recently many studies have focused on multimodal imaging techniques as a way for the precise characteristics of the cancer. This method merges two or more diagnostic techniques in one examination. For this reason, one should use a special marker suitable for various imaging techniques.The thesis presents the path of synthesis of the compound that combines markers for PET and optical imaging (OI). The preliminary studies evaluating the usefulness of the resulting substance as a new marker of cancer are also described.The resulting structure appears to be promising for tumor bimodal imaging PET-OI in the future. It has good photophysical and chemical properties such as high values of molar absorption coefficients, and a large Stokes shift. In vitro tests have shown a high uptake of the compound by living cells and its relatively low cytotoxicity

PD-L1 inhibitors : different classes, activities, and mechanisms of action

Targeting the programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) interaction has become an established strategy for cancer immunotherapy. Although hundreds of small-molecule, peptide, and peptidomimetic inhibitors have been proposed in recent years, only a limited number of drug candidates show good PD-1/PD-L1 blocking activity in cell-based assays. In this article, we compare representative molecules from different classes in terms of their PD-1/PD-L1 dissociation capacity measured by HTRF and in vitro bioactivity determined by the immune checkpoint blockade (ICB) co-culture assay. We point to recent discoveries that underscore important differences in the mechanisms of action of these molecules and also indicate one principal feature that needs to be considered, which is the eventual human PD-L1 specificity