Epitope Mapping and Binding Assessment by Solid-State NMR Provide a Way for the Development of Biologics under the Quality by Design Paradigm.

Multispecific biologics are an emerging class of drugs, in which antibodies and/or proteins designed to bind pharmacological targets are covalently linked or expressed as fusion proteins to increase both therapeutic efficacy and safety. Epitope mapping on the target proteins provides key information to improve the affinity and also to monitor the manufacturing process and drug stability. Solid-state NMR has been here used to identify the pattern of the residues of the programmed cell death ligand 1 (PD-L1) ectodomain that are involved in the interaction with a new multispecific biological drug.

Interfering with the Tumor-Immune Interface: Making Way for Triazine-Based Small Molecules as Novel PD-L1 Inhibitors.

The inhibition of the PD-1/PD-L1 axis by monoclonal antibodies has achieved remarkable success in treating a growing number of cancers. However, a novel class of small organic molecules, with BMS-202 () as the lead, is emerging as direct PD-L1 inhibitors. Herein, we report a series of 2,4,6-tri- and 2,4-disubstituted 1,3,5-triazines, which were synthesized and assayed for their PD-L1 binding by NMR and homogeneous time-resolved fluorescence.

Evaluation of the Higher Order Structure of Biotherapeutics Embedded in Hydrogels for Bioprinting and Drug Release.

Biocompatible hydrogels for tissue regeneration/replacement and drug release with specific architectures can be obtained by three-dimensional bioprinting techniques. The preservation of the higher order structure of the proteins embedded in the hydrogels as drugs or modulators is critical for their biological activity. Solution nuclear magnetic resonance (NMR) experiments are currently used to investigate the higher order structure of biotherapeutics in comparability, similarity, and stability studies.

SARS-CoV-2 M inhibition by a zinc ion: structural features and hints for drug design.

Structural data on the SARS-CoV-2 main protease in complex with a zinc-containing organic inhibitor are already present in the literature and gave hints on the presence of a zinc binding site involving the catalytically relevant cysteine and histidine residues. In this paper, the structural basis of ionic zinc binding to the SARS-CoV-2 main protease has been elucidated by X-ray crystallography. The zinc binding affinity and its ability to inhibit the SARS-CoV-2 main protease have been investigated.

Fucosylated ubiquitin and orthogonally glycosylated mutant A28C: conceptually new ligands for lectin (BambL).

Two orthogonal, metal free click reactions, enabled to glycosylate ubiquitin and its mutant A28C forming two protein scaffolds with high affinity for BambL, a lectin from the human pathogen . A new fucoside analogue, with high affinity with BambL, firstly synthetized and co-crystallized with the protein target, provided the insights for sugar determinants grafting onto ubiquitin. Three ubiquitin-based glycosides were thus assembled.

CXCR4 antagonism sensitizes cancer cells to novel indole-based MDM2/4 inhibitors in glioblastoma multiforme.

Glioblastoma Multiforme (GBM) is a highly invasive primary brain tumour characterized by chemo- and radio-resistance and poor overall survival. GBM can present an aberrant functionality of p53, caused by the overexpression of the murine double minute 2 protein (MDM2) and its analogue MDM4, which may influence the response to conventional therapies. Moreover, tumour resistance/invasiveness has been recently attributed to an overexpression of the chemokine receptor CXCR4, identified as a pivotal mediator of glioma neovascularization.

A Structurally Simple Vaccine Candidate Reduces Progression and Dissemination of Triple-Negative Breast Cancer.

The Tn antigen is a well-known tumor-associated carbohydrate determinant, often incorporated in glycopeptides to develop cancer vaccines. Herein, four copies of a conformationally constrained mimetic of the antigen TnThr (GalNAc-Thr) were conjugated to the adjuvant CRM197, a protein licensed for human use. The resulting vaccine candidate, mime[4]CRM elicited a robust immune response in a triple-negative breast cancer mouse model, correlated with high frequency of CD4+ T cells and low frequency of M2-type macrophages, which reduces tumor progression and lung metastasis growth.

Synthesis of 3-Alkylideneisoindolin-1-ones via Sonogashira Cyclocarbonylative Reactions of 2-Ethynylbenzamides.

Cyclocarbonylative Sonogashira reactions of -ethynylbenzamides have been investigated. The process is carried out under CO pressure, in the presence of a very small amount of PdCl(PPh) (0.4 mol %) as a catalytic precursor and without the need for a Cu salt as the co-catalyst.

HOPPI-NMR: Hot-Peptide-Based Screening Assay for Inhibitors of Protein-Protein Interactions by NMR.

Protein-protein interactions (PPIs) contribute to the onset and/or progression of several diseases, especially cancer, and this discovery has paved the way for considering disruption of the PPIs as an attractive anti-tumor strategy. In this regard, simple and efficient biophysical methods for detecting the interaction of the inhibitors with the protein counterpart are still in high demand. Herein, we describe a convenient NMR method for the screening of putative PPI inhibitors based on the use of "hot peptides" (HOPPI-NMR).

The Photocatalyzed Thiol-ene reaction: A New Tag to Yield Fast, Selective and reversible Paramagnetic Tagging of Proteins.

Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand.

Insights into telomeric G-quadruplex DNA recognition by HMGB1 protein.

HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes.

Structural characterization of a protein adsorbed on aluminum hydroxide adjuvant in vaccine formulation.

The heterogeneous composition of vaccine formulations and the relatively low concentration make the characterization of the protein antigens extremely challenging. Aluminum-containing adjuvants have been used to enhance the immune response of several antigens over the last 90 years and still remain the most commonly used. Here, we show that solid-state NMR and isotope labeling methods can be used to characterize the structural features of the protein antigen component of vaccines and to investigate the preservation of the folding state of proteins adsorbed on Alum hydroxide matrix, providing the way to identify the regions of the protein that are mainly affected by the presence of the inorganic matrix.

Real-Time Insights into Biological Events: In-Cell Processes and Protein-Ligand Interactions.

FlowNMR has the aim of continuously monitoring processes that occur in conditions that are not compatible with being carried out within a closed tube. However, it is sample intensive and not suitable for samples, such as proteins or living cells, that are often available in limited volumes and possibly low concentrations. We here propose a dialysis-based modification of a commercial flowNMR setup that allows for recycling the medium while confining the sample (proteins and cells) within the active volume of the tube.

Metal centers in biomolecular solid-state NMR.

Solid state NMR (SSNMR) has earned a substantial success in the characterization of paramagnetic systems over the last decades. Nowadays, the resolution and sensitivity of solid state NMR in biological molecules has improved significantly and these advancements can be translated into the study of paramagnetic biomolecules. However, the electronic properties of different metal centers affect the quality of their SSNMR spectra differently, and not all systems turn out to be equally easy to approach by this technique.

Non-crystallographic symmetry in proteins: Jahn-Teller-like and Butterfly-like effects?

Partial symmetry, i.e., the presence of more than one molecule in the asymmetric unit of a crystal, is a relatively rare phenomenon in small-molecule crystallography, but is quite common in protein crystallography, where it is typically known as non-crystallographic symmetry (NCS).

Characterization of PEGylated Asparaginase: New Opportunities from NMR Analysis of Large PEGylated Therapeutics.

Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X-ray crystallography.

A small heterobifunctional ligand provides stable and water dispersible core-shell CdSe/ZnS quantum dots (QDs).

We describe a simple method to prepare water dispersible core-shell CdSe/ZnS quantum dots (QDs) 1 by capping QDs with a new thiol-containing heterobifunctional dicarboxylic ligand 4 (DHLA-EDADA). This ligand, obtained on a gram scale through a few synthetic steps, provides a compact layer on the QDs, whose hydrodynamic size in H2O is 15 nm ± 3 nm. The colloidal stability is dramatically enhanced with respect to the well-known (±) α-lipoic acid (DHLA).

Protein Glycosylation through Sulfur Fluoride Exchange (SuFEx) Chemistry: The Key Role of a Fluorosulfate Thiolactoside.

Protein glycosylation is the most complex post-translational modification process. More than 50 % of human cells proteins are glycosylated, whereas bacteria such as E. coli do not have this modification machinery.

Simultaneous Targeting of RGD-Integrins and Dual Murine Double Minute Proteins in Glioblastoma Multiforme.

In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin α5β1 and p53 are part of convergent pathways in the control of glioma apoptosis. This observation prompted us to seek a molecule able to simultaneously modulate both target families. Analyzing the results of a previous virtual screening against murine double minute 2 protein (MDM2), we envisaged that Arg-Gly-Asp (RGD)-mimetic molecules could be inhibitors of MDM2/4.

Engineering l-asparaginase for spontaneous formation of calcium phosphate bioinspired microreactors.

Active bioinspired materials are appealing biotechnological targets, and their study is gaining momentum. These materials, which comprise of an inorganic matrix and one or more biomolecules, are extremely variable and therefore may result difficult to characterize in their intimate structure. In this work we have prepared a hydroxyapatite-l-asparaginase composite, with the perspective of using it in acute leukemia treatment.

Atomic structural details of a protein grafted onto gold nanoparticles.

The development of a methodology for the structural characterization at atomic detail of proteins conjugated to nanoparticles would be a breakthrough in nanotechnology. Solution and solid-state NMR spectroscopies are currently used to investigate molecules and peptides grafted onto nanoparticles, but the strategies used so far fall short in the application to proteins, which represent a thrilling development in theranostics. We here demonstrate the feasibility of highly-resolved multidimensional heteronuclear spectra of a large protein assembly conjugated to PEGylated gold nanoparticles.

Long-range paramagnetic NMR data can provide a closer look on metal coordination in metalloproteins.

Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metalloproteins or metal-substituted proteins, mostly as distance or angle restraints. However, they could in principle provide much more information, because the magnetic susceptibility of a paramagnetic metal ion is largely determined by its coordination sphere. This information can in turn be used to evaluate changes occurring in the coordination sphere of the metal when ligands (e.

Characterization of the Conjugation Pattern in Large Polysaccharide-Protein Conjugates by NMR Spectroscopy.

Carbohydrate-based vaccines are among the safest and most effective vaccines and represent potent tools for prevention of life-threatening bacterial infectious diseases, like meningitis and pneumonia. The chemical conjugation of a weak antigen to protein as a source of T-cell epitopes generates a glycoconjugate vaccine that results more immunogenic. Several methods have been used so far to characterize the resulting polysaccharide-protein conjugates.

Regulation of HuR structure and function by dihydrotanshinone-I.

The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide.