The Role of Lysozyme in the Formation of Bioinspired Silicon Dioxide.

Several organisms are able to polycondensate tetraoxosilicic(IV) acid to form silicon(IV) dioxide using polycationic molecules. According to an earlier mechanistic proposal, these molecules undergo a phase separation and recent experimental evidence appears to confirm this model. At the same time, polycationic proteins like lysozyme can also promote polycondensation of silicon(IV) dioxide, and they do so under conditions that are not compatible with liquid-liquid phase separation. In this manuscript we investigate this conundrum by molecular simulations.

Multivalent Calixarene Complexation of a Designed Pentameric Lectin.

We describe complex formation between a designed pentameric ß-propeller and the anionic macrocycle sulfonato-calix[8]arene (sclx8), as characterized by X-ray crystallography and NMR spectroscopy. Two crystal structures and 15N HSQC experiments reveal a single calixarene binding site in the concave pocket of the ß-propeller toroid. Despite the symmetry mismatch between the pentameric protein and the octameric macrocycle, they form a high affinity multivalent complex, with the largest protein-calixarene interface observed to date. This system provides a platform for investigating multivalency.

Machine Learning-Enhanced Quantum Chemistry-Assisted Refinement of the Active Site Structure of Metalloproteins.

Understanding the fine structural details of inhibitor binding at the active site of metalloenzymes can have a profound impact on the rational drug design targeted to this broad class of biomolecules. Structural techniques such as NMR, cryo-EM, and X-ray crystallography can provide bond lengths and angles, but the uncertainties in these measurements can be as large as the range of values that have been observed for these quantities in all the published structures. This uncertainty is far too large to allow for reliable calculations at the quantum chemical (QC) levels for developing precise structure-activity relationships or for improving the energetic considerations in protein-inhibitor studies. Therefore, the need arises to rely upon computational methods to refine the active site structures well beyond the resolution obtained with routine application of structural methods. In a recent paper, we have shown that it is possible to refine the active site of cobalt(II)-substituted MMP12, a metalloprotein that is a relevant drug target, by matching to the experimental pseudocontact shifts (PCS) those calculated using multireference ab initio QC methods. The computational cost of this methodology becomes a significant bottleneck when the starting structure is not sufficiently close to the final one, which is often the case with biomolecular structures. To tackle this problem, we have developed an approach based on a neural network (NN) and a support vector regression (SVR) and applied it to the refinement of the active site structure of oxalate-inhibited human carbonic anhydrase 2 (hCAII), another prototypical metalloprotein target. The refined structure gives a remarkably good agreement between the QC-calculated and the experimental PCS. This study not only contributes to the knowledge of CAII but also demonstrates the utility of combining machine learning (ML) algorithms with QC calculations, offering a promising avenue for investigating other drug targets and complex biological systems in general.

Integration of NMR Spectroscopy in an Analytical Workflow to Evaluate the Effects of Oxidative Stress on Abituzumab: Beyond the Fingerprint of mAbs.

The assessment of the higher-order structure (HOS) by NMR is a powerful methodology to characterize the structural features of biologics. Forced oxidative stress studies are used to investigate the stability profile, to develop pharmaceutical formulations and analytical methods. Here, the effects of forced oxidative stress by H2O2 on the monoclonal antibody Abituzumab have been characterized by a multianalytical approach combining NMR spectroscopy, mass spectrometry, differential scanning calorimetry, surface plasmon resonance, computational tools, and bioassays. This integrated strategy has provided qualitative and semiquantitative characterization of the samples and information at residue level of the effects that oxidation has on the HOS of Abituzumab, correlating them to the loss of the biological activity.

Interhelical interactions within the STIM1 CC1 domain modulate CRAC channel activation.

The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.

Site-Selective Functionalized PD-1 Mutant for a Modular Immunological Activity against Cancer Cells.

Targeting immune checkpoints is a well-established strategy in cancer therapy, and antibodies blocking PD-1/PD-L1 interactions to restore the immunological activity against cancer cells have been clinically validated. High-affinity mutants of the PD-1 ectodomain have recently been proposed as an alternative to antibodies to target PD-L1 on cancer cells, shedding new light on this research area. In this dynamic scenario, the PD-1 mutant, here reported, largely expands the chemical space of nonantibody and nonsmall-molecule inhibitor therapeutics that can be used to target cancer cells overexpressing PD-L1 receptors. The polyethylene glycol moieties and the immune response-stimulating carbohydrates, used as site-selective tags, represent the proof of concept for future applications.

Combining Solid-State NMR with Structural and Biophysical Techniques to Design Challenging Protein-Drug Conjugates.

Several protein-drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non-covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure-guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well-established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X-ray crystallography and NMR. In particular, we show that solid-state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes.

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. This is possible because the large size and the intrinsic flexibility of the complexes are not limiting factors for solid-state NMR.

Large Protein Assemblies for High-Relaxivity Contrast Agents: The Case of Gadolinium-Labeled Asparaginase.

Biologics are emerging as the most important class of drugs and are used to treat a large variety of pathologies. Most of biologics are proteins administered in large amounts, either by intramuscular injection or by intravenous infusion. Asparaginase is a large tetrameric protein assembly, currently used against acute lymphoblastic leukemia. Here, a gadolinium(III)-DOTA derivative has been conjugated to asparaginase, and its relaxation properties have been investigated to assess its efficiency as a possible theranostic agent. The field-dependent 1H longitudinal relaxation measurements of water solutions of gadolinium(III)-labeled asparaginase indicate a very large increase in the relaxivity of this paramagnetic protein complex with respect to small gadolinium chelates, opening up the possibility of its use as an MRI contrast agent.

Not only manganese, but fruit component effects dictate the efficiency of fruit juice as an oral magnetic resonance imaging contrast agent.

Several fruit juices are used as oral contrast agents to improve the quality of images in magnetic resonance cholangiopancreatography. They are often preferred to conventional synthetic contrast agents because of their very low cost, natural origin, intrinsic safety, and comparable image qualities. Pineapple and blueberry juices are the most employed in clinical practice due to their higher content of manganese(II) ions. The interest of pharmaceutical companies in these products is testified by the appearance in the market of fruit juice derivatives with improved contrast efficacy. Here, we investigate the origin of the contrast of blueberry juice, analyze the parameters that can effect it, and elucidate the differences with pineapple juice and manganese(II) solutions. It appears that, although manganese(II) is the paramagnetic ion responsible for the contrast, it is the interaction of manganese(II) with other juice components that modulates the efficiency of the juice as a magnetic resonance contrast agent. On these grounds, we conclude that blueberry juice concentrated to the same manganese concentration of pineapple juice would prove a more efficient contrast agent than pineapple juice.

Solid-state NMR methods for the characterization of bioconjugations and protein-material interactions.

Protein solid-state NMR has evolved dramatically over the last two decades, with the development of new hardware and sample preparation methodologies. This technique is now ripe for complex applications, among which one can count bioconjugation, protein chemistry and functional biomaterials. In this review, we provide our account on this aspect of protein solid-state NMR.

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. However, the size of pores in the gel, protein-matrix interactions, and the size of the embedded proteins often prevent the use of this methodology. The recent advancements of solid-state NMR allow for the comparison of the higher order structure of the matrix-embedded and free isotopically enriched proteins, allowing for the evaluation of the functionality of the material in several steps of hydrogel development. Moreover, the structural information at atomic detail on the matrix-protein interactions paves the way for a structure-based design of these biomaterials.

Automated Determination of Nuclear Magnetic Resonance Chemical Shift Perturbations in Ligand Screening Experiments: The PICASSO Web Server.

Nuclear magnetic resonance (NMR) is an effective, commonly used experimental approach to screen small organic molecules against a protein target. A very popular method consists of monitoring the changes of the NMR chemical shifts of the protein nuclei upon addition of the small molecule to the free protein. Multidimensional NMR experiments allow the interacting residues to be mapped along the protein sequence. A significant amount of human effort goes into manually tracking the chemical shift variations, especially when many signals exhibit chemical shift changes and when many ligands are tested. Some computational approaches to automate the procedure are available, but none of them as a web server. Furthermore, some methods require the adoption of a fairly specific experimental setup, such as recording a series of spectra at increasing small molecule:protein ratios. In this work, we developed a tool requesting a minimal amount of experimental data from the user, implemented it as an open-source program, and made it available as a web application. Our tool compares two spectra, one of the free protein and one of the small molecule:protein mixture, based on the corresponding peak lists. The performance of the tool in terms of correct identification of the protein-binding regions has been evaluated on different protein targets, using experimental data from interaction studies already available in the literature. For a total of 16 systems, our tool achieved between 79% and 100% correct assignments, properly identifying the protein regions involved in the interaction.

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. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.

A High-Resolution View of the Coordination Environment in a Paramagnetic Metalloprotein from its Magnetic Properties.

Metalloproteins constitute a significant fraction of the proteome of all organisms and their characterization is critical for both basic sciences and biomedical applications. A large portion of metalloproteins bind paramagnetic metal ions, and paramagnetic NMR spectroscopy has been widely used in their structural characterization. However, the signals of nuclei in the immediate vicinity of the metal center are often broadened beyond detection. In this work, we show that it is possible to determine the coordination environment of the paramagnetic metal in the protein at a resolution inaccessible to other techniques. Taking the structure of a diamagnetic analogue as a starting point, a geometry optimization is carried out by fitting the pseudocontact shifts obtained from first principles quantum chemical calculations to the experimental ones.

NMR quality control of fragment libraries for screening.

Fragment-based screening has evolved as a remarkable approach within the drug discovery process both in the industry and academia. Fragment screening has become a more structure-based approach to inhibitor development, but also towards development of pathway-specific clinical probes. However, it is often witnessed that the availability, immediate and long-term, of a high quality fragment-screening library is still beyond the reach of most academic laboratories. Within iNEXT (Infrastructure for NMR, EM and X-rays for Translational research), a EU-funded Horizon 2020 program, a collection of 782 fragments were assembled utilizing the concept of "poised fragments" with the aim to facilitate downstream synthesis of ligands with high affinity by fragment ligation. Herein, we describe the analytical procedure to assess the quality of this purchased and assembled fragment library by NMR spectroscopy. This quality assessment requires buffer solubility screening, comparison with LC/MS quality control and is supported by state-of-the-art software for high throughput data acquisition and on-the-fly data analysis. Results from the analysis of the library are presented as a prototype of fragment progression through the quality control process.

Single Peptide Backbone Surrogate Mutations to Regulate Angiotensin GPCR Subtype Selectivity.

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]6 -Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT2 R/AT1 R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21.

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. Here we describe a photo-catalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.

A New NT4 Peptide-Based Drug Delivery System for Cancer Treatment.

The development of selective tumor targeting agents to deliver multiple units of chemotherapy drugs to cancer tissue would improve treatment efficacy and greatly advance progress in cancer therapy. Here we report a new drug delivery system based on a tetrabranched peptide known as NT4, which is a promising cancer theranostic by virtue of its high cancer selectivity. We developed NT4 directly conjugated with one, two, or three units of paclitaxel and an NT4-based nanosystem, using NIR-emitting quantum dots, loaded with the NT4 tumor-targeting agent and conjugated with paclitaxel, to obtain a NT4-QD-PTX nanodevice designed to simultaneously detect and kill tumor cells. The selective binding and in vitro cytotoxicity of NT4-QD-PTX were higher than for unlabeled QD-PTX when tested on the human colon adenocarcinoma cell line HT-29. NT4-QD-PTX tumor-targeted nanoparticles can be considered promising for early tumor detection and for the development of effective treatments combining simultaneous therapy and diagnosis.

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. This approach is demonstrated in the specific cases of in-cell NMR and protein-based ligand studies.