Gonococcal Mimitope Vaccine Candidate Forms a Beta-Hairpin Turn and Binds Hydrophobically to a Therapeutic Monoclonal Antibody.

The spread of multidrug-resistant strains of Neisseria gonorrhoeae, the etiologic agent of gonorrhea, represents a global health emergency. Therefore, the development of a safe and effective vaccine against gonorrhea is urgently needed. In previous studies, murine monoclonal antibody (mAb) 2C7 was raised against gonococcal lipooligosaccharide (LOS). mAb 2C7 elicits complement-dependent bactericidal activity against gonococci, and its glycan epitope is expressed by almost every clinical isolate. Furthermore, we identified a peptide, cyclic peptide 2 (CP2) that mimicked the 2C7 LOS epitope, elicited bactericidal antibodies in mice, and actively protected in a mouse vaginal colonization model. In this study, we performed structural analyses of mAb 2C7 and its complex with the CP2 peptide by X-ray crystallography, NMR spectroscopy, and molecular dynamics (MD) simulations. The crystal structure of Fab 2C7 bound to CP2 showed that the peptide adopted a beta-hairpin conformation and bound the Fab primarily through hydrophobic interactions. We employed NMR spectroscopy and MD simulations to map the 2C7 epitope and identify the bioactive conformation of CP2. We also used small-angle X-ray scattering (SAXS) and native mass spectrometry to obtain further information about the shape and assembly state of the complex. Collectively, our new structural information suggests strategies for humanizing mAb 2C7 as a therapeutic against gonococcal infection and for optimizing peptide CP2 as a vaccine antigen.

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.

Molecular Insights into O-Linked Sialoglycans Recognition by the Siglec-Like SLBR-N (SLBRUB10712) of Streptococcus gordonii.

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3'SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections.

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.

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.

Theoretical and experimental studies on the interaction of biphenyl ligands with human and murine PD-L1: Up-to-date clues for drug design.

Today it is widely recognized that the PD-1/PD-L1 axis plays a fundamental role in escaping the immune system in cancers, so that anti-PD-1/PD-L1 antibodies have been evaluated for their antitumor properties in more than 1000 clinical trials. As a result, some of them have entered the market revolutionizing the treatment landscape of specific cancer types. Nonetheless, a new era based on the development of small molecules as anti PD-L1 drugs has begun. There are, however, some limitations to advancing these compounds into clinical stages including the possible difficulty in counteracting the PD-1/PD-L1 interaction in vivo, the discrepancy between the in vitro IC50 (HTFR assay) and cellular EC50 (immune checkpoint blockade co-culture assay), and the differences in ligands' affinity between human and murine PD-L1, which can affect their preclinical evaluation. Here, an extensive theoretical study, assisted by MicroScale Thermophoresis binding assays and NMR experiments, was performed to provide an atomistic picture of the binding event of three representative biphenyl-based compounds in both human and murine PD-L1. Structural determinants of the species' specificity were unraveled, providing unprecedented details useful for the design of next generation anti-PD-L1 molecules.

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.

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.

Cerebrospinal fluid lipoproteins inhibit α-synuclein aggregation by interacting with oligomeric species in seed amplification assays.

BACKGROUND: Aggregation of α-synuclein (α-syn) is a prominent feature of Parkinson's disease (PD) and other synucleinopathies. Currently, α-syn seed amplification assays (SAAs) using cerebrospinal fluid (CSF) represent the most promising diagnostic tools for synucleinopathies. However, CSF itself contains several compounds that can modulate the aggregation of α-syn in a patient-dependent manner, potentially undermining unoptimized α-syn SAAs and preventing seed quantification. METHODS: In this study, we characterized the inhibitory effect of CSF milieu on detection of α-syn aggregates by means of CSF fractionation, mass spectrometry, immunoassays, transmission electron microscopy, solution nuclear magnetic resonance spectroscopy, a highly accurate and standardized diagnostic SAA, and different in vitro aggregation conditions to evaluate spontaneous aggregation of α-syn. RESULTS: We found the high-molecular weight fraction of CSF (> 100,000 Da) to be highly inhibitory on α-syn aggregation and identified lipoproteins to be the main drivers of this effect. Direct interaction between lipoproteins and monomeric α-syn was not detected by solution nuclear magnetic resonance spectroscopy, on the other hand we observed lipoprotein-α-syn complexes by transmission electron microscopy. These observations are compatible with hypothesizing an interaction between lipoproteins and oligomeric/proto-fibrillary α-syn intermediates. We observed significantly slower amplification of α-syn seeds in PD CSF when lipoproteins were added to the reaction mix of diagnostic SAA. Additionally, we observed a decreased inhibition capacity of CSF on α-syn aggregation after immunodepleting ApoA1 and ApoE. Finally, we observed that CSF ApoA1 and ApoE levels significantly correlated with SAA kinetic parameters in n = 31 SAA-negative control CSF samples spiked with preformed α-syn aggregates. CONCLUSIONS: Our results describe a novel interaction between lipoproteins and α-syn aggregates that inhibits the formation of α-syn fibrils and could have relevant implications. Indeed, the donor-specific inhibition of CSF on α-syn aggregation explains the lack of quantitative results from analysis of SAA-derived kinetic parameters to date. Furthermore, our data show that lipoproteins are the main inhibitory components of CSF, suggesting that lipoprotein concentration measurements could be incorporated into data analysis models to eliminate the confounding effects of CSF milieu on α-syn quantification efforts.

Elucidating the concentration-dependent effects of thiocyanate binding to carbonic anhydrase.

Many proteins naturally carry metal centers, with a large share of them being in the active sites of several enzymes. Paramagnetic effects are a powerful source of structural information and, therefore, if the native metal is paramagnetic, or it can be functionally substituted with a paramagnetic one, paramagnetic effects can be used to study the metal sites, as well as the overall structure of the protein. One notable example is cobalt(II) substitution for zinc(II) in carbonic anhydrase. In this manuscript we investigate the effects of sodium thiocyanate on the chemical environment of the metal ion of the human carbonic anhydrase II. The electron paramagnetic resonance (EPR) titration of the cobalt(II) protein with thiocyanate shows that the EPR spectrum changes from A-type to C-type on passing from 1:1 to 1:1000-fold ligand excess. This indicates the occurrence of a change in the electronic structure, which may reflect a sizable change in the metal coordination environment in turn caused by a modification of the frozen solvent glass. However, paramagnetic nuclear magnetic resonance (NMR) data indicate that the metal coordination cage remains unperturbed even in 1:1000-fold ligand excess. This result proves that the C-type EPR spectrum observed at large ligand concentration should be ascribed to the low temperature at which EPR measurements are performed, which impacts on the structure of the protein when it is destabilized by a high concentration of a chaotropic agent.

A single amino acid deletion in the ER Ca2+ sensor STIM1 reverses the in vitro and in vivo effects of the Stormorken syndrome-causing R304W mutation.

Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1, which forms the Ca2+ release-activated Ca2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.

Solid-state NMR - a complementary technique for protein framework characterization.

Protein frameworks are an emerging class of biomaterial with medical and technological applications. Frameworks are studied mainly by X-ray diffraction or scattering techniques. Complementary strategies are required. Here, we report solid-state NMR analyses of a microcrystalline protein-macrocycle framework and the rehydrated freeze-dried protein. This methodology may aid the characterization of low-crystallinity frameworks.

Small-Molecule Ebselen Binds to YTHDF Proteins Interfering with the Recognition of N 6-Methyladenosine-Modified RNAs.

YTHDF proteins bind the N 6-methyladenosine (m6A)-modified mRNAs, influencing their processing, stability, and translation. Therefore, the members of this protein family play crucial roles in gene regulation and several physiological and pathophysiological conditions. YTHDF proteins contain a hydrophobic pocket that accommodates the m6A embedded in the RRACH consensus sequence on mRNAs. We exploited the presence of this cage to set up an m6A-competitive assay and performed a high-throughput screen aimed at identifying ligands binding in the m6A pocket. We report the organoselenium compound ebselen as the first-in-class inhibitor of the YTHDF m6A-binding domain. Ebselen, whose interaction with YTHDF proteins was validated via orthogonal assays, cannot discriminate between the binding domains of the three YTHDF paralogs but can disrupt the interaction of the YTHDF m6A domain with the m6A-decorated mRNA targets. X-ray, mass spectrometry, and NMR studies indicate that in YTHDF1 ebselen binds close to the m6A cage, covalently to the Cys412 cysteine, or interacts reversibly depending on the reducing environment. We also showed that ebselen engages YTHDF proteins within cells, interfering with their mRNA binding. Finally, we produced a series of ebselen structural analogs that can interact with the YTHDF m6A domain, proving that ebselen expansion is amenable for developing new inhibitors. Our work demonstrates the feasibility of drugging the YTH domain in YTHDF proteins and opens new avenues for the development of disruptors of m6A recognition.

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.

Functionalized Hyaluronic Acid for "In Situ" Matrix Metalloproteinase Inhibition: A Bioactive Material to Treat the Dry Eye Sydrome.

Hyaluronic acid (HA) is a naturally occurring polysaccharide with many molecular functions, including maintaining the structure and physiology of the tissues, tissue remodeling, and inflammation. HA is found naturally in physiological tear fluid, possesses excellent mucus-layer-adhesive properties, and is successfully employed in the treatment of dry eye syndrome (DES). However, HA has as major drawback: its rapid in vivo degradation by hyaluronidase. We report on a unique material, namely, HA-3, obtained by the functionalization of HA with the metalloproteinase inhibitor 3 (MMPI). This material is characterized by an increased resistance to hyaluronidase degradation, associated with MMP inhibition properties. The ability of HA-3 to prevent dehydration of human corneal epithelial cells in vitro and in vivo may accelerate the development of more efficient DES treatment and broaden the application of HA in human diseases.

Identification and Characterization of an RRM-Containing, RNA Binding Protein in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogen, known to acquire resistance to antibiotics used in the clinic. The RNA-binding proteome of this bacterium is poorly characterized, in particular for what concerns the proteins containing RNA Recognition Motif (RRM). Here, we browsed the A. baumannii proteome for homologous proteins to the human HuR(ELAVL1), an RNA binding protein containing three RRMs. We identified a unique locus that we called AB-Elavl, coding for a protein with a single RRM with an average of 34% identity to the first HuR RRM. We also widen the research to the genomes of all the bacteria, finding 227 entries in 12 bacterial phyla. Notably we observed a partial evolutionary divergence between the RNP1 and RNP2 conserved regions present in the prokaryotes in comparison to the metazoan consensus sequence. We checked the expression at the transcript and protein level, cloned the gene and expressed the recombinant protein. The X-ray and NMR structural characterization of the recombinant AB-Elavl revealed that the protein maintained the typical ß1α1ß2ß3α2ß4 and three-dimensional organization of eukaryotic RRMs. The biochemical analyses showed that, although the RNP1 and RNP2 show differences, it can bind to AU-rich regions like the human HuR, but with less specificity and lower affinity. Therefore, we identified an RRM-containing RNA-binding protein actually expressed in A. baumannii.

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.

HuR-targeted agents: An insight into medicinal chemistry, biophysical, computational studies and pharmacological effects on cancer models.

The Human antigen R (HuR) protein is an RNA-binding protein, ubiquitously expressed in human tissues, that orchestrates target RNA maturation and processing both in the nucleus and in the cytoplasm. A survey of known modulators of the RNA-HuR interactions is followed by a description of its structure and molecular mechanism of action - RRM domains, interactions with RNA, dimerization, binding modes with naturally occurring and synthetic HuR inhibitors. Then, the review focuses on HuR as a validated molecular target in oncology and briefly describes its role in inflammation. Namely, we show ample evidence for the involvement of HuR in the hallmarks and enabling characteristics of cancer, reporting findings from in vitro and in vivo studies; and we provide abundant experimental proofs of a beneficial role for the inhibition of HuR-mRNA interactions through silencing (CRISPR, siRNA) or pharmacological inhibition (small molecule HuR inhibitors).

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.

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 (1) 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. Among them, compound 10 demonstrated to strongly bind with the PD-L1 protein and challenged it in a co-culture of PD-L1 expressing cancer cells (PC9 and HCC827 cells) and peripheral blood mononuclear cells enhanced antitumor immune activity of the latter. Compound 10 significantly increased interferon γ release and apoptotic induction of cancer cells, with low cytotoxicity in healthy cells when compared to 1, thus paving the way for subsequent preclinical optimization and medical applications.