Discovery of a Druggable, Cryptic Pocket in SARS-CoV-2 nsp16 Using Allosteric Inhibitors.

A collaborative, open-science team undertook discovery of novel small molecule inhibitors of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase using a high throughput screening approach with the potential to reveal new inhibition strategies. This screen yielded compound 5a, a ligand possessing an electron-deficient double bond, as an inhibitor of SARS-CoV-2 nsp16 activity. Surprisingly, X-ray crystal structures revealed that 5a covalently binds within a previously unrecognized cryptic pocket near the S-adenosylmethionine binding cleft in a manner that prevents occupation by S-adenosylmethionine. Using a multidisciplinary approach, we examined the mechanism of binding of compound 5a to the nsp16 cryptic pocket and developed 5a derivatives that inhibited nsp16 activity and murine hepatitis virus replication in rat lung epithelial cells but proved cytotoxic to cell lines canonically used to examine SARS-CoV-2 infection. Our study reveals the druggability of this newly discovered SARS-CoV-2 nsp16 cryptic pocket, provides novel tool compounds to explore the site, and suggests a new approach for discovery of nsp16 inhibition-based pan-coronavirus therapeutics through structure-guided drug design.

Photochemical synthesis of pink silver and its use for monitoring peptide nitration via surface enhanced Raman spectroscopy (SERS).

Oxidative stress may cause extended tyrosine posttranslational modifications of peptides and proteins. The 3-nitro-L-tyrosine (Nit), which is typically formed, affects protein behavior during neurodegenerative processes, such as Alzheimer's and Parkinson's diseases. Such metabolic products may be conveniently detected at very low concentrations by surface enhanced Raman spectroscopy (SERS). Previously, we have explored the SERS detection of the Nit NO2 bending vibrational bands in a presence of hydrogen chloride (Niederhafner et al., Amino Acids 53:517-532, 2021, ibid). In this article, we describe performance of a new SERS substrate, "pink silver", synthesized photochemically. It provides SERS even without the HCl induction, and the acid further decreases the detection limit about 9 times. Strong SERS bands were observed in the asymmetric (1550-1475 cm-1) and symmetric (1360-1290 cm-1) NO stretching in the NO2 group. The bending vibration was relatively weak, but appeared stronger when HCl was added. The band assignments were supported by density functional theory modeling.

Monitoring peptide tyrosine nitration by spectroscopic methods.

Oxidative stress can lead to various derivatives of the tyrosine residue in peptides and proteins. A typical product is 3-nitro-L-tyrosine residue (Nit), which can affect protein behavior during neurodegenerative processes, such as those associated with Alzheimer's and Parkinson's diseases. Surface enhanced Raman spectroscopy (SERS) is a technique with potential for detecting peptides and their metabolic products at very low concentrations. To explore the applicability to Nit, we use SERS to monitor tyrosine nitration in Met-Enkephalin, rev-Prion protein, and α-synuclein models. Useful nitration indicators were the intensity ratio of two tyrosine marker bands at 825 and 870 cm-1 and a bending vibration of the nitro group. During the SERS measurement, a conversion of nitrotyrosine to azobenzene containing peptides was observed. The interpretation of the spectra has been based on density functional theory (DFT) simulations. The CAM-B3LYP and ωB97XD functionals were found to be most suitable for modeling the measured data. The secondary structure of the α-synuclein models was monitored by electronic and vibrational circular dichroism (ECD and VCD) spectroscopies and modeled by molecular dynamics (MD) simulations. The results suggest that the nitration in these peptides has a limited effect on the secondary structure, but may trigger their aggregation.

Binding of Lanthanide Complexes to Histidine-Containing Peptides Probed by Raman Optical Activity Spectroscopy.

Lanthanide complexes are used as convenient spectroscopic probes for many biomolecules. Their binding to proteins is believed to be enhanced by the presence of histidine, but the strength of the interaction significantly varies across different systems. To understand the role of peptide length and sequence, short histidine-containing peptides have been synthesized (His-Gly, His-Gly-Gly, His-Gly-Gly-Gly, Gly-His, Gly-His-Gly, His-His, and Gly-Gly-His) and circularly polarized luminescence (CPL) induced at the [Eu(dpa)3 ]3- complex has been measured by means of a Raman optical activity (ROA) spectrometer. The obtained data indicate relatively weak binding of the histidine residue to the complex, with a strong participation of other parts of the peptide. Longer peptides, low pH, and a histidine residue close to the N-peptide terminus favor the binding. The binding strengths are approximately proportional to the CPL intensity and roughly correlate with predictions based on molecular dynamics (MD) simulations. The specificity of lanthanide binding to the peptide structure and its intense luminescence and high optical activity make the ROA/CPL technique suitable for probing secondary and tertiary structures of peptides and proteins.

Reactivity of 9-aminoacridine drug quinacrine with glutathione limits its antiprion activity.

Quinacrine-the drug based on 9-aminoacridine-failed in clinical trials for prion diseases, whereas it was active in in vitro studies. We hypothesize that aromatic nucleophilic substitution at C9 could be contributing factor responsible for this failure because of the transfer of acridine moiety from quinacrine to abundant glutathione. Here, we described the semi-large-scale synthesis of the acridinylated glutathione and the consequences of its formation on biological and biophysical activities. The acridinylated glutathione is one order of magnitude weaker prion protein binder than the parent quinacrine. Moreover, according to log DpH 7.4 , the glutathione conjugate is two orders of magnitude more hydrophilic than quinacrine. Its higher hydrophilicity and higher dsDNA binding potency will significantly decrease its bioavailability in membrane-like environment. The glutathione deactivates quinacrine not only directly but also decreases its bioavailability. Furthermore, the conjugate can spontaneously decompose to practically insoluble acridone, which is precipitated out from the living systems.

Rapid acidolysis of benzyl group as a suitable approach for syntheses of peptides naturally produced by oxidative stress and containing 3-nitrotyrosine.

3-Nitrotyrosine (Nit) belongs to products of oxidative stress and could probably influence conformation of neurodegenerative proteins. Syntheses of peptides require availability of suitable synthon for introduction of Nit residue. Common phenolic protection groups are more acid labile, when they are attached to Nit residue. We have found that Fmoc-Nit(Bn)-OH is a good building block for syntheses of Nit containing peptides by Fmoc/tBu strategy. Interestingly, the peptides containing multiple Nit residues can be available solely by use of Fmoc-Nit(Bn)-OH synthon. Bn is removed rapidly with ca 80 % trifluoroacetic acid in dark. The cleavage of Bn from Fmoc-Nit(Bn)-OH proceeds via pseudo-first order mechanism with activation barrier 32 kcal mol(-1) and rate k = 15.3 s(-1) at 20 °C. This rate is more than 2,000,000 times faster than that for cleavage of benzyl from Tyr(Bn).

Nonplanar tertiary amides in rigid chiral tricyclic dilactams. Peptide group distortions and vibrational optical activity.

We investigate amide nonplanarity in vibrational optical activity (VOA) spectra of tricyclic spirodilactams 5,8-diazatricyclo[6,3,0,0(1,5)]undecan-4,9-dione (I) and its 6,6',7,7'-tetradeuterio derivative (II). These rigid molecules constrain amide groups to nonplanar geometries with twisted pyramidal arrangements of bonds to amide nitrogen atoms. We have collected a full range vibrational circular dichroism (VCD) and Raman optical activity (ROA) spectra including signals of C-H and C-D stretching vibrations. We report normal-mode analysis and a comparison of calculated to experimental VCD and ROA. The data provide band-to-band assignment and offer a possibility to evaluate roles of constrained nonplanar tertiary amide groups and rigid chiral skeletons. Nonplanarity shows as single-signed VCD and ROA amide I signals, prevailing the couplets expected to arise from the amide-amide interaction. Amide-amide coupling dominates amide II (mainly C'-N stretching, modified in tertiary amides by the absence of a N-H bond) transitions (strong couplet in VCD, no significant ROA) probably due to the close proximity of amide nitrogen atoms. At lower wavenumbers, ROA spectra exhibit another likely manifestation of amide nonplanarity, showing signals of amide V (δ(oop)(N-C) at ~570 cm(-1)) and amide VI (δ(oop)(C'═O) at ~700 cm(-1) and ~650 cm(-1)) vibrations.

Glutamate carboxypeptidase II does not process amyloid-ß peptide.

The accumulation of amyloid-ß (Aß) peptide is thought to be a major causative mechanism of Alzheimer's disease. Aß accumulation could be caused by dysregulated processing of amyloid precursor protein, yielding excessive amounts of Aß, and/or by inefficient proteolytic degradation of the peptide itself. Several proteases have been described as Aß degradation enzymes, most notably metalloendopeptidases, aspartic endopeptidases, and some exopeptidases. Recently a report suggested that another metallopeptidase, glutamate carboxypeptidase II (GCPII), can also cleave Aß. GCPII is a zinc exopeptidase that cleaves glutamate from N-acetyl-L-aspartyl-L-glutamate in the central nervous system and from pteroylpoly-γ-glutamate in the jejunum. GCPII has been proposed as a promising therapeutic target for disorders caused by glutamate neurotoxicity. However, an Aß-degrading activity of GCPII would compromise potential pharmaceutical use of GCPII inhibitors, because the enzyme inhibition might lead to increased Aß levels and consequently to Alzheimer's disease. Therefore, we analyzed the reported Aß-degrading activity of GCPII using highly purified recombinant enzyme and synthetic Aß. We did not detect any Aß degradation activity of GCPII or its homologue even under prolonged incubation at a high enzyme to substrate ratio. These results are in good agreement with the current detailed structural understanding of the substrate specificity and enzyme-ligand interactions of GCPII.

Quinacrine reactivity with prion proteins and prion-derived peptides.

Quinacrine is a drug that is known to heal neuronal cell culture infected with prions, which are the causative agents of neurodegenerative diseases called transmissible spongiform encephalopathies. However, the drug fails when it is applied in vivo. In this work, we analyzed the reason for this failure. The drug was suggested to "covalently" modify the prion protein via an acridinyl exchange reaction. To investigate this hypothesis more closely, the acridine moiety of quinacrine was covalently attached to the thiol groups of cysteines belonging to prion-derived peptides and to the full-length prion protein. The labeled compounds were conveniently monitored by fluorescence and absorption spectroscopy in the ultraviolet and visible spectral regions. The acridine moiety demonstrated characteristic UV-vis spectrum, depending on the substituent at the C-9 position of the acridine ring. These results confirm that quinacrine almost exclusively reacts with the thiol groups present in proteins and peptides. The chemical reaction alters the prion properties and increases the concentration of the acridine moiety in the prion protein.

Theoretical Modeling of the Surface-Enhanced Raman Optical Activity.

Surface-enhanced Raman optical activity (SEROA) is a new technique combining the sensitivity of the surface-enhanced Raman scattering (SERS) with the detailed information about molecular structure provided by the chiral spectroscopies. So far, experimental SEROA spectra have been reported in several studies, but the interpretation and theoretical background are rather limited. In this work, general expressions for the electromagnetic contribution to SEROA are derived using the matrix polarization theory and used to investigate the enhancement in model systems. The results not only reveal a strong dependence of the enhancement on the distance between the molecule and a metal part but also the dependence of the ratio of ROA and Raman intensities (circular intensity difference, CID) on the distance and rotational averaging. For a ribose model, an optimal molecule-colloid distance was predicted which provided the highest CIDs. However, the CID maximum disappeared after a rotational averaging. For cysteine zwitterion, the simulated SEROA and SERS spectra provided a qualitative agreement with previous experiments.

Bifunctional phenolic-choline conjugates as anti-oxidants and acetylcholinesterase inhibitors.

Because of the complex cascade of molecular events that can occur in the brain of an Alzheimer's disease (AD) patient, the therapy of this neurodegenerative disease seems more likely to be achieved by multifunctional drugs. Herein, a new series of dual-targeting ligands have been developed and in vitro bioevaluated. Their architecture is based on conjugating the acetylcholinesterase inhibition and anti-oxidant properties in one molecular entity. Specifically, a series of naturally occurring phenolic acids with recognized anti-oxidant properties (derivatives of caffeic acid, rosmarinic acid, and trolox) have been conjugated with choline to account for the recognition by acetylcholinesterase (AChE). The synthesized hybrid compounds evidenced AChE inhibitory capacity of micromolar range (rationalized by molecular modeling studies) and good antioxidant properties. Their effects on human neuroblastoma cells, previously treated with beta-amyloid peptides and 1-methyl-4-phenylpyridinium ion neurotoxins (to simulate AD and Parkinson's disease, respectively), also demonstrated a considerable capacity for protection against the cytotoxicity of these stressors.

Peptide and glycopeptide dendrimers and analogous dendrimeric structures and their biomedical applications.

The size of information that can be stored in nucleic acids, proteins, and carbohydrates was calculated. The number of hexamers for peptides is 64,000,000 (20(6)) and seems to be impressive in comparison with 4,096 (4(6)) hexanucleotides, but the number of isomers of hexasaccharides is 1.44 × 10(15). Carbohydrates are therefore the best high-density coding system. This language has been named glycocode resp. sugar code. In comparison with peptide dendrimers, the amount of information carried by glycopeptide dendrimers or glycodendrimers is therefore much higher. This is reflected by the variability of structures and functions (activities). This review is about the broad area of peptide and glycopeptide dendrimers. The dendrimeric state and physicochemical properties and general consequences are described, together with a cluster effect. The impact of cluster effect to biological, chemical, and physical properties is discussed. Synthesis of dendrimers by convergent and divergent approaches, "Lego" chemistry, ligation strategies, and click chemistry is given with many examples. Purification and characterization of dendrimers by chromatographic methods, electromigration methods, and mass spectrometry are briefly mentioned. Different types of dendrimers with cyclic core, i.e. RAFTs, TASPs and analogous cyclic structures, carbopeptides, carboproteins, octopus glycosides, inositol-based dendrimers, cyclodextrins, calix[4]arenes, resorcarenes, cavitands, and porphyrins are given. Dendrimers can be used for creation of libraries, catalysts, and solubilizing agents. Biocompatibility and toxicity of dendrimers is discussed, as well as their applications in nanoscience, nanotechnology, drug delivery, and gene delivery. Carbohydrate interactions of glycopeptide dendrimers (bacteria, viruses, and cancer) are described. Examples of dendrimers as anti-prion agents are given. Dendrimers represent a fast developing area which partly overlaps with nanoparticles and nanotechnologies.

Glycopeptide dendrimers, part III: a review. Use of glycopeptide dendrimers in immunotherapy and diagnosis of cancer and viral diseases.

Glycopeptide dendrimers containing different types of tumor associated-carbohydrate antigens (T(N), TF, sialyl-T(N), sialyl-TF, sialyl-Le(x), sialyl-Le(a) etc.) were used in diagnosis and therapy of different sorts of cancer. These dendrimeric structures with incorporated T-cell epitopes and adjuvants can be used as antitumor vaccines. Best results were obtained with multiantigenic vaccines, containing, e.g. five or six different TAAs. The topic of TAAs and their dendrimeric forms at molecular level are reviewed, including structure, syntheses, and biological activities. Use of glycopeptide dendrimers as antiviral vaccines against HIV and influenza is also described. Their syntheses, physico-chemical properties, and biological activities are given with many examples.

Glycopeptide dendrimers. Part II.

Glycopeptide dendrimers are regularly branched structures containing both carbohydrates and peptides. Various types of these compounds differing in composition and structure are mentioned, together with their practical use spanning from catalysis, transport vehicles to synthetic vaccines. This Part II (for Part I see JeZek J, et al., J. Pept. Sci. 2008; 14: 2-43) covers linear oligomers with variable valency (brush dendrimers, comb dendrimers), sequential oligopeptide carriers SOCn-I and SOCn-II, chitosan-based dendrimers, and brush dendrimers. Other types of glycopeptide dendrimers are self-immolative dendrimers (cascade release dendrimers, domino dendrimers), dendrimers containing omega-amino acids (Gly, beta-Ala, gamma-Abu and epsilon-aminohexanoic acid), etc. Microwave-assisted synthesis of dendrimers and libraries of glycopeptides and glycopeptide dendrimers are also included. Characterization of dendrimers by electromigration methods, mass spectrometry, and time-resolved and nonlinear optical spectroscopy, etc. plays an important role in purity assessment and structure characterization. Physicochemical properties of dendrimers including chirality are given. Stability of dendrimers, their biocompatibility and toxicity are reviewed. Finally, biomedical applications of dendrimers including imaging agents (contrast agents), site-specific drug delivery systems, artificial viruses, synthetic antibacterial, antiviral, and anticancer vaccines, inhibitors of cell surface protein-carbohydrate interactions, intervention with bacterial adhesion, etc. are given. Glycopeptide dendrimers were used also for studying recognition processes, as diagnostics and mimetics, for complexation of different cations, for therapeutic purposes, as immunodiagnostics, and in drug design.

Glycopeptide dendrimers. Part I.

Glycopeptide dendrimers are branched structures containing both carbohydrates and peptides. Various classes of these compounds differing in composition and structure are mentioned, together with their practical use spanning from catalysis, transport vehicles to synthetic vaccines. The main stress is given to glycopeptide dendrimers, namely multiple antigen glycopeptides (MAGs). In MAGs, the core, branches or both are composed of amino acids or peptides. Other classes of glycodendrimers (PAMAM, polypropylene imine, cyclodextrin, calixarene, etc.) are mentioned too, but to a smaller extent. Their syntheses, physicochemical properties and biological activities are given with many examples. Glycopeptide dendrimers can be used as inhibitors of cell surface protein-carbohydrate interactions, intervention with bacterial adhesion, for studying of recognition processes, diagnostics, imaging and contrast agents, mimetics, for complexation of different cationts, as site-specific molecular delivery systems, for therapeutic purposes, as immunodiagnostics and in drug design. Biomedical applications of glycopeptide dendrimers as drug and gene delivery systems are also given.

A role of the 9-aminoacridines and their conjugates in a life science.

The 9-aminoacridines play an important role in medicine. They were applied first in a treatment of protozoal infections in the beginning of the last century. Recently, it has been shown that the 9-aminoacridines are successful candidates for treatment of cancer, viral and prion diseases. Their conjugation with biomolecules such as peptides and proteins may modulate their activity, bioavailability and applicability. This review deals with the synthesis of 9-aminoacridine, its conjugation with variety of molecules and utilization of such conjugates in several fields of science.

Interpretation of synchrotron radiation circular dichroism spectra of anionic, cationic, and zwitterionic dialanine forms.

Electronic absorption and synchrotron radiation circular dichroism (SRCD) spectra of the anionic, cationic, and zwitterionic forms of L-alanyl-L-alanine (AA) in aqueous solutions were measured and interpreted by molecular dynamics (MD) and ab initio computations. Time-dependent density functional theory (TD DFT) was applied to predict the electronic excited states. The modeling enabled the assessment of the role of molecular conformation, charge, and interaction with the polar environment in the formation of the spectral shapes. Particularly, inclusion of explicit solvent molecules in the computations appeared to be imperative because of the participation of water orbitals in the amide electronic structure. Implicit dielectric continuum solvent models gave inferior results for clusters, especially at low-energy transitions. Because of the dispersion of transition energies, tens of water/AA clusters had to be averaged in order to obtain reasonable spectral shapes with a more realistic inhomogeneous broadening. The modeling explained most of the observed differences, as the anionic and zwitterionic SRCD spectra were similar and significantly different from the cationic spectrum. The greatest deviation between the experimental and theoretical curves observed for the lowest-energy negative anion signal can be explained by the limited precision of the TD DFT method, but also by the complex dynamics of the amine group. The results also indicate that differences in the experimental spectral shapes do not directly correlate with the peptide main-chain conformation. Future peptide and protein conformational studies based on circular dichroic spectroscopy can be reliable only if such effects of molecular dynamics, solvent structure, and polar solvent-solute interactions are taken into account.

Peptide dendrimers.

Dendrimers are branched structures and represent a fast growing field covering many areas of chemistry. Various types of dendrimers differing in composition and structure are mentioned, together with their practical use spanning from catalysis, transport vehicles to synthetic vaccines. The main stress is given to peptide dendrimers, namely, multiple antigenic peptides (MAPs). Their synthesis, physicochemical properties, biological activities, etc. have been described with many examples. MAPs can be used as diagnostics, mimetics, for complexation of different cations, as vaccines against parasites, bacteria, viruses, etc.