Gausemycin Antibiotic Family Acts via Ca2+-Dependent Membrane Targeting.

We report the molecular mechanism of action of gausemycins and the isolation of new members of the family, gausemycins C (1c), D (1d), E (1e), and F (1f), the minor components of the mixture. To elucidate the mechanism of action of gausemycins, we investigated the antimicrobial activity of the most active compounds, gausemycins A and B, in the presence of Ca2+, other metal ions, and phosphate. Gausemycins require a significantly higher Ca2+ concentration for maximum activity than daptomycin but lower than that required for malacidine and cadasides. Species-specific antimicrobial activity was found upon testing against a wide panel of Gram-positive bacteria. Membranoactivity of gausemycins was demonstrated upon their interactions with model lipid bilayers and micelles. The pore-forming ability was found to be dramatically dependent on the Ca2+ concentration and the membrane lipid composition. An NMR study of gausemycin B in zwitterionic and anionic micelles suggested the putative structure of the gausemycin/membrane complex and revealed the binding of Ca2+ by the macrocyclic domain of the antibiotic.

Recent Advances in Molecular Mechanisms of Nucleoside Antivirals.

The search for new drugs has been greatly accelerated by the emergence of new viruses and drug-resistant strains of known pathogens. Nucleoside analogues (NAs) are a prospective class of antivirals due to known safety profiles, which are important for rapid repurposing in the fight against emerging pathogens. Recent improvements in research methods have revealed new unexpected details in the mechanisms of action of NAs that can pave the way for new approaches for the further development of effective drugs. This review accounts advanced techniques in viral polymerase targeting, new viral and host enzyme targeting approaches, and prodrug-based strategies for the development of antiviral NAs.

Aminooxy Click Modification of a Periodate-Oxidized Immunoglobulin G: A General Approach to Antibody-Drug Conjugates with Dye-Mediated Expeditious Stoichiometry Control.

A universal approach to the construction of antibody-drug conjugates (ADCs) has been developed. It relies on periodate oxidation of naturally present glycans of immunoglobulin G, followed by oxime ligation and, optionally, copper(I)-catalyzed alkyne-azide cycloaddition for conjugation with a toxic payload. The introduction of highly absorbing cyanine dyes into the linker allows for facile determination of the drug-antibody ratio. We applied this methodology to the synthesis of cytotoxic conjugates of an antibody against the tumor-associated antigen PRAME with doxorubicin and monomethyl auristatin E (MMAE). The resultant conjugates retained their affinity to a large extent, yet their cytotoxicity in vitro varied dramatically: while the doxorubicin-based conjugate did not produce any effect on cells, the MMAE-based one demonstrated specific activity against PRAME-expressing cancer cell lines. Importantly, the latter conjugate constitutes the first reported example of a PRAME-targeting ADC.

Alkyl Derivatives of Perylene Photosensitizing Antivirals: Towards Understanding the Influence of Lipophilicity.

Amphipathic perylene derivatives are broad-spectrum antivirals against enveloped viruses that act as fusion inhibitors in a light-dependent manner. The compounds target the lipid bilayer of the viral envelope using the lipophilic perylene moiety and photogenerating singlet oxygen, thereby causing damage to unsaturated lipids. Previous studies show that variation of the polar part of the molecule is important for antiviral activity. Here, we report modification of the lipophilic part of the molecule, perylene, by the introduction of 4-, 8-, and 12-carbon alkyls into position 9(10) of the perylene residue. Using Friedel-Crafts acylation and Wolff-Kishner reduction, three 3-acetyl-9(10)-alkylperylenes were synthesized from perylene and used to prepare 9 nucleoside and 12 non-nucleoside amphipathic derivatives. These compounds were characterized as fluorophores and singlet oxygen generators, as well as tested as antivirals against herpes virus-1 (HSV-1) and vesicular stomatitis virus (VSV), both known for causing superficial skin/mucosa lesions and thus serving as suitable candidates for photodynamic therapy. The results suggest that derivatives with a short alkyl chain (butyl) have strong antiviral activity, whereas the introduction of longer alkyl substituents (n = 8 and 12) to the perylenyethynyl scaffold results in a dramatic reduction of antiviral activity. This phenomenon is likely attributable to the increased lipophilicity of the compounds and their ability to form insoluble aggregates. Moreover, molecular dynamic studies revealed that alkylated perylene derivatives are predominately located closer to the middle of the bilayer compared to non-alkylated derivatives. The predicted probability of superficial positioning correlated with antiviral activity, suggesting that singlet oxygen generation is achieved in the subsurface layer of the membrane, where the perylene group is more accessible to dissolved oxygen.

Introduction of Carbonyl Groups into Antibodies.

Antibodies and their derivatives (scFv, Fabs, etc.) represent a unique class of biomolecules that combine selectivity with the ability to target drug delivery. Currently, one of the most promising endeavors in this field is the development of molecular diagnostic tools and antibody-based therapeutic agents, including antibody-drug conjugates (ADCs). To meet this challenge, it is imperative to advance methods for modifying antibodies. A particularly promising strategy involves the introduction of carbonyl groups into the antibody that are amenable to further modification by biorthogonal reactions, namely aliphatic, aromatic, and α-oxo aldehydes, as well as aliphatic and aryl-alkyl ketones. In this review, we summarize the preparation methods and applications of site-specific antibody conjugates that are synthesized using this approach.

Branched Linkers for Site-Specific Fluorescent Labeling of Antibodies.

Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry.

Membrane-Targeting Perylenylethynylphenols Inactivate Medically Important Coronaviruses via the Singlet Oxygen Photogeneration Mechanism.

Perylenylethynyl derivatives have been recognized as broad-spectrum antivirals that target the lipid envelope of enveloped viruses. In this study, we present novel perylenylethynylphenols that exhibit nanomolar or submicromolar antiviral activity against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and feline infectious peritonitis virus (FIPV) in vitro. Perylenylethynylphenols incorporate into viral and cellular membranes and block the entry of the virus into the host cell. Furthermore, these compounds demonstrate an ability to generate singlet oxygen when exposed to visible light. The rate of singlet oxygen production is positively correlated with antiviral activity, confirming that the inhibition of fusion is primarily due to singlet-oxygen-induced damage to the viral envelope. The unique combination of a shape that affords affinity to the lipid bilayer and the capacity to generate singlet oxygen makes perylenylethynylphenols highly effective scaffolds against enveloped viruses. The anticoronaviral activity of perylenylethynylphenols is strictly light-dependent and disappears in the absence of daylight (under red light). Moreover, these compounds exhibit negligible cytotoxicity, highlighting their significant potential for further exploration of the precise antiviral mechanism and the broader scope and limitations of this compound class.

Near-Infrared Dyes: Towards Broad-Spectrum Antivirals.

Broad antiviral activity in vitro is known for many organic photosensitizers generating reactive oxygen species under irradiation with visible light. Low tissue penetration of visible light prevents further development of antiviral therapeutics based on these compounds. One possible solution to this problem is the development of photosensitizers with near-infrared absorption (NIR dyes). These compounds found diverse applications in the photodynamic therapy of tumors and bacterial infections, but they are scarcely mentioned as antivirals. In this account, we aimed to evaluate the therapeutic prospects of various NIR-absorbing and singlet oxygen-generating chromophores for the development of broad-spectrum photosensitizing antivirals.

Radiochemical Synthesis of 4-[18F]FluorobenzylAzide and Its Conjugation with EGFR-Specific Aptamers.

Central nervous system tumors related to gliomas are of neuroectodermal origin and cover about 30% of all primary brain tumors. Glioma is not susceptible to any therapy and surgical attack remains one of the main approaches to its treatment. Preoperative tumor imaging methods, such as positron emission tomography (PET), are currently used to distinguish malignant tissue to increase the accuracy of glioma removal. However, PET is lacking a specific visualization of cells possessing certain molecular markers. Here, we report an application of aptamers to enhancing specificity in imaging tumor cells bearing the epidermal growth factor receptor (EGFR). Glioblastoma is characterized by increased EGFR expression, as well as mutations of this receptor associated with active division, migration, and adhesion of tumor cells. Since 2021, EGFR has been included into the WHO classification of gliomas as a molecular genetic marker. To obtain conjugates of aptamers GR20 and GOL1-specific to EGFR, a 4-[18F]fluorobenzylazide radiotracer was used as a synthon. For the production of the synthon, a method of automatic synthesis on an Eckert & Ziegler research module was adapted and modified using spirocyclic iodonium ylide as a precursor. Conjugation of 4-[18F]fluorobenzylazide and alkyne-modified aptamers was carried out using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with/without the TBTA ligand. As a result, it was possible to obtain 18F-labelled conjugates with 97% radiochemical purity for [18F]FB-GR20 and 98% for [18F]FB-GOL1. The obtained conjugates can be used for further studies in PET analysis on model animals with grafted glioblastoma.

Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach.

Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.

Photosensitizing Antivirals.

Antiviral action of various photosensitizers is already summarized in several comprehensive reviews, and various mechanisms have been proposed for it. However, a critical consideration of the matter of the area is complicated, since the exact mechanisms are very difficult to explore and clarify, and most publications are of an empirical and "phenomenological" nature, reporting a dependence of the antiviral action on illumination, or a correlation of activity with the photophysical properties of the substances. Of particular interest is substance-assisted photogeneration of highly reactive singlet oxygen (1O2). The damaging action of 1O2 on the lipids of the viral envelope can probably lead to a loss of the ability of the lipid bilayer of enveloped viruses to fuse with the lipid membrane of the host cell. Thus, lipid bilayer-affine 1O2 photosensitizers have prospects as broad-spectrum antivirals against enveloped viruses. In this short review, we want to point out the main types of antiviral photosensitizers with potential affinity to the lipid bilayer and summarize the data on new compounds over the past three years. Further understanding of the data in the field will spur a targeted search for substances with antiviral activity against enveloped viruses among photosensitizers able to bind to the lipid membranes.

Gausemycins†A,B: Cyclic Lipoglycopeptides from Streptomyces sp.*.

We report a novel family of natural lipoglycopeptides produced by Streptomyces sp. INA-Ac-5812. Two major components of the mixture, named gausemycins A and B, were isolated, and their structures were elucidated. The compounds are cyclic peptides with a unique peptide core and several remarkable structural features, including unusual positions of d-amino acids, lack of the Ca2+ -binding Asp-X-Asp-Gly (DXDG) motif, tyrosine glycosylation with arabinose, presence of 2-amino-4-hydroxy-4-phenylbutyric acid (Ahpb) and chlorinated kynurenine (ClKyn), and N-acylation of the ornithine side chain. Gausemycins have pronounced activity against Gram-positive bacteria. Mechanistic studies highlight significant differences compared to known glyco- and lipopeptides. Gausemycins exhibit only slight Ca2+ -dependence of activity and induce no pore formation at low concentrations. Moreover, there is no detectable accumulation of cell wall biosynthesis precursors under treatment with gausemycins.

Excimer-FRET Cascade in Dual DNA Probes: Open Access to Large Stokes Shift, Enhanced Acceptor Light up, and Robust RNA Sensing.

The efficacy of fluorescent hybridization assays is often limited by the low signal-to-background ratio of the probes that can be partially overcome by sophisticated signal amplification methods. Deep understanding of the mechanisms of fluorescence quenching and energy transfer in complex DNA probes and the choice of optimal donor/acceptor pairs along with rational design can significantly enhance the performance of DNA probes. Here, we proposed and studied novel Förster resonance energy transfer (FRET) dual DNA probes with the excimer-forming pyrene pair as a donor and sulfo-Cy3 dye as an acceptor, which demonstrated remarkable 75-fold enhancement of sulfo-Cy3 fluorescence upon target capturing. Stokes shift up to 220 nm minimizes fluorescence crosstalk. Time-correlated single-photon counting revealed two excited states of pyrene excimer wherein only one is directly involved in the resonance energy transfer to sulfo-Cy3. Optimized DNA probes demonstrated high sensitivity with excellent signal-to-background ratio, which were applied for visualization of 18S rRNA by fluorescent in situ hybridization in HEK-293T cells.

Toehold-Mediated Selective Assembly of Compact Discrete DNA Nanostructures.

Production of small discrete DNA nanostructures containing covalent junctions requires reliable methods for the synthesis and assembly of branched oligodeoxynucleotide (ODN) conjugates. This study reports an approach for self-assembly of hard-to-obtain primitive discrete DNA nanostructures-"nanoethylenes", dimers formed by double-stranded oligonucleotides using V-shaped furcate blocks. We scaled up the synthesis of V-shaped oligonucleotide conjugates using pentaerythritol-based diazide and alkyne-modified oligonucleotides using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and optimized the conditions for "nanoethylene" formation. Next, we designed nanoethylene-based "nanomonomers" containing pendant adapters. They demonstrated smooth and high-yield spontaneous conversion into the smallest cyclic product, DNA tetragon aka "nano-methylcyclobutane". Formation of DNA nanostructures was confirmed using native polyacrylamide gel electrophoresis (PAGE) and atomic force microscopy (AFM) and additionally studied by molecular modeling. The proposed facile approach to discrete DNA nanostructures using precise adapter-directed association expands the toolkit for the realm of DNA origami.

Simplistic perylene-related compounds as inhibitors of tick-borne encephalitis virus reproduction.

Rigid amphipathic fusion inhibitors are potent broad-spectrum antivirals based on the perylene scaffold, usually decorated with a hydrophilic group linked via ethynyl or triazole. We have sequentially simplified these structures by removing sugar moiety, then converting uridine to aniline, then moving to perylenylthiophenecarboxylic acids and to perylenylcarboxylic acid. All these polyaromatic compounds, as well as antibiotic heliomycin, still showed pronounced activity against tick-borne encephalitis virus (TBEV) with limited toxicity in porcine embryo kidney (PEK) cell line. 5-(Perylen-3-yl)-2-thiophenecarboxylic acid (5a) showed the highest antiviral activity with 50% effective concentration of approx. 1.6 nM.

Rigid amphipathic fusion inhibitors demonstrate antiviral activity against African swine fever virus.

Rigid amphipathic fusion inhibitors (RAFIs) are a family of nucleoside derivatives that inhibit the infectivity of several enveloped viruses by interacting with virion envelope lipids and inhibiting fusion between viral and cellular membranes. Here we tested the antiviral activity of two RAFIs, 5-(Perylen-3-ylethynyl)-arabino-uridine (aUY11) and 5-(Perylen-3-ylethynyl)uracil-1-acetic acid (cm1UY11) against African swine fever virus (ASFV), for which no effective vaccine is available. Both compounds displayed a potent, dose-dependent inhibitory effect on ASFV infection in Vero cells. The major antiviral effect was observed when aUY11 and cm1UY11 were added at early stages of infection and maintained during the complete viral cycle. Furthermore, virucidal assay revealed a significant extracellular anti-ASFV activity for both compounds. We also found decrease in the synthesis of early and late viral proteins in Vero cells treated with cm1UY11. Finally, the inhibitory effect of aUY11 and cm1UY11 on ASFV infection in porcine alveolar macrophages was confirmed. Overall, our study has identified novel anti-ASFV compounds with potential for future therapeutic developments.

4-Chloro-L-kynurenine as fluorescent amino acid in natural peptides.

4-Chloro-L-kynurenine (3-(4-chloroanthraniloyl)-L-alanine, L-4-ClKyn), an amino acid known as a prospective antidepressant, was recently for the first time found in nature in the lipopeptide antibiotic taromycin. Here, we report another instance of its identification in a natural product: 4-chloro-L-kynurenine was isolated from acidic hydrolysis of a new complex peptide antibiotic INA-5812. L-4-ClKyn is a fluorescent compound responsible for the fluorescence of the above antibiotic. Whereas fluorescence of 4-chlorokynurenine was not reported before, we synthesized the racemic compound and studied its emission in various solvents. Next, we prepared conjugates of DL-4-ClKyn with two suitable energy acceptors, BODIPY FL and 3-(phenylethynyl)perylene (PEPe), and studied fluorescence of the derivatives. 4-Chloro-DL-kynurenine emission is not detected in both conjugates, thus evidencing effective energy transfer. However, BODIPY FL emission in the conjugate is substantially reduced, probably due to collisional or photoinduced charge-transfer-mediated quenching. The intrinsic fluorescence of L-4-ClKyn amino acid in antibiotics paves the way for spectral studies of their mode of action.

Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N-Acetylgalactosamine Clusters.

We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt)3 as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.

Fine Tuning of Pyrene Excimer Fluorescence in Molecular Beacons by Alteration of the Monomer Structure.

Oligonucleotide probes labeled with pyrene pairs that form excimers have a number of applications in hybridization analysis of nucleic acids. A long excited state lifetime, large Stokes shift, and chemical stability make pyrene excimer an attractive fluorescent label. Here we report synthesis of chiral phosphoramidite building blocks based on (R)-4-amino-2,2-dimethylbutane-1,3-diol, easily available from an inexpensive d-(-)-pantolactone. 1-Pyreneacetamide, 1-pyrenecarboxamide, and DABCYL derivatives have been used in preparation of molecular beacon (MB) probes labeled with one or two pyrenes/quenchers. We observed significant difference in the excimer emission maxima (475-510 nm; Stokes shifts 125-160 nm or 7520-8960 cm-1) and excimer/monomer ratio (from 0.5 to 5.9) in fluorescence spectra depending on the structure and position of monomers in the pyrene pair. The pyrene excimer formed by two rigid 1-pyrenecarboxamide residues showed the brightest emission. This is consistent with molecular dynamics data on excimer stability. Increase of the excimer fluorescence for MBs after hybridization with DNA was up to 24-fold.

A triphenylcyclopropenylium mass tag: synthesis and application to ultrasensitive LC/MS analysis of amines.

Thiol adducts of triphenylcyclopropenylium undergo efficient heterolytic dissociation under conditions of both electrospray (ESI) and laser desorption ionization (LDI) mass spectrometry giving rise to a prominent signal of an aromatic C3Ph3(+) cation. A functionalized mass tagging reagent, an activated ester carrying an S-linked C3Ph3 unit, has been developed and used for the derivatization of amines and their subsequent HPLC/ESI-MS detection in low attomolar amounts.