Alexey Ustinov

A series of hemicyanine derivatives was synthesized via condensation of 1,2,3,3‐tetramethyl‐3H‐indolium and its 5‐iodo and 4,6‐diiodo derivatives with polyaromatic aldehydes. Their spectral properties, singlet oxygen photogeneration, and inhibitory activity against vesicular stomatitis virus (VSV) were investigated. The aim of this study was to combine a positive charge for improved solubility with either a pyrene or perylene polycycle and heavy iodine atoms to enhance singlet oxygen generation and induce a bathochromic shift in the dye absorption. It was found that the synthesized hemicyanines are unstable both toward various nucleophiles, particularly the cyanide anion, and toward light. Although the perylene‐based derivatives demonstrate antiviral activity against VSV, the potential application of such compounds lies not in the antiviral domain but rather in the field of chemical sensors and pH indicators.

The vast majority of viruses causing human and animal diseases are enveloped—their virions contain an outer lipid bilayer originating from a host cell. Small molecule antivirals targeting the lipid bilayer cover the broadest spectrum of viruses. In this context, we consider the chemical nature and mechanisms of action of membrane-targeting antivirals. They can affect virions by (1) physically modulating membrane properties to inhibit fusion of the viral envelope with the cell membrane, (2) physically affecting envelope lipids and proteins leading to membrane damage, pore formation and lysis, (3) causing photochemical damage of unsaturated membrane lipids resulting in integrity loss and fusion arrest. Other membrane-active compounds can target host cell membranes involved in virion’s maturation, coating, and egress (endoplasmic reticulum, Golgi apparatus, and outer membrane) affecting these last stages of viral reproduction. Both virion- and host-targeting membrane-active molecules are promising concepts for broad-spectrum antivirals. A panel of approved antivirals would be a superior weapon to respond to and control emerging disease outbreaks caused by new viral strains and variants.

The development of broad‐spectrum antiviral drugs effective against a wide range of viruses is of significant practical importance. Derivatives of perylene, a pentacyclic aromatic hydrocarbon, demonstrate pronounced antiviral activity. These compounds act primarily as membrane‐active singlet oxygen photogenerators, disrupting virions and inhibiting their fusion with the host cell membrane. Modification of the perylene core allows for chemical diversification of antiviral photosensitizers. Additionally, achieving a bathochromic shift of the absorption band is crucial for effective treatment of superficial lesions, as it facilitates deeper tissue penetration of therapeutic light. In this work, donor‐acceptor perylenylethylenes and (perylenethienyl)ethylenes were synthesized and evaluated for their spectral properties, singlet oxygen photogeneration, and inhibitory activity against vesicular stomatitis virus (VSV), a representative enveloped virus. Incorporation of a thiophene moiety into the molecule significantly enhanced both the singlet oxygen generation ability and the antiviral activity. These findings provide useful insights into the relationship between the structure, spectral/photochemical properties, and biological activity of perylene‐based photosensitizers.

Enveloped viruses, such as flaviviruses and coronaviruses, are pathogens of significant medical concern that cause severe infections in humans. Some photosensitizers are known to possess virucidal activity against enveloped viruses, targeting their lipid bilayer. Here we report a series of halogenated difluoroboron-dipyrromethene (BODIPYs) photosensitizers with strong virus-inactivating activity. Our structure-activity relationship analysis revealed that BODIPY scaffolds with a heavy halogen atom demonstrate significant efficacy against both tick-borne encephalitis virus (TBEV; Flaviviridae family) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; Coronaviridae family) along with high singlet oxygen quantum yields. Moreover, select compounds also inactivated other enveloped viruses, such as herpes simplex virus type 1 and monkeypox virus. The nature and length of the alkyl side chain notably influenced the virus-inactivating activity of BODIPY molecules. Furthermore, molecular dynamics studies highlighted the critical importance of the positioning of the chromophore moiety within the lipid bilayer. As membrane-targeting photosensitizers, BODIPYs interact directly with virus particles, causing damage to the viral envelope membranes. Thus, TBEV pretreated with BODIPY was completely noninfective for lab mice. Consequently, BODIPY-based photosensitizers hold potential either as broad-spectrum virus-inactivating antivirals against a variety of phylogenetically unrelated enveloped viruses or as potent inactivators of viruses for the development of vaccines for preventing life-threatening emerging viral diseases.

Sulfonated indocyanines 3 and 5 (sCy3, sCy5) are widely used to label biomolecules. Their high molar absorption coefficients and lack of spectral overlap with biopolymers make them ideal as linker components for rapid assessment of bioconjugate stoichiometry. We recently found that the determination of the sCy3:sCy5 molar ratio in a conjugate from its optical absorption spectrum is not straightforward, as the sCy3:sCy5 absorbance ratio at the maxima tends to be larger than expected. In this work, we have investigated this phenomenon in detail by studying the spectral properties of a series of sCy3-sCy5 conjugates in which the dyes are separated by linkers of various lengths, including DNA duplexes. It was found that when sCy3 and sCy5 are located in close proximity, they consistently exhibit an “abnormal” absorbance ratio. However, when the two dyes are separated by long rigid DNA-based spacers, the absorbance ratio becomes consistent with their individual molar absorption coefficients. This phenomenon should be taken into account when assessing the molar ratio of the dyes by UV-Vis spectroscopy.

Homogeneous antibody–drug conjugates (ADCs) exhibit significantly improved pharmacological properties compared to their heterogeneous counterparts. Site-specific conjugation of the payload to the IgG required for homogeneity can be achieved using enzymes. One example is microbial transglutaminase (MTGase), which can selectively perform transamidation on the Q295 residue of human Fc when N297 glycans are removed. As a result, two modifications can be introduced per IgG molecule; however, achieving higher drug-to-antibody ratios (DARs) requires the use of branched linkers. While several such linkers have been reported, little information is available on the relationship between linker structure and ADC properties. To address this gap, we synthesized two branched amino triazide linkers, differing by a PEG₄ fragment inserted after the branching point, which were used to prepare two homogeneous trastuzumab-based DAR 6 ADCs (a “short” and a “long” one). This was achieved by a two-step process consisting of enzymatic linker conjugation followed by bioorthogonal coupling with a cleavable linker bearing monomethyl auristatin E (MMAE). Two other trastuzumab–MMAE conjugates were used as controls: a heterogeneous DAR 6 ADC, made using conventional thiol–maleimide chemistry, and a homogeneous DAR 2 ADC. We found that, while the four conjugates had identical affinity for HER2, their cytotoxicity differed significantly: the “long” homogeneous DAR 6 ADC was just as active as its heterogeneous counterpart, but the “short” DAR 6 ADC was an order of magnitude less potent, inferior even to the DAR 2 conjugate. Our findings indicate that the length of the branched linker critically affects the cytotoxic activity of ADCs, possibly due to steric hindrance influencing the rate of linker cleavage by lysosomal enzymes.

The development of broad-spectrum antiviral drugs effective against a wide variety of viruses is of significant practical importance. Perylene derivatives, which are pentacyclic aromatic hydrocarbons, have demonstrated strong antiviral activity. These compounds primarily act as membranoactive singlet oxygen photogenerators, disrupting virions and inhibiting fusion in enveloped viruses. Modifying the perylene core allows for chemical diversification of antiviral photosensitizers. Additionally, achieving a bathochromic shift in absorbance is crucial for the effective treatment of superficial lesions, as it facilitates deeper tissue penetration of therapeutic light. In this work, donor–acceptor perylenylethylenes and (perylenethienyl)ethylenes were synthesized and evaluated for their spectral properties, singlet oxygen photogeneration, and antiviral activity against vesicular stomatitis virus (VSV), a representative enveloped virus. The incorporation of a thiophene unit into the molecule significantly enhanced both the quantum yield of singlet oxygen generation and the antiviral activity. These findings provide valuable insights into the relationship between the structure, spectral/photochemical properties, and biological activity of perylene-based photosensitizers.

Background/Objectives: Targeted delivery of chemotherapeutic agents is a well-established approach to cancer therapy. Antibody–drug conjugates (ADCs) typically carry toxic payloads attached to a tumor-associated antigen-targeting IgG antibody via an enzyme-cleavable linker that releases the drug inside the cell. Aptamers are a promising alternative to antibodies in terms of antigen targeting; however, their polynucleotide nature and smaller size result in a completely different PK/PD profile compared to an IgG. This may prove advantageous: owing to their lower molecular weight, aptamer-drug conjugates may achieve better penetration of solid tumors compared to ADCs. Methods: On the way to therapeutic aptamer–drug conjugates, we aimed to develop a versatile and modular approach for the assembly of aptamer–enzymatically cleavable payload conjugates of various drug–aptamer ratios. We chose the epidermal growth factor receptor (EGFR), a transmembrane protein often overexpressed in brain tumors, as the target antigen. We used the 46 mer EGFR-targeting DNA sequence GR-20, monomethylauristatin E (MMAE) on the cathepsin-cleavable ValCit-p-aminobenzylcarbamate linker as the payload, and pentaerythritol-based tetraazide as the branching point for the straightforward synthesis of aptamer–drug conjugates by means of a stepwise Cu-catalyzed azide–alkyne cycloaddition (CuAAC) click reaction. Results: Branched aptamer conjugates of 1:3, 2:2, and 3:1 stoichiometry were synthesized and showed higher cytotoxic activity compared to a 1:1 conjugate, particularly on several glioma cell lines. Conclusions: This approach is convenient and potentially applicable to any aptamer sequence, as well as other payloads and cleavable linkers, thus paving the way for future development of aptamer–drug therapeutics by easily providing a range of branched conjugates for in vitro and in vivo testing.

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.