Single-stranded oligonucleotides have been explored in the field of precision medicine both as therapeutics and diagnostic tools. Oligonucleotides that function in a sequence-dependent manner have been used to address diseases that range from neurological to metabolic, as well as to develop vaccines. Until now, their use has been limited by their short half-life in the biological environment. These limiting aspects can now be overcome by resorting to chemical modifications in the drug and using appropriate nanocarriers. Thus, synthetic analogues of oligonucleotides are exploited to increase their application. Peptide nucleic acid (PNA) represents a promising class of synthetic DNA analogues in which the favoured sugar backbone is replaced by N2 aminoethylglycine repeats held together by peptide bonds. In this context, we investigated the relevance of PNAs in therapy and diagnostics. In the first case, we synthesized an antigene anti-Bcl-2 PNA, and we have developed a new delivery system never used before for the transport of PNA based on oncolytic adenoviruses. This promising transport system has already demonstrated its extraordinary effectiveness, as evidenced by the recent development of SARS-CoV vaccines.

Furthermore, we have also used PNAs to support the functionalization of the biosensor development. We covalently bound the PNA to the surface of ZnO nanowires for mRNA CD5 detection, a diagnostic marker of Leukemia. The PNA-based biosensor has also been developed to detect mutations responsible for pathologies such as Brugada syndrome. The wide range of applications denotes the versatility of single-stranded oligonucleotides as a robust therapeutic and diagnostic platform.

Diarylpentanoids are chalcone analogues with two aromatic rings connected by a five-carbon bridge, showing a wide range of biological activities, being the in vitro growth inhibitory activity against cancer cells one of the most studied. However, the underlying mechanisms by which these compounds suppress cancer cell growth is still unclear. Our research group has reported several chalcones with notable antiproliferative activity in human cancer cells. Particularly, chalcone CM-M345 showed potent growth inhibitory activity against a panel of cancer cells, being this associated with the interference with the interaction between the tumor suppressor protein p53 and its endogenous negative regulator MDM2. In this communication, the design, synthesis, and biological evaluation of analogues of CM-M345 with diarylpentanoid scaffold showing promising antitumor activity are explored. From these studies new diarylpentanoids with promising antitumor effect through the interference with p53 pathway and mitosis have emerged. Our results show, for the first time, that exploration of diarylpentanoid scaffold can result in the discovery of new potential p53-MDM2/X dual inhibitors as well as promising antimitotic agents, which may be used as starting point for the discovery of new drug candidates for cancer therapy.

Recent data revealed that the combined administration of histone deacetylase inhibitors such as vorinostat (suberoylanilide hydroxamic acid, SAHA), with genotoxic agents, such as doxorubicin (DOX), enhances the antitumoral effects of both drugs against solid tumors. Herein we designed nanoparticles based on copolymer of lactic and glycolic acids (PLGA) simultaneously loaded with DOX and SAHA to provide synergy against tumor cells.

We obtained the nanoparticles via double emulsion solvent evaporation technique with dichloromethane as the organic solvent and polyvinyl alcohol (PVA) as the emulsion stabilizer. To optimize the nanoformulation, a 12-run, three-factor, three-level Box-Behnken design was used. We investigated the influence of PLGA amount (X1), dichloromethane volume (X2), and PVA concentration (X3) on the nanoparticle size (Y1) and SAHA drug loading (Y2). Next, we optimize the factors via desirability function. After optimization the calculated values for nanoparticle size was 203 nm and for SAHA drug loading was 0.5 %. Experimental data revealed that optimized conditions provided the nanoparticles with a size of 207±8 nm and SAHA drug loading of 0.9 %, which is close to calculated data. Besides these parameters nanoparticles had ζ-potential of -18.0±4.6 mV and DOX drug loading of 2.1 %.

The optimization approach used in this work allowed the determination of the factors required to produce nanoparticles with minimum size and maximum drug loading of SAHA. Furthermore, the calculated responses were close to the experimental data. Thus, the obtained dual-drug loaded PLGA nanoparticles had suitable physical properties for promising further studies in vitro and in vivo.

Funding: This study was supported by the Russian Science Foundation research grant No. 22-25-00293, https://rscf.ru/project/22-25-00293/

Increase in drug resistance of pathogenic fungi as well as a growing immunocompromised patient population in risk of fungal infections create a need for novel anti-infective drugs [1].

HSP90 forms homodimers and is an essential chaperone involved in a plethora of protein-protein-interactions (PPI) [2]. The HSP90 conformational cycle is driven by the binding and hydrolysis of ATP by HSP90. Whereas HSP90 is highly conserved across species, co-chaperones of HSP90 like Sba1 are less conserved, providing a potential target structure for the development of selective antifungal drugs [3]. A prerequisite of HSP90 – Sba1 interaction is the prior binding of ATP to HSP90.

In this study, a high-throughput capable in vitro Förster Resonance Energy Transfer (FRET) based assay for the identification of HSP90 - Sba1 interaction inhibitors was developed. The assay is also suitable to identify ATP-competitive HSP90 inhibitors. HSP90-mNeonGreen (donor) and Sba1-mScarlet-I (acceptor) fusion protein constructs showed specific interaction in the assay. Known ATP-competitive HSP90 inhibitors such as geldanamycin can be reliably identified and characterized with the assay. This demonstrates the applicability of the assay to identify and characterize the activity of small molecule inhibitors. Competition experiments with Sba1 for Sba1-mScarlet-I binding to HSP90-mNeonGreen provide evidence for the suitability as a screening assay to also identify direct PPI inhibitors. Analogous setups for the homologous human HSP90 – p23 interaction enable the determination of selectivity for C. albicans PPI inhibition. A small nucleoside-mimetic library of 320 compounds was screened for inhibition of the HSP90 – Sba1 interaction. Overall, the developed assay showed low data variability and a robust separation, resulting in a Z-factor of consistently > 0.5 [4].

References
1. Aldholmi, M.; Marchand, P.; Ourliac-Garnier, I.; Le Pape, P.; Ganesan, A. A Decade of Antifungal Leads from Natural Products: 2010-2019. Pharmaceuticals (Basel) 2019, 12, doi:10.3390/ph12040182.
2. Schopf, F.H.; Biebl, M.M.; Buchner, J. The HSP90 chaperone machinery. Nat. Rev. Mol. Cell Biol. 2017, 18, 345–360, doi:10.1038/nrm.2017.20.
3. Gu, X.; Xue, W.; Yin, Y.; Liu, H.; Li, S.; Sun, X. The Hsp90 Co-chaperones Sti1, Aha1, and P23 Regulate Adaptive Responses to Antifungal Azoles. Front. Microbiol. 2016, 7, 1571, doi:10.3389/fmicb.2016.01571.
4. Ji-Hu Zhang, Thomas D. Y. Chung, and Kevin R. Oldenburg. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.

Chemotherapy remains one of the main options for cancer treatment. But the accompanying side effects often have a strong impact on the patient’s body condition and seriously disrupt their quality of life. Using of synergistic adjuvants in combination with therapeutic drugs may be an option to solve this problem. We found that some small-molecule isoxazole and isothiazole derivatives can act in this way. They enhance the effect of first-line antitumor drugs cisplatin, temobel, cytarabine against neuroepithelial tumors like glioma C6 or medulloblastoma. Addition of adjuvante in non-active dose to a reduced dose of toxic chemotherapy drug allows to achieve higher level of tumor cell death in vitro than the chemotherapy itself causes at this dose. We decided to begin the explanation of this phenomenon with a quantum chemical study of the interaction of morpholinium and 4-methylpiperazinium 4,5-dichloroisothiazol-3-carboxylates with cisplatin. DFT calculations showed that these compounds interact with cisplatin, which leads to a redistribution of electron density and an increase in the dipole moment. We assume that due to it the resulting system has increased bioactivity, which enhances the efficiency of its interaction with DNA. The data obtained are the basis for the search and design of new effective adjuvants.

Purpose This study aimed to develop a neural network model (ANN) and molecular similarity (MS) to screen PD-L1 inhibitors, which helps the immune system reactivate tumor destruction.

Methods This research collected 2,044 substances from Google Patents, splitting them into training, validation, and test sets. These sets were used to build MS and ANN models. MS model utilized five fingerprints (AVALON, MACCS, ECFP4, RDK5, and MAP4), and BMS-1166 was a query molecule. The decoys were generated by the DeepCoy library. The ANN model was employed using SECFP fingerprint. A support vector machine (SVC) and Random Forest (RF) were implemented in a benchmarking analysis, which was based on Wilcoxon signed rank test, to compare performance with the ANN model. F1 score and average precision were evaluation metrics due to the imbalanced dataset. Subsequently, 15235 compounds from the Drugbank database underwent screening through medicinal chemistry filters, MS, and the ANN model.

Results The decoy generation achieved promising results, with AUC-ROC 1NN of 0.52, AUC-ROC RF of 0.65, Doppelganger scores mean of 0.24, and Doppelganger scores max of 0.346, indicating that the decoys closely resemble the active set. In MS establishment, the AVALON fingerprint was the best nominee for similarity searching, with EF1% of 10.99%, AUC-ROC of 0.963, and a similarity threshold of 0.32. The ANN model attained average precision of 0.863±0.032 and F1 score of 0.745±0.039 in cross-validation, higher than those of the SVC and RF models, although without a significant difference. In external evaluation, the ANN model exhibited average precision of 0.854 and F1 score of 0.799, also higher than those of the SVC and RF models. Finally, only 7 molecules from the Drugbank database fulfilled three filters, with CHEM431 emerging as the most optimistic candidate, possessing active probability of 75% and Tanimoto coefficient of 0.34.

Conclusions Virtual screening pinpointed CHEM431 as the most potential candidate, suggesting follow-up steps, including molecular docking, molecular dynamics, synthesis, and bioactivity testing.

Some highly pathogenic human viruses, such as influenza A virus, human immunodeficiency virus type 1, hepatitis C virus, modern coronaviruses, etc. produce proteins capable of forming ion-conducting pores in the membrane, namely, viroporins. They disrupt the ionic homeostasis of the target cell in favor of the virus to ensure proper replication and assembly of viral particles.

Pyrazine-2-carboxylic acid derivatives have found use as pharmaceutical molecules for the treatment of various diseases. The idea of the proposed work is to study antiviral compounds of pyrazine-2-carboxylic acid with amino acid esters similar to those that we obtained by condensation of aminoadamantane with amino acid and peptide residues. It is important to note that viroporins are an attractive and reliable target for the treatment of viral infections because ion channel activity is highly conserved and the virus is unlikely to become stable through mutational variability.

Derivatives of pyrazine-2-carboxylic acid with amino acid esters containing an aromatic and aliphatic side group were successfully synthesized (Ser-OMe, Thr-OMe, His-OMe, Trp-OMe). In biological experiments in vitro, the target compounds were used as complexes with iron(II) chloride formed in situ at the Fe : L = 1 : 2 ratio to give transparent colorless water solutions. Their antiviral activity against influenza A/H1N1 and SARS-CoV-2 viruses in vitro was studied. Using the MDCK cell culture, it was shown that only one of the presented amino acid derivatives with 2-pyrazinecarboxylic acid (namely, Trp-OMe) has the ability to suppress the replication of the pandemic strain of influenza A virus.

The cytotoxicity of the compounds was found to be about 62.5 μg/ml for a monolayer of Vero-E6 cells, and about 80 μg/ml for MDCK cells, i.e. these compounds are moderately toxic. The proposed method of attaching functional groups to molecules of heterocyclic carboxylic acids and creating chelated compounds based on these ligands makes it possible to obtain bioavailable forms of antiviral drugs. The proposed compounds of amino acids with heterocyclic carboxylic acids, due to their significant activity and low toxicity, can be considered a model for the creation of new antiviral drugs based on them. On the other hand, these compounds were found to have no antiviral properties against the modern strain of coronavirus SARS-CoV-2 in in vitro experiments.

This work was supported by the Russian Science Foundation, grant no. 23-23-00142 (Russia).

Multidrug resistance (MDR) remains a significant challenge in cancer therapy, primarily due to the overexpression of transmembrane drug transporters, with P-glycoprotein (P-gp) encoded by the human ABCB1/MDR1 gene being a central focus. Reduced intracellular drug levels lead to decreased chemosensitivity of cancer cells, culminating in drug resistance. Consequently, the development of P-gp inhibitors has emerged as a promising strategy to combat MDR. In this study, we evaluated eight soloxolone amides for their potential to inhibit P-gp-mediated efflux in MDR tumor cells. Using molecular docking, all compounds were shown to have a direct interaction with the P-gp transmembrane domain characterized by low binding energies (< -9 kcal/mol). Validation of P-gp inhibitory activity was performed on KB-8-5 human cervical cancer cells and RLS40 murine lymphosarcoma cells with P-gp-mediated MDR. The lead compound sg-650 at non-toxic concentration of 40 μM significantly increased the intracellular accumulation of the P-gp substrates rhodamine-123 and doxorubicin by 10.4- and 1.5-fold, respectively, in KB-8-5 cells. Kinetic studies demonstrated an uncompetitive manner of doxorubicin efflux inhibition. In addition, sg-650 synergistically enhanced doxorubicin cytotoxicity in a dose-dependent manner, demonstrating MDR reversal activity. Similar effects were observed in sg-650-treated RLS40 cells. These results underscore the potential of sg-650 as a potent small molecule P-gp inhibitor that holds promise for overcoming MDR in cancer treatment. This work was supported by the Russian Science Foundation (grant no. 23-14-00374).

Transfersomes, a novel lipid-based vesicular system, have emerged as an innovative tool in the field of drug delivery due to their remarkable ability to enhance the skin delivery of bioactive compounds. These deformable lipid vesicles are composed of phospholipids and edge activators, which play a crucial role in conferring flexibility and deformability to the vesicles, thereby enabling efficient drug loading as well as an improved drug transport across biological barriers. [1,2]

Traditionally, transfersomes have been designed using a single edge activator; however, recent research has highlighted the potential benefits of adjusting their composition by mixing different edge activators to further optimize their performance. Having this in mind, quality-by-design strategies, like Box-Behnken factorial design (BBD) appear as a useful approach to enhance the development of new formulations. [3]

In this context, the main goals of this work were the analysis of the impact of single or mixed edge activators on transfersomes physicochemical properties and to assess their cytotoxicity profile. These nanosystems were prepared in presence and absence of two model drugs – ibuprofen sodium salt and caffeine.

To evaluate the effect of the edge activators on transfersomes’ physicochemical properties the formulations were prepared and characterized in terms of vesicle size (Vs), polydispersity index (PDI), encapsulation efficiency (EE), and loading capacity (LC) for 60 days, under storage. Moreover, In vitro cytotoxicity studies were performed using the HaCaT cell line.

All formulations composed of single or mixed surfactants presented interesting results (Vs < 300 nm, and PDI < 0.3), considering the cutaneous delivery target. The EE and LC results were reasonably good for IBU, while caffeine encapsulation still needs to be improved. These results were maintained over time under refrigerated conditions, showing that transfersomes are stable for at least 30 days. Additionally, according to the cell viability results, transfersomes were found to be compatible with human keratinocytes.

Overall, by combining the distinct physicochemical properties of diverse edge activators, it was possible to tailor transfersomes as skin delivery carriers, enhancing their characteristics and addressing challenges related to the colloidal stability.

Acknowledgements: This study was financially supported by Fundação para an Ciência e Tecnologia, I.P., through funding EXPL/BTM-MAT/0112/2021, UIDB/04567/2020, and UIDP/04567/2020, as well as by the research grant attributed to J.V.

References:

[1] – Opatha, S.A.T.; Titapiwatanakun, V.; Chutoprapat, R. Transfersomes: A Promising Nanoencapsulation Technique for Transdermal Drug Delivery. Pharmaceutics 2020, 12, 855. https://doi.org/10.3390/pharmaceutics12090855

[2] – Fernández-García, R.; Lalatsa, A.; Statts, L.; Bolás-Fernández, F.; Ballesteros, M.P.; Serrano, D.R. Transferosomes as nanocarriers for drugs across the skin: Quality by design from lab to industrial scale. Int. J. Pharm. 2020, 573, https://doi.org/10.1016/j.ijpharm.2019.118817

[3] – Vieira, J.; Castelo, J.; Martins, M.; Saraiva, N.; Rosado, C.; Pereira-Leite, C. Mixed Edge Activators in Ibuprofen-Loaded Transfersomes: An Innovative Optimization Strategy Using Box–Behnken Factorial Design. Pharmaceutics 2023, 15, 1209. https://doi.org/10.3390/pharmaceutics15041209

Human pathogenic fungi pose a growing threat to global health, with the emerging challenge of antimicrobial resistance. Epigenetics, the field that investigates chemical modifications to DNA and regulatory proteins governing gene expression, offers a promising approach to control these microorganisms. Epigenetic modulators can selectively influence the gene activity of fungi, disrupting their virulence and addressing drug resistance. Among human fungal pathogens, the critical priority fungi currently include Aspergillus fumigatus and Candida species. Candida species and Aspergillus species are invasive fungal pathogens responsible for systemic infections and high mortality rates, especially in immunocompromised populations, and are resistant to major classes of antifungal drugs. Histone Deacetylase (HDAC) Inhibitors have proven effective in countering virulence and have potent, synergistic effects with fluconazole against resistant Candida albicans infection. Bromodomain and Extraterminal domain (BET) inhibitors reduce the virulence of A. fumigatus. In particular, BET inhibitors reduce the expression of proteins associated with fungal virulence, both intracellular and extracellular. These results highlight the revolutionary potential of this new strategy in combating fungal infections, providing a promising outlook for the future of the treatment of diseases caused by human pathogenic fungi. The use of epigenetics as a therapeutic alternative represents a novel approach in the fight against fungal infections, with the potential for significant impacts on public health and the management of infectious diseases.