Bioprospecting, Synergistic Antifungal and Toxicological Aspects of the Hydroxychalcones and Their Association with Azole Derivates against Candida spp. for Treating Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC) remains a prevalent fungal disease, characterized by challenges, such as increased fungal resistance, side effects of current treatments, and the rising prevalence of non-albicans Candida spp. naturally more resistant. This study aimed to propose a novel therapeutic approach by investigating the antifungal properties and toxicity of 2-hydroxychalcone (2-HC) and 3'-hydroxychalcone (3'-HC), both alone and in combination with fluconazole (FCZ) and clotrimazole (CTZ). A lipid carrier (LC) was also developed to deliver these molecules. The study evaluated in vitro anti-Candida activity against five Candida species and assessed cytotoxicity in the C33-A cell line. The safety and therapeutic efficacy of in vivo were tested using an alternative animal model, Galleria mellonella. The results showed antifungal activity of 2-HC and 3'-HC, ranging from 7.8 to 31.2 as fungistatic and 15.6 to 125.0 mg/L as fungicide effect, with cell viability above 80% from a concentration of 9.3 mg/L (2-HC). Synergistic and partially synergistic interactions of these chalcones with FCZ and CTZ demonstrated significant improvement in antifungal activity, with MIC values ranging from 0.06 to 62.5 mg/L. Some combinations reduced cytotoxicity, achieving 100% cell viability in many interactions. Additionally, two LCs with suitable properties for intravaginal application were developed. These formulations demonstrated promising therapeutic efficacy and low toxicity in Galleria mellonella assays. These results suggest the potential of this approach in developing new therapies for VVC.

Acridones as promising drug candidates against Oropouche virus.

Oropouche virus (OROV) is an emerging vector-borne arbovirus found in South America that causes Oropouche fever, a febrile infection similar to dengue fever. It has a high epidemic potential, causing illness in over 500,000 cases diagnosed since the virus was first discovered in 1955. Currently, the prevention of human viral infection depends on vaccination, but availability for many viruses is limited, and they are classified as neglected viruses. At present, there are no vaccines or antiviral treatments available. An alternative approach to limiting the spread of the virus is to selectively disrupt viral replication mechanisms. Here, we demonstrate the inhibitory effect of acridones, which efficiently inhibited viral replication by 99.9 % in vitro. To evaluate possible mechanisms of action, we conducted tests with dsRNA, an intermediate in virus replication, as well as MD simulations, docking, and binding free energy analysis. The results showed a strong interaction between FAC21 and the OROV endonuclease, which possibly limits the interaction of viral RNA with other proteins. Therefore, our results suggest a dual mechanism of antiviral action, possibly caused by ds-RNA intercalation. In summary, our findings demonstrate that a new generation of antiviral drugs could be developed based on the selective optimization of molecules.

Chalcone T4 Inhibits RANKL-Induced Osteoclastogenesis and Stimulates Osteogenesis In Vitro.

Chalcones are phenolic compounds produced during the biosynthesis of flavonoids that have numerous biological activities, including anti-inflammatory, antioxidant and anticancer. In this in vitro study, we investigate a newly synthesized chalcone (Chalcone T4) in the context of bone turnover, specifically on the modulation of osteoclast differentiation and activity and osteoblast differentiation. Murine macrophages (RAW 264.7) and pre-osteoblasts (MC3T3-E1) were used as models of osteoclasts and osteoblasts, respectively. Differentiation and activity osteoclasts were induced by RANKL in the presence and absence of non-cytotoxic concentrations of Chalcone T4, added in different periods during osteoclastogenesis. Osteoclast differentiation and activity were assessed by actin ring formation and resorption pit assay, respectively. Expression of osteoclast-specific markers (Nfatc1, Oscar, Acp5, Mmp-9 and Ctsk) was determined by RT-qPCR, and the activation status of relevant intracellular signaling pathways (MAPK, AKT and NF-kB) by Western blot. Osteoblast differentiation and activity was induced by osteogenic culture medium in the presence and absence of the same concentrations of Chalcone T4. Outcomes assessed were the formation of mineralization nodules via alizarin red staining and the expression of osteoblast-related genes (Alp e Runx2) by RT-qPCR. Chalcone T4 reduced RANKL-induced osteoclast differentiation and activity, suppressed Oscar, Acp5 and Mmp-9 expression, and decreased ERK and AKT activation in a dose-dependent manner. Nfact1 expression and NF-kB phosphorylation were not modulated by the compound. Mineralized matrix formation and the expression of Alp and Runx2 by MC3T3-E1 cells were markedly stimulated by Chalcone T4. Collectively, these results demonstrate that Chalcone T4 inhibits in osteoclast differentiation and activity and stimulates osteogenesis, which indicates a promising therapeutic potential in osteolytic diseases.

2-Hydroxychalcone as a Potent Compound and Photosensitizer Against Dermatophyte Biofilms.

Dermatophytes, fungi that cause dermatophytosis, can invade keratinized tissues in humans and animals. The biofilm-forming ability of these fungi was described recently, and it may be correlated with the long treatment period and common recurrences of this mycosis. In this study, we evaluated the anti-dermatophytic and anti-biofilm activity of 2-hydroxychalcone (2-chalcone) in the dark and photodynamic therapy (PDT)-mediated and to determine its mechanism of action. Trichophyton rubrum and Trichophyton mentagrophytes strains were used in the study. The antifungal susceptibility test of planktonic cells, early-stage biofilms, and mature biofilms were performed using colorimetric methods. Topographies were visualized by scanning electron microscopy (SEM). Human skin keratinocyte (HaCat) monolayers were also used in the cytotoxicity assays. The mechanisms of action of 2-chalcone in the dark and under photoexcitation were investigated using confocal microscopy and the quantification of ergosterol, reactive oxygen species (ROS), and death induction by apoptosis/necrosis. All strains, in the planktonic form, were inhibited after treatment with 2-chalcone (minimum inhibitory concentration (MIC) = 7.8-15.6 mg/L), terbinafine (TRB) (MIC = 0.008-0.03 mg/L), and fluconazole (FLZ) (1-512 mg/L). Early-stage biofilm and mature biofilms were inhibited by 2-chalcone at concentrations of 15.6 mg/L and 31.2 mg/L in all tested strains. However, mature biofilms were resistant to all the antifungal drugs tested. When planktonic cells and biofilms (early-stage and mature) were treated with 2-chalcone-mediated PDT, the inhibitory concentrations were reduced by four times (2-7.8 mg/L). SEM images of biofilms treated with 2-chalcone showed cell wall collapse, resulting from a probable extravasation of cytoplasmic content. The toxicity of 2-chalcone in HaCat cells showed higher IC50 values in the dark than under photoexcitation. Further, 2-chalcone targets ergosterol in the cell and promotes the generation of ROS, resulting in cell death by apoptosis and necrosis. Overall, 2-chalcone-mediated PDT is a promising and safe drug candidate against dermatophytes, particularly in anti-biofilm treatment.

Flavonoids from Pterogyne nitens as Zika virus NS2B-NS3 protease inhibitors.

Although the widespread epidemic of Zika virus (ZIKV) and its neurological complications are well-known there are still no approved drugs available to treat this arboviral disease or vaccine to prevent the infection. Flavonoids from Pterogyne nitens have already demonstrated anti-flavivirus activity, although their target is unknown. In this study, we virtually screened an in-house database of 150 natural and semi-synthetic compounds against ZIKV NS2B-NS3 protease (NS2B-NS3p) using docking-based virtual screening, as part of the OpenZika project. As a result, we prioritized three flavonoids from P. nitens, quercetin, rutin and pedalitin, for experimental evaluation. We also used machine learning models, built with Assay Central® software, for predicting the activity and toxicity of these flavonoids. Biophysical and enzymatic assays generally agreed with the in silico predictions, confirming that the flavonoids inhibited ZIKV protease. The most promising hit, pedalitin, inhibited ZIKV NS2B-NS3p with an IC50 of 5 µM. In cell-based assays, pedalitin displayed significant activity at 250 and 500 µM, with slight toxicity in Vero cells. The results presented here demonstrate the potential of pedalitin as a candidate for hit-to-lead (H2L) optimization studies towards the discovery of antiviral drug candidates to treat ZIKV infections.