The infusion of Baccharis spicata (Lam. ) Baill (Asteraceae) has antiviral activity against hepatitis B virus without cytotoxic effects against several cell lines / La infusión de Baccharis spicata (Lam. ) Baill (Asteraceae) posee actividad antiviral contra el virus de la hepatitis B sin efectos citotóxicos contra varias líneas celulares

Medicinal plants are used to cure diseases, and their replacement is frequent and affects public health. The genus Baccharis has representatives within the medicinal flora of Argentina, although the replacement of the species of this genus known under the vulgar name of "carqueja" by Baccharis spicata has been detected i n herbalists or markets of herbal products. The genotoxic safety of this species has been established in previous work of our group. The aim of this study was to evaluate the antiviral activity of an infusion made from B. spicata leaves against hepatitis B virus with the HepG2.2.15 cellular system and to determine cytotoxicity in HepG2.2,15, A549 and Vero cell lines. Infusion of B. spicata was active to inhibit HBV replication with an EC 50 of 22.54 µg/mL and a CC 50 of 190 µg/mL.

Las plantas medicinales son empleadas para la cura de enfermedades, y su sustituc ión es frecuente y afecta a la salud pública. El género Baccharis posee representantes dentro de la flora medicinal de Argentina, aunque se ha detectado la sustitución de las especies de dicho género conocidas bajo el nombre vulgar de "carqueja" por Baccha ris spicata en herboristerías o mercados de productos herb arios . Se ha establecido la seguridad genotóxica de esta especie en trabajos previos de nuestro grupo. Este estudio buscó evaluar la actividad antiviral de una infusión elaborada a partir de hojas de B. spicata frente al virus de la hepatitis B con el sistema celular HepG2.2.15 y determinar la citotoxicidad en las líneas celulares HepG2.2.15, A549 y Vero. La infusión de B. spicata fue activa para inhibir la replicación del virus con un EC 50 de 22.54 µg/mL y un CC 50 de 190 µg/mL.

Development of novel cytoprotective small compounds inhibiting mitochondria-dependent cell death.

We identified cytoprotective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in ocular disease mouse models. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only mouse embryonic fibroblasts. M109S also inhibited apoptosis induced by staurosporine, etoposide, and obatoclax. M109S decreased maximal mitochondrial oxygen consumption rate and reactive oxygen species production, whereas it increased glycolysis. These effects on cellular metabolism may contribute to the cytoprotective activity of M109S. M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both in vitro and in vivo. M109S has the potential to become a research tool for studying cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway.

Galanin receptor 3 - A new pharmacological target in retina degeneration.

The neuropeptide galanin receptor 3 (GALR3) is a class A G protein-coupled receptor (GPCR) broadly expressed in the nervous system, including the retina. GALR3 is involved in the modulation of immune and inflammatory responses. Tight control of these processes is critical for maintaining homeostasis in the retina and is required to sustain vision. Here, we investigated the role of GALR3 in retina pathologies triggered by bright light and P23H mutation in the rhodopsin (RHO) gene, associated with the activation of oxidative stress and inflammatory responses. We used a multiphase approach involving pharmacological inhibition of GALR3 with its antagonist SNAP-37889 and genetic depletion of GALR3 to modulate the GALR3 signaling. Our in vitro experiments in the retinal pigment epithelium-derived cells (ARPE19) susceptible to all-trans-retinal toxicity indicated that GALR3 could be involved in the cellular stress response to this phototoxic product. Indeed, blocking the GALR3 signaling in Abca4-/-/Rdh8-/- and wild-type Balb/cJ mice, sensitive to bright light-induced retina damage, protected retina health in these mice exposed to light. The retina morphology and function were substantially improved, and stress response processes were reduced in these mouse models compared to the controls. Furthermore, in P23H Rho knock-in mice, a model of retinitis pigmentosa (RP), both pharmacological inhibition and genetic ablation of GALR3 prolonged the survival of photoreceptors. These results indicate that GALR3 signaling contributes to acute light-induced and chronic RP-linked retinopathies. Together, this work provides the pharmacological knowledge base to evaluate GALR3 as a potential target for developing novel therapies to combat retinal degeneration.

Molecular basis for variations in the sensitivity of pathogenic rhodopsin variants to 9-cis-retinal.

Over 100 mutations in the rhodopsin gene have been linked to a spectrum of retinopathies that include retinitis pigmentosa and congenital stationary night blindness. Though most of these variants exhibit a loss of function, the molecular defects caused by these underlying mutations vary considerably. In this work, we utilize deep mutational scanning to quantitatively compare the plasma membrane expression of 123 known pathogenic rhodopsin variants in the presence and absence of the stabilizing cofactor 9-cis-retinal. We identify 69 retinopathy variants, including 20 previously uncharacterized variants, that exhibit diminished plasma membrane expression in HEK293T cells. Of these apparent class II variants, 67 exhibit a measurable increase in expression in the presence of 9-cis-retinal. However, the magnitude of the response to this molecule varies considerably across this spectrum of mutations. Evaluation of the observed shifts relative to thermodynamic estimates for the coupling between binding and folding suggests underlying differences in stability constrains the magnitude of their response to retinal. Nevertheless, estimates from computational modeling suggest that many of the least sensitive variants also directly compromise binding. Finally, we evaluate the functional properties of three previous uncharacterized, retinal-sensitive variants (ΔN73, S131P, and R135G) and show that two of these retain residual function in vitro. Together, our results provide a comprehensive experimental characterization of the proteostatic properties of retinopathy variants and their response to retinal.

Chromenone derivatives as novel pharmacological chaperones for retinitis pigmentosa-linked rod opsin mutants.

The correct expression of folded, functional rhodopsin (Rho) is critical for visual perception. However, this seven-transmembrane helical G protein-coupled receptor is prone to mutations with pathological consequences of retinal degeneration in retinitis pigmentosa (RP) due to Rho misfolding. Pharmacological chaperones that stabilize the inherited Rho variants by assisting their folding and membrane targeting could slow the progression of RP. In this study, we employed virtual screening of synthetic compounds with a natural product scaffold in conjunction with in vitro and in vivo evaluations to discover a novel chromenone-containing small molecule with favorable pharmacological properties that stabilize rod opsin. This compound reversibly binds to unliganded bovine rod opsin with an EC50 value comparable to the 9-cis-retinal chromophore analog and partially rescued membrane trafficking of multiple RP-related rod opsin variants in vitro. Importantly, this novel ligand of rod opsin was effective in vivo in murine models, protecting photoreceptors from deterioration caused by either bright light or genetic insult. Together, our current study suggests potential broad therapeutic implications of the new chromenone-containing non-retinoid small molecule against retinal diseases associated with photoreceptor degeneration.

Rhodopsin as a Molecular Target to Mitigate Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a group of hereditary degenerative diseases affecting 1 of 4000 people worldwide and being the most prevalent cause of visual handicap among working populations in developed countries. These disorders are mainly related to the abnormalities in the rod G protein-coupled receptor (GPCR), rhodopsin reflected in the dysregulated membrane trafficking, stability and phototransduction processes that lead to progressive loss of retina function and eventually blindness. Currently, there is no cure for RP, and the therapeutic options are limited. Targeting rhodopsin with small molecule chaperones to improve the folding and stability of the mutant receptor is one of the most promising pharmacological approaches to alleviate the pathology of RP. This review provides an update on the current knowledge regarding small molecule compounds that have been evaluated as rhodopsin modulators to be considered as leads for the development of novel therapies for RP.

Systematic profiling of temperature- and retinal-sensitive rhodopsin variants by deep mutational scanning.

Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of "temperature-sensitive" variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. There are many examples of temperature-sensitive rhodopsin variants, the misfolding of which is associated with the molecular basis of retinitis pigmentosa. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and 9-cis-retinal, an investigational corrector, on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures correlates with the response to 9-cis-retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants appear to disrupt a native helical kink within this transmembrane domain. By comparison, mutants that alter the structure of a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest that this observed insensitivity cannot be attributed to a single variable, but likely arises from the composite effects of mutations on the energetics of membrane integration, the stability of the native conformation, and the integrity of the retinal-binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings highlight several biophysical constraints that appear to influence the sensitivity of genetic variants to temperature and small-molecule correctors.

Protective Effects of Flavonoids in Acute Models of Light-Induced Retinal Degeneration.

Degeneration of photoreceptors caused by excessive illumination, inherited mutations, or aging is the principal pathology of blinding diseases. Pharmacological compounds that stabilize the visual receptor rhodopsin and modulate the cellular pathways triggering death of photoreceptors could avert this pathology. Interestingly, flavonoids can modulate the cellular processes, such as oxidative stress, inflammatory responses, and apoptosis, that are activated during retinal degeneration. As we found previously, flavonoids also bind directly to unliganded rod opsin, enhancing its folding, stability, and regeneration. In addition, flavonoids stimulate rhodopsin gene expression. Thus, we evaluated the effect of two main dietary flavonoids, quercetin and myricetin, in ATP-binding cassette subfamily A member 4 -/- /retinol dehydrogenase 8 -/- and wild-type BALB/c mice susceptible to light-induced photoreceptor degeneration. Using in vivo imaging, such as optical coherence tomography, scanning laser ophthalmoscopy, and histologic assessment of retinal morphology, we found that treatment with these flavonoids prior to light insult remarkably protected retina from deterioration and preserved its function. Using high-performance liquid chromatography-mass spectrometry analysis, we detected these flavonoids in the eye upon their intraperitoneal administration. The molecular events associated with the protective effect of quercetin and myricetin were related to the elevated expression of photoreceptor-specific proteins, rhodopsin and cone opsins, decreased expression of the specific inflammatory markers, and the shift of the equilibrium between cell death regulators BCL2-associated X protein (BAX) and B-cell lymphoma 2 toward an antiapoptotic profile. These results were confirmed in photoreceptor-derived 661W cells treated with either H2O2 or all-trans-retinal stressors implicated in the mechanism of retinal degeneration. Altogether, flavonoids could have significant prophylactic value for retinal degenerative diseases. SIGNIFICANCE STATEMENT: Flavonoids commonly present in food exhibit advantageous effects in blinding diseases. They bind to and stabilize unliganded rod opsin, which in excess accelerates degenerative processes in the retina. Additionally, flavonoids enhance the expression of the visual receptors, rod and cone opsins; inhibit the inflammatory reactions; and induce the expression of antiapoptotic markers in the retina, preventing the degeneration in vivo. Thus, flavonoids could have a prophylactic value for retinal degenerative diseases.

Understanding Severe Acute Respiratory Syndrome Coronavirus 2 Replication to Design Efficient Drug Combination Therapies.

BACKGROUND: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its disease CO-VID-19 has strongly encouraged the search for antiviral compounds. Most of the evaluated drugs against SARS-CoV-2 derive from drug repurposing of Food and Drug Administration-approved molecules. These drugs have as target three major processes: (1) early stages of virus-cell interaction, (2) viral proteases, and (3) the viral RNA-dependent RNA polymerase. SUMMARY: This review focused on the basic principles of virology and pharmacology to understand the importance of early stages of virus-cell interaction as therapeutic targets and other main processes vital for SARS-CoV-2 replication. Furthermore, we focused on describing the main targets associated with SARS-CoV-2 antiviral therapy and the rationale of drug combinations for efficiently suppressing viral replication. Key Messages: We hypothesized that blocking of both entry mechanisms could allow a more effective antiviral effect compared to the partial results obtained with chloroquine or its derivatives alone. This approach, already used to achieve an antiviral effect higher than that offered by every single drug administered separately, has been successfully applied in several viral infections such as HIV and HCV. This review will contribute to expanding the perception of the possible therapeutic targets in SARS-CoV-2 infection and highlight the benefits of using combination therapies.

Class A G Protein-Coupled Receptor Antagonist Famotidine as a Therapeutic Alternative Against SARS-CoV2: An In Silico Analysis.

The pandemic associated with Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV2) and its disease named COVID-19 challenged the scientific community to discover effective therapeutic solutions in a short period. Repurposing existing drugs is one viable approach that emphasizes speed during these urgent times. Famotidine, a class A G protein-coupled receptor antagonist used for the treatment of gastroesophageal reflux was recently identified in an in silico screening. Additionally, a recent retrospective clinical report showed that the treatment with famotidine provided a good outcome in patients infected with SARS-CoV2. A clinical trial testing effectiveness of famotidine in combination with hydroxychloroquine is currently ongoing in the United States (US). In the 1990s, famotidine was described as an antiviral agent against human immunodeficiency virus (HIV). Interestingly, some HIV protease inhibitors are presently being used against SARS-CoV2. However, it is not clear if famotidine could be effective against SARS-CoV2. Thus, by using a computational analysis, we aimed to examine if the antiviral effect of famotidine could be related to the inhibition of proteases involved in the virus replication. Our results showed that famotidine could interact within the catalytic site of the three proteases associated with SARS-CoV2 replication. However, weak binding affinity of famotidine to these proteases suggests that a successful famotidine therapy could likely be achieved only in combination with other antiviral drugs. Finally, analysis of famotidine's pharmacokinetic parameters indicated that its effect against SARS-CoV2 infection could be reached only upon intravenous administration. This work will contribute to the pharmacological knowledge of famotidine as an antiviral agent against SARS-CoV2.

Retinoid analogs and polyphenols as potential therapeutics for age-related macular degeneration.

IMPACT STATEMENT: Age-related macular degeneration (AMD) is a devastating retinal degenerative disease. Epidemiological reports showed an expected increasing prevalence of AMD in the near future. The only one existing FDA-approved pharmacological treatment involves an anti-vascular endothelial growth factor (VEGF) therapy with serious disadvantages. This limitation emphasizes an alarming need to develop new therapeutic approaches to prevent and treat AMD. In this review, we summarize scientific data unraveling the therapeutic potential of the specific retinoid and natural compounds. The experimental results reported by us and other research groups demonstrated that retinoid analogs and compounds with natural product scaffolds could serve as lead compounds for the development of new therapeutic agents with potential to prevent or slow down the pathogenesis of AMD.

The Retinoid and Non-Retinoid Ligands of the Rod Visual G Protein-Coupled Receptor.

G protein-coupled receptors (GPCRs) play a predominant role in the drug discovery effort. These cell surface receptors are activated by a variety of specific ligands that bind to the orthosteric binding pocket located in the extracellular part of the receptor. In addition, the potential binding sites located on the surface of the receptor enable their allosteric modulation with critical consequences for their function and pharmacology. For decades, drug discovery focused on targeting the GPCR orthosteric binding sites. However, finding that GPCRs can be modulated allosterically opened a new venue for developing novel pharmacological modulators with higher specificity. Alternatively, focus on discovering of non-retinoid small molecules beneficial in retinopathies associated with mutations in rhodopsin is currently a fast-growing pharmacological field. In this review, we summarize the accumulated knowledge on retinoid ligands and non-retinoid modulators of the light-sensing GPCR, rhodopsin and their potential in combating the specific vision-related pathologies. Also, recent findings reporting the potential of biologically active compounds derived from natural products as potent rod opsin modulators with beneficial effects against degenerative diseases related to this receptor are highlighted here.

Flavonoids enhance rod opsin stability, folding, and self-association by directly binding to ligand-free opsin and modulating its conformation.

Rhodopsin (Rho) is a visual G protein-coupled receptor expressed in the rod photoreceptors of the eye, where it mediates transmission of a light signal into a cell and converts this signal into a nerve impulse. More than 100 mutations in Rho are linked to various ocular impairments, including retinitis pigmentosa (RP). Accordingly, much effort has been directed toward developing ligands that target Rho and improve its folding and stability. Natural compounds may provide another viable approach to such drug discovery efforts. The dietary polyphenol compounds, ubiquitously present in fruits and vegetables, have beneficial effects in several eye diseases. However, the underlying mechanism of their activity is not fully understood. In this study, we used a combination of computational methods, biochemical and biophysical approaches, including bioluminescence resonance energy transfer, and mammalian cell expression systems to clarify the effects of four common bioactive flavonoids (quercetin, myricetin, and their mono-glycosylated forms quercetin-3-rhamnoside and myricetrin) on rod opsin stability, function, and membrane organization. We observed that by directly interacting with ligand-free opsin, flavonoids modulate its conformation, thereby causing faster entry of the retinal chromophore into its binding pocket. Moreover, flavonoids significantly increased opsin stability, most likely by introducing structural rigidity and promoting receptor self-association within the biological membranes. Of note, the binding of flavonoids to an RP-linked P23H opsin variant partially restored its normal cellular trafficking. Together, our results suggest that flavonoids could be utilized as lead compounds in the development of effective nonretinoid therapeutics for managing RP-related retinopathies.

Specificity of the chromophore-binding site in human cone opsins.

The variable composition of the chromophore-binding pocket in visual receptors is essential for vision. The visual phototransduction starts with the cis-trans isomerization of the retinal chromophore upon absorption of photons. Despite sharing the common 11-cis-retinal chromophore, rod and cone photoreceptors possess distinct photochemical properties. Thus, a detailed molecular characterization of the chromophore-binding pocket of these receptors is critical to understanding the differences in the photochemistry of vision between rods and cones. Unlike for rhodopsin (Rh), the crystal structures of cone opsins remain to be determined. To obtain insights into the specific chromophore-protein interactions that govern spectral tuning in human visual pigments, here we harnessed the unique binding properties of 11-cis-6-membered-ring-retinal (11-cis-6mr-retinal) with human blue, green, and red cone opsins. To unravel the specificity of the chromophore-binding pocket of cone opsins, we applied 11-cis-6mr-retinal analog-binding analyses to human blue, green, and red cone opsins. Our results revealed that among the three cone opsins, only blue cone opsin can accommodate the 11-cis-6mr-retinal in its chromophore-binding pocket, resulting in the formation of a synthetic blue pigment (B6mr) that absorbs visible light. A combination of primary sequence alignment, molecular modeling, and mutagenesis experiments revealed the specific amino acid residue 6.48 (Tyr-262 in blue cone opsins and Trp-281 in green and red cone opsins) as a selectivity filter in human cone opsins. Altogether, the results of our study uncover the molecular basis underlying the binding selectivity of 11-cis-6mr-retinal to the cone opsins.

The role of the glycosyl moiety of myricetin derivatives in anti-HIV-1 activity in vitro.

BACKGROUND: Plant extracts are sources of valuable compounds with biological activity, especially for the anti-proliferative activity against pathogens or tumor cells. Myricetin is a flavonoid found in several plants that has been described as an inhibitor of Human immunodeficiency virus type 1 (HIV-1) through its action against the HIV reverse transcriptase, but myricetin derivatives have not been fully studied. The aim of this study was to evaluate the anti-HIV-1 activity of glycosylated metabolites obtained from Marcetia taxifolia and derived from myricetin: myricetin rhamnoside and myricetin 3-(6-rhamnosylgalactoside). METHODS: Compounds were obtained from organic extracts by maceration of aerial parts of M. taxifolia. All biological assays were performed in the MT4 cell line. Antiviral activity was measured as inhibition of p24 and reverse transcriptase with a fluorescent assay. RESULTS: Both flavonoids have antiviral activity in vitro, with an EC50 of 120 µM for myricetin 3-rhamnoside (MR) and 45 µM for myricetin 3-(6-rhamnosylgalactoside) (MRG), both significantly lower than the EC50 of myricetin (230 µM). Although both compounds inhibited the reverse transcriptase activity, with an IC50 of 10.6 µM for MR and 13.8 µM for MRG, myricetin was the most potent, with an IC50 of 7.6 µM, and an inhibition greater than 80%. Molecular docking approach showed correlation between the free energy of binding with the assays of enzyme inhibition. CONCLUSIONS: The results suggest that glycosylated moiety might enhance the anti-HIV-1 activity of myricetin, probably by favoring the internalization of the flavonoid into the cell. The inhibition of the HIV-1 reverse transcriptase is likely responsible for the antiviral activity.