In Silico and In Vitro Screening of 50 Curcumin Compounds as EGFR and NF-κB Inhibitors

The improvement of cancer chemotherapy remains a major challenge, and thus new drugs are urgently required to develop new treatment regimes. Curcumin, a polyphenolic antioxidant derived from the rhizome of turmeric (Curcuma longa L.), has undergone extensive preclinical investigations and, thereby, displayed remarkable efficacy in vitro and in vivo against cancer and other disorders. However, pharmacological limitations of curcumin stimulated the synthesis of numerous novel curcumin analogs, which need to be evaluated for their therapeutic potential. In the present study, we calculated the binding affinities of 50 curcumin derivatives to known cancer-related target proteins of curcumin, i.e., epidermal growth factor receptor (EGFR) and nuclear factor κB (NF-κB) by using a molecular docking approach. The binding energies for EGFR were in a range of −12.12 (±0.21) to −7.34 (±0.07) kcal/mol and those for NF-κB ranged from −12.97 (±0.47) to −6.24 (±0.06) kcal/mol, indicating similar binding affinities of the curcumin compounds for both target proteins. The predicted receptor-ligand binding constants for EGFR and curcumin derivatives were in a range of 0.00013 (±0.00006) to 3.45 (±0.10) µM and for NF-κB in a range of 0.0004 (±0.0003) to 10.05 (±4.03) µM, indicating that the receptor-ligand binding was more stable for EGFR than for NF-κB. Twenty out of 50 curcumin compounds showed binding energies to NF-κB smaller than −10 kcal/mol, while curcumin as a lead compound revealed free binding energies of >−10 kcal/mol. Comparable data were obtained for EGFR: 15 out of 50 curcumin compounds were bound to EGFR with free binding energies of <−10 kcal/mol, while the binding affinity of curcumin itself was >−10 kcal/mol. This indicates that the derivatization of curcumin may indeed be a promising strategy to improve targe specificity and to obtain more effective anticancer drug candidates. The in silico results have been exemplarily validated using microscale thermophoresis. The bioactivity has been further investigated by using resazurin cell viability assay, lactate dehydrogenase assay, flow cytometric measurement of reactive oxygen species, and annexin V/propidium iodide assay. In conclusion, molecular docking represents a valuable approach to facilitate and speed up the identification of novel targeted curcumin-based drugs to treat cancer.     

Inhibitory Effect of Avenanthramides (Avn) on Tyrosinase Activity and Melanogenesis in α-MSH-Activated SK-MEL-2 Cells: In Vitro and In Silico Analysis

Melanin causes melasma, freckles, age spots, and chloasma. Anti-melanogenic agents can prevent disease-related hyperpigmentation. In the present study, the dose-dependent tyrosinase inhibitory activity of Avenanthramide (Avn)-A-B-C was demonstrated, and 100 µM Avn-A-B-C produced the strongest competitive inhibition against inter-cellular tyrosinase and melanin synthesis. Avn-A-B-C inhibits the expression of melanogenesis-related proteins, such as TRP1 and 2. Molecular docking simulation revealed that AvnC (−7.6 kcal/mol) had a higher binding affinity for tyrosinase than AvnA (−7.3 kcal/mol) and AvnB (−6.8 kcal/mol). AvnC was predicted to interact with tyrosinase through two hydrogen bonds at Ser360 (distance: 2.7 Å) and Asn364 (distance: 2.6 Å). In addition, AvnB and AvnC were predicted to be skin non-sensitizers in mammals by the Derek Nexus Quantitative Structure–Activity Relationship system.     

The Potential Role of Phenolic Acids from Salvia miltiorrhiza and Cynara scolymus and Their Derivatives as JAK Inhibitors: An In Silico Study

JAK inhibition is a new strategy for treating autoimmune and inflammatory diseases. Previous studies have shown the immunoregulatory and anti-inflammatory effects of Salvia miltiorrhiza and Cynara scolymus and suggest that the bioactivity of their phenolic acids involves the JAK-STAT pathway, but it is unclear whether these effects occur through JAK inhibition. The JAK binding affinities obtained by docking Rosmarinic acid (RosA), Salvianolic acid A (SalA), Salvianolic acid C (SalC), Lithospermic acid, Salvianolic acid B and Cynarin (CY) to JAK (PDB: 6DBN) with AutoDock Vina are −8.8, −9.8, −10.7, −10.0, −10.3 and −9.7 kcal/mol, respectively. Their predicted configurations enable hydrogen bonding with the hinge region and N- and C-terminal lobes of the JAK kinase domain. The benzofuran core of SalC, the compound with the greatest binding affinity, sits near Leu959, such as Tofacitinib’s pyrrolopyrimidine. A SalC derivative with a binding affinity of −12.2 kcal/mol was designed while maintaining this relationship. The docking results show follow-up studies of these phenolic acids as JAK inhibitors may be indicated. Furthermore, derivatives of SalC, RosA, CY and SalA can yield better binding affinity or bioavailability scores, indicating that their structures may be suitable as scaffolds for the design of new JAK inhibitors.     

Caffeine Inhibits Acetylcholinesterase,But Not Butyrylcholinesterase

Caffeine is an alkaloid with a stimulant effect in the body. It can interfere in transmissions based on acetylcholine, epinephrine, norepinephrine, serotonin, dopamine and glutamate. Clinical studies indicate that it can be involved in the slowing of Alzheimer disease pathology and some other effects. The effects are not well understood. In the present work, we focused on the question whether caffeine can inhibit acetylcholinesterase (AChE) and/or, butyrylcholinesterase (BChE), the two enzymes participating in cholinergic neurotransmission. A standard Ellman test with human AChE and BChE was done for altering concentrations of caffeine. The test was supported by an in silico examination as well. Donepezil and tacrine were used as standards. In compliance with Dixon’s plot, caffeine was proved to be a non-competitive inhibitor of AChE and BChE. However, inhibition of BChE was quite weak, as the inhibition constant, K_i, was 13.9 ± 7.4 mol/L. Inhibition of AChE was more relevant, as K_i was found to be 175 ± 9 μmol/L. The predicted free energy of binding was −6.7 kcal/mol. The proposed binding orientation of caffeine can interact with Trp86, and it can be stabilize by Tyr337 in comparison to the smaller Ala328 in the case of human BChE; thus, it can explain the lower binding affinity of caffeine for BChE with reference to AChE. The biological relevance of the findings is discussed.     

Toward the Discovery of a Novel Class of Leads for High Altitude Disorders by Virtual Screening and Molecular Dynamics Approaches Targeting Carbonic Anhydrase

For decades, carbonic anhydrase (CA) inhibitors, most notably the acetazolamide-bearing 1,3,4-thiadiazole moiety, have been exploited at high altitudes to alleviate acute mountain sickness, a syndrome of symptomatic sensitivity to the altitude characterized by nausea, lethargy, headache, anorexia, and inadequate sleep. Therefore, inhibition of CA may be a promising therapeutic strategy for high-altitude disorders. In this study, co-crystallized inhibitors with 1,3,4-thiadiazole, 1,3-benzothiazole, and 1,2,5-oxadiazole scaffolds were employed for pharmacophore-based virtual screening of the ZINC database, followed by molecular docking and molecular dynamics simulation studies against CA to find possible ligands that may emerge as promising inhibitors. Compared to the co-crystal ligands of PDB-1YDB, 6BCC, and 6IC2, ZINC12336992, ZINC24751284, and ZINC58324738 had the highest docking scores of −9.0, −9.0, and −8.9 kcal/mol, respectively. A molecular dynamics (MD) simulation analysis of 100 ns was conducted to verify the interactions of the top-scoring molecules with CA. The system’s backbone revealed minor fluctuations, indicating that the CA–ligand complex was stable during the simulation period. Simulated trajectories were used for the MM-GBSA analysis, showing free binding energies of −16.00 ± 0.19, −21.04 ± 0.17, and −19.70 ± 0.18 kcal/mol, respectively. In addition, study of the frontier molecular orbitals of these compounds by DFT-based optimization at the level of B3LYP and the 6-311G(d,p) basis set showed negative values of the HOMO and LUMO, indicating that the ligands are energetically stable, which is essential for forming a stable ligand–protein complex. These molecules may prove to be a promising therapy for high-altitude disorders, necessitating further investigations.     

Kisspeptin-10 Rescues Cholinergic Differentiated SHSY-5Y Cells from α-Synuclein-Induced Toxicity In Vitro

The neuropathological substrate of dementia with Lewy bodies (DLB) is defined by the inextricable cross-seeding accretion of amyloid-β (Aβ) and α-synuclein (α-syn)-laden deposits in cholinergic neurons. The recent revelation that neuropeptide kisspeptin-10 (KP-10) is able to mitigate Aβ toxicity via an extracellular binding mechanism may provide a new horizon for innovative drug design endeavors. Considering the sequence similarities between α-syn’s non-amyloid-β component (NAC) and Aβ’s C-terminus, we hypothesized that KP-10 would enhance cholinergic neuronal resistance against α-syn’s deleterious consequences through preferential binding. Here, human cholinergic SH-SY5Y cells were transiently transformed to upsurge the mRNA expression of α-syn while α-syn-mediated cholinergic toxicity was quantified utilizing a standardized viability-based assay. Remarkably, the E46K mutant α-syn displayed elevated α-syn mRNA levels, which subsequently induced more cellular toxicity compared with the wild-type α-syn in choline acetyltransferase (ChAT)-positive cholinergic neurons. Treatment with a high concentration of KP-10 (10 µM) further decreased cholinergic cell viability, while low concentrations of KP-10 (0.01–1 µM) substantially suppressed wild-type and E46K mutant α-syn-mediated toxicity. Correlating with the in vitro observations are approximations from in silico algorithms, which inferred that KP-10 binds favorably to the C-terminal residues of wild-type and E46K mutant α-syn with CDOCKER energy scores of −118.049 kcal/mol and −114.869 kcal/mol, respectively. Over the course of 50 ns simulation time, explicit-solvent molecular dynamics conjointly revealed that the docked complexes were relatively stable despite small-scale fluctuations upon assembly. Taken together, our findings insinuate that KP-10 may serve as a novel therapeutic scaffold with far-reaching implications for the conceptualization of α-syn-based treatments.     

Metal-Bound Methisazone; Novel Drugs Targeting Prophylaxis and Treatment of SARS-CoV-2, a Molecular Docking Study

SARS-CoV-2 currently lacks effective first-line drug treatment. We present promising data from in silico docking studies of new Methisazone compounds (modified with calcium, Ca; iron, Fe; magnesium, Mg; manganese, Mn; or zinc, Zn) designed to bind more strongly to key proteins involved in replication of SARS-CoV-2. In this in silico molecular docking study, we investigated the inhibiting role of Methisazone and the modified drugs against SARS-CoV-2 proteins: ribonucleic acid (RNA)-dependent RNA polymerase (RdRp), spike protein, papain-like protease (PlPr), and main protease (MPro). We found that the highest binding interactions were found with the spike protein (6VYB), with the highest overall binding being observed with Mn-bound Methisazone at −8.3 kcal/mol, followed by Zn and Ca at −8.0 kcal/mol, and Fe and Mg at −7.9 kcal/mol. We also found that the metal-modified Methisazone had higher affinity for PlPr and MPro. In addition, we identified multiple binding pockets that could be singly or multiply occupied on all proteins tested. The best binding energy was with Mn–Methisazone versus spike protein, and the largest cumulative increases in binding energies were found with PlPr. We suggest that further studies are warranted to identify whether these compounds may be effective for treatment and/or prophylaxis.     

Development of Effective Therapeutic Molecule from Natural Sources against Coronavirus Protease

The SARS-CoV-2 main protease (M^pro) is one of the molecular targets for drug design. Effective vaccines have been identified as a long-term solution but the rate at which they are being administered is slow in several countries, and mutations of SARS-CoV-2 could render them less effective. Moreover, remdesivir seems to work only with some types of COVID-19 patients. Hence, the continuous investigation of new treatments for this disease is pivotal. This study investigated the inhibitory role of natural products against SARS-CoV-2 M^pro as repurposable agents in the treatment of coronavirus disease 2019 (COVID-19). Through in silico approach, selected flavonoids were docked into the active site of M^pro. The free energies of the ligands complexed with M^pro were computationally estimated using the molecular mechanics-generalized Born surface area (MM/GBSA) method. In addition, the inhibition process of SARS-CoV-2 Mpro with these ligands was simulated at 100 ns in order to uncover the dynamic behavior and complex stability. The docking results showed that the selected flavonoids exhibited good poses in the binding domain of M^pro. The amino acid residues involved in the binding of the selected ligands correlated well with the residues involved with the mechanism-based inhibitor (N3) and the docking score of Quercetin-3-O-Neohesperidoside (−16.8 Kcal/mol) ranked efficiently with this inhibitor (−16.5 Kcal/mol). In addition, single-structure MM/GBSA rescoring method showed that Quercetin-3-O-Neohesperidoside (−87.60 Kcal/mol) is more energetically favored than N3 (−80.88 Kcal/mol) and other ligands (Myricetin 3-Rutinoside (−87.50 Kcal/mol), Quercetin 3-Rhamnoside (−80.17 Kcal/mol), Rutin (−58.98 Kcal/mol), and Myricitrin (−49.22 Kcal/mol). The molecular dynamics simulation (MDs) pinpointed the stability of these complexes over the course of 100 ns with reduced RMSD and RMSF. Based on the docking results and energy calculation, together with the RMSD of 1.98 ± 0.19 Å and RMSF of 1.00 ± 0.51 Å, Quercetin-3-O-Neohesperidoside is a better inhibitor of M^pro compared to N3 and other selected ligands and can be repurposed as a drug candidate for the treatment of COVID-19. In addition, this study demonstrated that in silico docking, free energy calculations, and MDs, respectively, are applicable to estimating the interaction, energetics, and dynamic behavior of molecular targets by natural products and can be used to direct the development of novel target function modulators.     

In Silico Discovery of Potential VEGFR-2 Inhibitors from Natural Derivatives for Anti-Angiogenesis Therapy

Angiogenesis is the growth of new capillaries from existing blood vessels that supply oxygen and nutrients and provide gateways for immune surveillance. Abnormal vessel growth in term of excessive angiogenesis is a hallmark of cancer, inflammatory and eye diseases. VEGFR-2 (vascular endothelial growth factor receptor 2) dominating the process of angiogenesis has led to approval of therapeutic inhibitors and is becoming a promising target for anti-angiogenic drugs. Notwithstanding these successes, the clinical use of current VEGFR-2 blockers is more challenging than anticipated. Taking axitinib as a reference drug, in our study we found three potent VEGFR-2 inhibitors (ZINC08254217, ZINC08254138, and ZINC03838680) from natural derivatives. Each of the three inhibitors acquired a better grid score than axitinib (−62.11) when docked to VEGFR-2. Molecular dynamics simulations demonstrated that ZINC08254217– and ZINC08254138–VEGFR-2 complexes were more stable than axitinib. Similar to bind free energy for axitinib (−54.68 kcal/mol), such for ZINC03838680, ZINC08254217, and ZINC08254138 was −49.37, −43.32, and −32.73 kcal/mol respectively. These results suggested these three compounds could be candidate drugs against angiogenesis, with comparable VEGFR-2 binding affinity of axitinib. Hence findings in our study are able to provide valuable information on discovery of effective anti-angiogenesis therapy.     

Computational Analysis of Molnupiravir

In this work, we report in-depth computational studies of three plausible tautomeric forms, generated through the migration of two acidic protons of the N⁴-hydroxylcytosine fragment, of molnupiravir, which is emerging as an efficient drug to treat COVID-19. The DFT calculations were performed to verify the structure of these tautomers, as well as their electronic and optical properties. Molecular docking was applied to examine the influence of the structures of the keto-oxime, keto-hydroxylamine and hydroxyl-oxime tautomers on a series of the SARS-CoV-2 proteins. These tautomers exhibited the best affinity behavior (−9.90, −7.90, and −9.30 kcal/mol, respectively) towards RdRp-RTR and Nonstructural protein 3 (nsp3_range 207–379-MES).     

The Novel benzamide Derivative,VKNG-2, Restores the Efficacy of Chemotherapeutic Drugs in Colon Cancer Cell Lines by Inhibiting the ABCG2 Transporter

The overexpression of ATP-binding cassette transporter, ABCG2, plays an important role in mediating multidrug resistance (MDR) in certain types of cancer cells. ABCG2-mediated MDR can significantly attenuate or abrogate the efficacy of anticancer drugs by increasing their efflux from cancer cells. In this study, we determined the efficacy of the novel benzamide derivative, VKNG-2, to overcome MDR due to the overexpression of the ABCG2 transporter in the colon cancer cell line, S1-M1-80. In vitro, 5 μM of VKNG-2 reversed the resistance of S1-M1-80 cell line to mitoxantrone (70-fold increase in efficacy) or SN-38 (112-fold increase in efficacy). In contrast, in vitro, 5 μM of VKNG-2 did not significantly alter either the expression of ABCG2, AKT, and PI3K p110β protein or the subcellular localization of the ABCG2 protein compared to colon cancer cells incubated with the vehicle. Molecular docking data indicated that VKNG-2 had a high docking score (−10.2 kcal/mol) for the ABCG2 transporter substrate-drug binding site whereas it had a low affinity on ABCB1 and ABCC1 transporters. Finally, VKNG-2 produced a significant concentration-dependent increase in ATPase activity (EC₅₀ = 2.3 µM). In conclusion, our study suggests that in vitro, VKNG-2 reverses the resistance of S1-M1-80, a cancer cell line resistant to mitoxantrone and SN-38, by inhibiting the efflux function of the ABCG2 transporter.     

Antibacterial and Sporicidal Activity Evaluation of Theaflavin-3,3′-digallate

Theaflavin-3,3′-digallate (TFDG), a polyphenol derived from the leaves of Camellia sinensis, is known to have many health benefits. In this study, the antibacterial effect of TFDG against nine bacteria and the sporicidal activities on spore-forming Bacillus spp. have been investigated. Microplate assay, colony-forming unit, BacTiter-Glo^TM, and Live/Dead Assays showed that 250 µg/mL TFDG was able to inhibit bacterial growth up to 99.97%, while 625 µg/mL TFDG was able to inhibit up to 99.92% of the spores from germinating after a one-hour treatment. Binding analysis revealed the favorable binding affinity of two germination-associated proteins, GPR and Lgt (GerF), to TFDG, ranging from −7.6 to −10.3 kcal/mol. Semi-quantitative RT-PCR showed that TFDG treatment lowered the expression of gpr, ranging from 0.20 to 0.39 compared to the control in both Bacillus spp. The results suggest that TFDG not only inhibits the growth of vegetative cells but also prevents the germination of bacterial spores. This report indicates that TFDG is a promising broad-spectrum antibacterial and anti-spore agent against Gram-positive, Gram-negative, acid-fast bacteria, and endospores. The potential anti-germination mechanism has also been elucidated.     

Spectrofluorometric and Molecular Docking Studies on the Binding of Curcumenol and Curcumenone to Human Serum Albumin

Curcumenol and curcumenone are two major constituents of the plants of medicinally important genus of Curcuma, and often govern the pharmacological effect of these plant extracts. These two compounds, isolated from C. zedoaria rhizomes were studied for their binding to human serum albumin (HSA) using the fluorescence quench titration method. Molecular docking was also performed to get a more detailed insight into their interaction with HSA at the binding site. Additions of these sesquiterpenes to HSA produced significant fluorescence quenching and blue shifts in the emission spectra of HSA. Analysis of the fluorescence data pointed toward moderate binding affinity between the ligands and HSA, with curcumenone showing a relatively higher binding constant (2.46 × 10⁵ M⁻¹) in comparison to curcumenol (1.97 × 10⁴ M⁻¹). Cluster analyses revealed that site I is the preferred binding site for both molecules with a minimum binding energy of −6.77 kcal·mol⁻¹. However, binding of these two molecules to site II cannot be ruled out as the binding energies were found to be −5.72 and −5.74 kcal·mol⁻¹ for curcumenol and curcumenone, respectively. The interactions of both ligands with HSA involved hydrophobic interactions as well as hydrogen bonding.     

Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome bcc-aa3 Supercomplex

Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa₃ supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatis aa₃-type terminal oxidase. Strikingly, we found that the enzyme in turnover with O₂ and reductants is resistant to inhibition by the ligand, being able to metabolize NO at 25 °C with an apparent turnover number as high as ≈303 mol NO (mol enzyme)⁻¹ min⁻¹ at 30 µM NO. The rate of NO consumption proved to be proportional to that of O₂ consumption, with 2.65 ± 0.19 molecules of NO being consumed per O₂ molecule by the mycobacterial bcc-aa₃. The enzyme was found to metabolize the ligand even under anaerobic reducing conditions with a turnover number of 2.8 ± 0.5 mol NO (mol enzyme)⁻¹ min⁻¹ at 25 °C and 8.4 µM NO. These results suggest a protective role of mycobacterial bcc-aa₃ supercomplexes against NO stress.     

Design of Rational JAK3 Inhibitors Based on the Parent Core Structure of 1,7-Dihydro-Dipyrrolo [2,3-b:3′,2′-e] Pyridine

JAK3 differs from other JAK family members in terms of tissue distribution and functional properties, making it a promising target for autoimmune disease treatment. However, due to the high homology of these family members, targeting JAK3 selectively is difficult. As a result, exploiting small changes or selectively boosting affinity within the ATP binding region to produce new tailored inhibitors of JAK3 is extremely beneficial. PubChem CID 137321159 was used as the lead inhibitor in this study to preserve the characteristic structure and to collocate it with the redesigned new parent core structure, from which a series of 1,7-dihydro-dipyrrolo [2,3-b:3′,2′-e] pyridine derivatives were obtained using the backbone growth method. From the proposed compounds, 14 inhibitors of JAK3 were found based on the docking scoring evaluation. The RMSD and MM/PBSA methods of molecular dynamics simulations were also used to confirm the stable nature of this series of complex systems, and the weak protein–ligand interactions during the dynamics were graphically evaluated and further investigated. The results demonstrated that the new parent core structure fully occupied the hydrophobic cavity, enhanced the interactions of residues LEU828, VAL836, LYS855, GLU903, LEU905 and LEU956, and maintained the structural stability. Apart from this, the results of the analysis show that the binding efficiency of the designed inhibitors of JAK3 is mainly achieved by electrostatic and VDW interactions and the order of the binding free energy with JAK3 is: 8 (−70.286 kJ/mol) > 11 (−64.523 kJ/mol) > 6 (−51.225 kJ/mol) > 17 (−42.822 kJ/mol) > 10 (−40.975 kJ/mol) > 19 (−39.754 kJ/mol). This study may provide a valuable reference for the discovery of novel JAK3 inhibitors for those patients with immune diseases.     

Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is activated by the small G-protein, Ras homolog enriched in brain (RHEB–GTPase). On lysosome, RHEB activates mTORC1 by binding the domains of N-heat, M-heat, and the focal adhesion targeting (FAT) domain, which allosterically regulates ATP binding in the active site for further phosphorylation. The crucial role of RHEB in regulating growth and survival through mTORC1 makes it a targetable site for anti-cancer therapeutics. However, the binding kinetics of RHEB to mTORC1 is still unknown at the molecular level. Therefore, we studied the kinetics by in vitro and in-cell protein–protein interaction (PPI) assays. To this end, we used the split-luciferase system (NanoBiT^®) for in-cell studies and prepared proteins for the in vitro measurements. Consequently, we demonstrated that RHEB binds to the whole mTOR both in the presence or absence of GTPγS, with five-fold weaker affinity in the presence of GTPγS. In addition, RHEB bound to the truncated mTOR fragments of N-heat domain (∆N, aa 60–167) or M-heat domain (∆M, aa 967–1023) with the same affinity in the absence of GTP. The reconstructed binding site of RHEB, ∆N-FAT-M, however, bound to RHEB with the same affinity as ∆N-M, indicating that the FAT domain (∆FAT, aa 1240–1360) is dispensable for RHEB binding. Furthermore, RHEB bound to the truncated kinase domain (∆ATP, aa 2148–2300) with higher affinity than to ∆N-FAT-M. In conclusion, RHEB engages two different binding sites of mTOR, ∆N-FAT-M and ∆ATP, with higher affinity for ∆ATP, which likely regulates the kinase activity of mTOR through multiple different biding modes.     

The Systems of Naringenin with Solubilizers Expand Its Capability to Prevent Neurodegenerative Diseases

Background: Naringenin (NAR) is a flavonoid with excellent antioxidant and neuroprotective potential that is limited by its low solubility. Thus, solid dispersions with β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (HP-β-CD), hydroxypropylmethylcellulose (HPMC), and microenvironmental pH modifiers were prepared. Methods: The systems formation analysis was performed by X-Ray Powder Diffraction (XRPD) and Fourier-transform infrared spectroscopy (FT-IR). Water solubility and dissolution rates were studied with a pH of 1.2 and 6.8. In vitro permeability through the gastrointestinal tract (GIT) and the blood-brain barrier (BBB) was assessed with the parallel artificial membrane permeability assay (PAMPA) assay. The antioxidant activity was studied with the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and cupric ion reducing antioxidant capacity (CUPRAC) assays, while in vitro enzymes studies involved the inhibition of acetylcholinesterase, butyrylcholinesterase, and tyrosinase. For the most promising system, in silico studies were conducted. Results: NAR solubility was increased 458-fold by the solid dispersion NAR:HP-β-CD:NaHCO₃ in a mass ratio of 1:3:1. The dissolution rate was elevated from 8.216% to 88.712% in a pH of 1.2 and from 11.644% to 88.843% in a pH of 6.8 (within 3 h). NAR GIT permeability, described as the apparent permeability coefficient, was increased from 2.789 × 10⁻⁶ cm s⁻¹ to 2.909 × 10⁻⁵ cm s⁻¹ in an acidic pH and from 1.197 × 10⁻⁶ cm s⁻¹ to 2.145 × 10⁻⁵ cm s⁻¹ in a basic pH. NAR BBB permeability was established as 4.275 × 10⁻⁶ cm s⁻¹. The antioxidant activity and enzyme inhibition were also increased. Computational studies confirmed NAR:HP-β-CD inclusion complex formation. Conclusions: A significant improvement in NAR solubility was associated with an increase in its biological activity.     

Effect of Silver Nanoparticles on the In Vitro Regeneration,Biochemical, Genetic,and Phenotype Variation in Adventitious Shoots Produced from Leaf Explants in Chrysanthemum

Novel and unique properties of nanomaterials, which are not apparent in larger-size forms of the same material, encourage the undertaking of studies exploring the multifaced effects of nanomaterials on plants. The results of such studies are not only scientifically relevant but, additionally, can be implemented to plant production and/or breeding. This study aimed to verify the applicability of silver nanoparticles (AgNPs) as a mutagen in chrysanthemum breeding. Chrysanthemum × grandiflorum (Ramat.) Kitam. ‘Lilac Wonder’ and ‘Richmond’ leaf explants were cultured on the modified MS medium supplemented with 0.6 mg·L⁻¹ 6-benzylaminopurine (BAP) and 2 mg·L⁻¹ indole-3-acetic acid (IAA) and treated with AgNPs (spherical; 20 nm in diameter size; 0, 50, and 100 mg·L⁻¹). AgNPs strongly suppressed the capability of leaf explants to form adventitious shoots and the efficiency of shoot regeneration. The content of primary and secondary metabolites (chlorophyll a, chlorophyll b, total chlorophylls, carotenoids, anthocyanins, phenolic compounds) and the activity of enzymatic antioxidants (superoxide dismutase and guaiacol peroxide) in leaf explants varied depending on the AgNPs treatment and age of culture. Phenotype variations of ex vitro cultivated chrysanthemums, covering the color and pigment content in the inflorescence, were detected in one 50 mg·L⁻¹ AgNPs-derived and five 100 mg·L⁻¹ AgNPs-derived ‘Lilac Wonder’ plants and were manifested as the color change from pink to burgundy-gold. However, no changes in inflorescence color/shape were found among AgNPs-treated ‘Richmond’ chrysanthemums. On the other hand, the stem height, number of leaves, and chlorophyll content in leaves varied depending on the AgNPs treatment and the cultivar analyzed. A significant effect of AgNPs on the genetic variation occurrence was found. A nearly two-fold higher share of polymorphic products, in both cultivars studied, was generated by RAPD markers than by SCoTs. To conclude, protocols using leaf explant treatment with AgNPs can be used as a novel breeding technique in chrysanthemum. However, the individual cultivars may differ in biochemical response, the efficiency of in vitro regeneration, genetic variation, and frequency of induced mutations in flowering plants.     

Identification of Potential Antiviral Inhibitors from Hydroxychloroquine and 1,2,4,5-Tetraoxanes Analogues and Investigation of the Mechanism of Action in SARS-CoV-2

This study aimed to identify potential inhibitors and investigate the mechanism of action on SARS-CoV-2 ACE2 receptors using a molecular modeling study and theoretical determination of biological activity. Hydroxychloroquine was used as a pivot structure and antimalarial analogues of 1,2,4,5 tetraoxanes were used for the construction and evaluation of pharmacophoric models. The pharmacophore-based virtual screening was performed on the Molport^® database (~7.9 million compounds) and obtained 313 structures. Additionally, a pharmacokinetic study was developed, obtaining 174 structures with 99% confidence for human intestinal absorption and penetration into the blood–brain barrier (BBB); posteriorly, a study of toxicological properties was realized. Toxicological predictions showed that the selected molecules do not present a risk of hepatotoxicity, carcinogenicity, mutagenicity, and skin irritation. Only 54 structures were selected for molecular docking studies, and five structures showed binding affinity (ΔG) values satisfactory for ACE2 receptors (PDB 6M0J), in which the molecule MolPort-007-913-111 had the best ΔG value of −8.540 Kcal/mol, followed by MolPort-002-693-933 with ΔG = −8.440 Kcal/mol. Theoretical determination of biological activity was realized for 54 structures, and five molecules showed potential protease inhibitors. Additionally, we investigated the Mpro receptor (6M0K) for the five structures via molecular docking, and we confirmed the possible interaction with the target. In parallel, we selected the TopsHits 9 with antiviral potential that evaluated synthetic accessibility for future synthesis studies and in vivo and in vitro tests.     

Synthesis,DNA Binding,and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 10⁴ to 1.0 × 10⁶ M⁻¹ and quenching constants from −0.2 × 10⁴ to 2.18 × 10⁴ M⁻¹ indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.