Bryophyllum pinnatum Inhibits Oxytocin and Vasopressin Signaling in Myometrial Cells.

The medicinal plant Bryophyllum pinnatum was previously shown to block oxytocin (OT)-induced signals in myometrial cells, consistent with its tocolytic effect observed in patients. OT activates not only OT receptors but also V1A receptors, two receptors with high receptor homology that are both expressed in the myometrium and play a crucial role in myometrial contraction signaling. We aimed to study the molecular pharmacology of B. pinnatum herbal preparations using specific receptor ligands, the human myometrial cell line hTERT-C3, and cell lines expressing recombinant human OT and V1A receptors.We found that press juice from B. pinnatum (BPJ) inhibits both OT- and vasopressin (AVP)-induced intracellular calcium increases in hTERT-C3 myometrial cells. In additional assays performed with cells expressing recombinant receptors, BPJ also inhibited OT and V1A receptor-mediated signals with a similar potency (IC50 about 0.5 mg/mL). We further studied endogenous OT- and AVP-sensitive receptors in hTERT-C3 cells and found that OT and AVP stimulated those receptors with similar potency (EC50 of ~ 1 nM), suggesting expression of both receptor subtypes. This interpretation was corroborated by the antagonist potencies of atosiban and relcovaptan that we found. However, using qPCR, we almost exclusively found expression of OT receptors suggesting a pharmacological difference between recombinant OT receptors and native receptors expressed in hTERT-C3 cells.In conclusion, we show that B. pinnatum inhibits both OT and AVP signaling, which may point beyond its tocolytic effects to other indications involving a disbalance in the vasopressinergic system.

The Utility of Peripherally Restricted Kappa-Opioid Receptor Agonists for Inhibiting Below-Level Pain After Spinal Cord Injury in Mice.

Pain after spinal cord injury (SCI) can be difficult to treat. Drugs that target the opioid receptor (OR) outside the central nervous system (CNS) have gained increasing interest in pain control owing to their low risk of central side effects. Asimadoline and ICI-204448 are believed to be peripherally restricted KOR agonists withlimited access to the CNS. This study examined whether they can attenuate pain hypersensitivity in mice subjected to a contusive T10 SCI. Subcutaneous (s.c.) injection of asimadoline (5, 20 mg/kg) and ICI-204448 (1, 10 mg/kg) inhibited heat hypersensitivity at both doses, but only attenuated mechanical hypersensitivity at the high dose. However, the high-dose asimadoline adversely affected animals' exploratory performance in SCI mice and caused aversion, suggesting CNS drug penetration. In contrast, high-dose ICI-204448 did not impair exploration and remained effective in reducing both mechanical and heat hypersensitivities after SCI. Accordingly, we chose to examine the potential peripheral neuronal mechanism for ICI-204448-induced pain inhibition by conducting in vivo calcium imaging of dorsal root ganglion (DRG) in Pirt-GCaMP6s+/- mice. High-dose ICI-204448 (10 mg/kg, s.c.) attenuated the increased fluorescence intensity of lumbar DRG neurons activated by a noxious pinch (400 g) stimulation in SCI mice. In conclusion, systemic administration of ICI-204448 achieved SCI pain inhibition at doses that did not induce notable side effects and attenuated DRG neuronal excitability which may partly contribute to its pain inhibition. These findings suggest that peripherally restricted KOR agonists may be useful for treating SCI pain, but the therapeutic window must be carefully examined.

Targeted isolation and identification of bioactive pyrrolidine alkaloids from Codonopsis pilosula using characteristic fragmentation-assisted mass spectral networking.

Codonopsis pilosula (CP), a well-known food medicine homology plant, is commonly used in many countries. In our preliminary study, a series of pyrrolidine alkaloids with high MS responses were detected as characteristic absorbed constituents in rat plasma after oral administration of CP extract. However, their structures were unclear due to the presence of various isomers and the lack of reference standards. In the present study, an MS-guided targeted isolation of pyrrolidine alkaloids of CP extract was performed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF MS). For data analysis under fast data directed acquisition mode (Fast-DDA), an effective approach named characteristic fragmentation-assisted mass spectral networking was successfully applied to discover new pyrrolidine alkaloids with high MS response in CP extract. As a result, seven new pyrrolizidine alkaloids [codonopyrrolidiums C-I (3-9)], together with two known ones (1 and 2), were isolated and identified by NMR spectral analysis. Among them, codonopyrrolidium B (1), codonopyrrolidium D (4) and codonopyrrolidium E (5) were evaluated for lipid-lowering activity, and they could improve high fructose-induced lipid accumulation in HepG2 cells. In addition, the characteristic MS/MS fragmentation patterns of these pyrrolizidine alkaloids were investigated, and 17 pyrrolidine alkaloids were identified. This approach could accelerate novel natural products discovery and characterize a class of natural products with MS/MS fragmentation patterns from similar chemical scaffolds. The research also provides a chemical basis for revealingin vivo effective substances in CP.

Phosphorus-nitrogen compounds: part 53-synthesis, characterization, cytotoxic and antimicrobial activity, DNA interaction and molecular docking studies of new mono- and dispirocyclotriphosphazenes with pendant arm(s).

Mono-/dispirocyclotriphosphazenes with pendant arm(s) are robust, but they are less investigated inorganic ring systems. In this study, a series of mono (3 and 4)- and dispirocyclotriphosphazenes with 4-chloro-benzyl pendant arm(s) (13-16) was obtained from the Cl exchange reactions of hexachlorocyclotriphosphazene with sodium (N-benzyl)aminopropanoxides (1 and 2). When compound (3) reacted with excess pyrrolidine, morpholine, tetra-1,4-dioxa-8-azaspiro[4,5]decane (DASD) and piperidine, the fully substituted monospirocyclotriphosphazenes (7, 9, 10 and 12) occurred. But, the reactions of 4 with excess piperidine and morpholine produced the gem-piperidino (5)- and morpholino (6)-substituted monospirocyclotriphosphazenes, whereas the reactions of 4 with excess pyrrolidine and DASD gave the fully substituted monospirocyclotriphosphazenes (8) and (11). However, it should be indicated that these derivatives were obtained to be used for the investigation of their spectral, stereogenic and biological properties. The structures of 5, 7 and 14 were determined crystallographically. X-ray data of 5 and 14 displayed that both of compounds were chiral in solid state, and their absolute configurations were assigned as R and RR. Additionally, the antimicrobial activities of phosphazenes were investigated. Minimum inhibitory concentrations, minimal bacterial concentrations and minimum fungicidal concentrations of phosphazenes were determined. The interactions of phosphazenes with plasmid DNA were evaluated by agarose gel electrophoresis. The cytotoxic activities of compounds were studied against L929 fibroblast and DLD-1 colon cancer cells. In addition, density functional theory calculations of 5, 7 and 14 were reported, and their molecular docking studies with DNA, E. coli DNA gyrase and topoisomerase IV were presented.

A fluorescence-based, gain-of-signal, live cell system to evaluate SARS-CoV-2 main protease inhibition.

The likelihood of continued circulation of COVID-19 and its variants, and novel coronaviruses due to future zoonotic transmissions, combined with the current paucity of coronavirus antivirals, emphasize the need for improved screening in developing effective antivirals for the treatment of infection by SARS-CoV-2 (CoV2) and other coronaviruses. Here we report the development of a live-cell based assay for evaluating the intracellular function of the critical, highly-conserved CoV2 target, the Main 3C-like protease (Mpro). This assay is based on expression of native wild-type mature CoV2 Mpro, the function of which is quantitatively evaluated in living cells through cleavage of a biosensor leading to loss of fluorescence. Evaluation does not require cell harvesting, allowing for multiple measurements from the same cells facilitating quantification of Mpro inhibition, as well as recovery of function upon removal of inhibitory drugs. The pan-coronavirus Mpro inhibitor, GC376, was utilized in this assay and effective inhibition of intracellular CoV2 Mpro was found to be consistent with levels required to inhibit CoV2 infection of human lung cells. We demonstrate that GC376 is an effective inhibitor of intracellular CoV2 Mpro at low micromolar levels, while other predicted Mpro inhibitors, bepridil and alverine, are not. Results indicate this system can provide a highly effective high-throughput coronavirus Mpro screening system.

An update on the progress of galidesivir (BCX4430), a broad-spectrum antiviral.

Galidesivir (BCX4430) is an adenosine nucleoside analog that is broadly active in cell culture against several RNA viruses of various families. This activity has also been shown in animal models of viral disease associated with Ebola, Marburg, yellow fever, Zika, and Rift Valley fever viruses. In many cases, the compound is more efficacious in animal models than cell culture activity would predict. Based on favorable data from in vivo animal studies, galidesivir has recently undergone evaluation in several phase I clinical trials, including against severe acute respiratory syndrome coronavirus 2, and as a medical countermeasure for the treatment of Marburg virus disease.

GC-MS, LC-MS/MS, Docking and Molecular Dynamics Approaches to Identify Potential SARS-CoV-2 3-Chymotrypsin-Like Protease Inhibitors from Zingiber officinale Roscoe.

This study aims to identify and isolate the secondary metabolites of Zingiber officinale using GC-MS, preparative TLC, and LC-MS/MS methods, to evaluate the inhibitory potency on SARS-CoV-2 3 chymotrypsin-like protease enzyme, as well as to study the molecular interaction and stability by using docking and molecular dynamics simulations. GC-MS analysis suggested for the isolation of terpenoids compounds as major compounds on methanol extract of pseudostems and rhizomes. Isolation and LC-MS/MS analysis identified 5-hydro-7, 8, 2'-trimethoxyflavanone (9), (E)-hexadecyl-ferulate (1), isocyperol (2), N-isobutyl-(2E,4E)-octadecadienamide (3), and nootkatone (4) from the rhizome extract, as well as from the leaves extract with the absence of 9. Three known steroid compounds, i.e., spinasterone (7), spinasterol (8), and 24-methylcholesta-7-en-3ß-on (6), were further identified from the pseudostem extract. Molecular docking showed that steroids compounds 7, 8, and 6 have lower predictive binding energies (MMGBSA) than other metabolites with binding energy of -87.91, -78.11, and -68.80 kcal/mole, respectively. Further characterization on the single isolated compound by NMR showed that 6 was identified and possessed 75% inhibitory activity on SARS-CoV-2 3CL protease enzyme that was slightly different with the positive control GC376 (77%). MD simulations showed the complex stability with compound 6 during 100 ns simulation time.

Pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs as possible candidates against neglected tropical diseases (NTDs): identification of hit compounds towards development of potential treatment of Leishmania donovani.

A rational-based process was adopted for repurposing pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs bearing variable acyl chains, different stereochemical configuration and/or positional relationships. Structural features were highly influential on activity. Amongst, enantiomer 1e having 1,2-vicinal relationship for the -CH2O- and the N-acyl moieties, a saturated palmitoyl chain and an opposite stereochemical configuration to natural sphingolipids was the most potent hit compound against promastigotes showing IC50 value of 28.32 µM. The corresponding enantiomer 1a was 2-fold less potent showing a eudismic ratio of 0.54 in promastigotes. Compounds 1a and 1e inhibited the growth of amastigotes more potently relative to promastigotes. Amongst, enantiomer 1a as the more selective and safer. In silico docking study using a homology model of Leishmania donovani inositol phosphoceramide synthase (IPCS) provided plausible reasoning for the molecular factors underlying the found activity. Collectively, this study suggests compounds 1a and 1e as potential hit compounds for further development of new antileishmanial agents.

Qing-Fei-Pai-Du decoction and wogonoside exert anti-inflammatory action through down-regulating USP14 to promote the degradation of activating transcription factor 2.

COVID-19 is often characterized by dysregulated inflammatory and immune responses. It has been shown that the Traditional Chinese Medicine formulation Qing-Fei-Pai-Du decoction (QFPDD) is effective in the treatment of the disease, especially for patients in the early stage. Our network pharmacology analyses indicated that many inflammation and immune-related molecules were the targets of the active components of QFPDD, which propelled us to examine the effects of the decoction on inflammation. We found in the present study that QFPDD effectively alleviated dextran sulfate sodium-induced intestinal inflammation in mice. It inhibited the production of pro-inflammatory cytokines IL-6 and TNFα, and promoted the expression of anti-inflammatory cytokine IL-10 by macrophagic cells. Further investigations found that QFPDD and one of its active components wogonoside markedly reduced LPS-stimulated phosphorylation of transcription factor ATF2, an important regulator of multiple cytokines expression. Our data revealed that both QFPDD and wogonoside decreased the half-life of ATF2 and promoted its proteasomal degradation. Of note, QFPDD and wogonoside down-regulated deubiquitinating enzyme USP14 along with inducing ATF2 degradation. Inhibition of USP14 with the small molecular inhibitor IU1 also led to the decrease of ATF2 in the cells, indicating that QFPDD and wogonoside may act through regulating USP14 to promote ATF2 degradation. To further assess the importance of ubiquitination in regulating ATF2, we generated mice that were intestinal-specific KLHL5 deficiency, a CUL3-interacting protein participating in substrate recognition of E3s. In these mice, QFPDD mitigated inflammatory reaction in the spleen, but not intestinal inflammation, suggesting CUL3-KLHL5 may function as an E3 for ATF2 degradation.

Single Exposure to the Cathinones MDPV and α-PVP Alters Molecular Markers of Neuroplasticity in the Adult Mouse Brain.

Synthetic cathinones have gained popularity among young drug users and are widely used in the clandestine market. While the cathinone-induced behavioral profile has been extensively investigated, information on their neuroplastic effects is still rather fragmentary. Accordingly, we have exposed male mice to a single injection of MDPV and α-PVP and sacrificed the animals at different time points (i.e., 30 min, 2 h, and 24 h) to have a rapid readout of the effect of these psychostimulants on neuroplasticity in the frontal lobe and hippocampus, two reward-related brain regions. We found that a single, low dose of MDPV or α-PVP is sufficient to alter the expression of neuroplastic markers in the adult mouse brain. In particular, we found increased expression of the transcription factor Npas4, increased ratio between the vesicular GABA transporter and the vesicular glutamate transporter together with changes in the expression of the neurotrophin Bdnf, confirming the widespread impact of these cathinones on brain plasticity. To sum up, exposure to low dose of cathinones can impair cortical and hippocampal homeostasis, suggesting that abuse of these cathinones at much higher doses, as it occurs in humans, could have an even more profound impact on neuroplasticity.

Selective estrogen receptor modulator lasofoxifene suppresses spondyloarthritis manifestation and affects characteristics of gut microbiota in zymosan-induced SKG mice.

Ankylosing spondylitis is a male-predominant disease and previous study revealed that estrogens have an anti-inflammatory effect on the spondyloarthritis (SpA) manifestations in zymosan-induced SKG mice. This study aimed to evaluate the effect of selective estrogen receptor modulator (SERM) lasofoxifene (Laso) on disease activity of SpA. Mice were randomized into zymosan-treated, zymosan + 17ß-estradiol (E2)-treated, and zymosan + Laso-treated groups. Arthritis was assessed by 18F-fluorodeoxyglucose (18F-FDG) small-animal positron emission tomography/computed tomography and bone mineral density (BMD) was measured. Fecal samples were collected and 16S ribosomal RNA gene sequencing was used to determine gut microbiota differences. Both zymosan + E2-treated mice and zymosan + Laso-treated mice showed lower arthritis clinical scores and lower 18F-FDG uptake than zymosan-treated mice. BMD was significantly higher in zymosan + E2-treated mice and zymosan + Laso-treated mice than zymosan-treated mice, respectively. Fecal calprotectin levels were significantly elevated at 8 weeks after zymosan injection in zymosan-treated mice, but it was not significantly changed in zymosan + E2-treated mice and zymosan + Laso-treated mice. Gut microbiota diversity of zymosan-treated mice was significantly different from zymosan + E2-treated mice and zymosan + Laso-treated mice, respectively. There was no significant difference in gut microbiota diversity between zymosan + E2-treated mice and zymosan + Laso -treated mice. Laso inhibited joint inflammation and enhanced BMD in SKG mice, a model of SpA. Laso also affected the composition and biodiversity of gut microbiota. This study provides new knowledge regarding that selected SpA patients could benefit from SERM treatment.

Zinc supplementation augments the suppressive effects of repurposed NF-κB inhibitors on ACE2 expression in human lung cell lines.

AIMS: Angiotensin-converting enzyme 2 (ACE2) is a key negative regulator of the renin-angiotensin system and also a major receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal a role for NF-κB in human lung cell expression of ACE2, and we further explore the potential utility of repurposing NF-κB inhibitors to downregulate ACE2. MAIN METHODS: Expression of ACE2 was assessed by Western blotting and RT-qPCR in multiple human lung cell lines with or without NF-κB inhibitor treatment. Surface ACE2 expression and intracellular reactive oxygen species (ROS) levels were measured with flow cytometry. p50 was knocked down with siRNA. Cytotoxicity was monitored by PARP cleavage and MTS assay. KEY FINDINGS: Pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, suppressed endogenous ACE2 mRNA and protein expression in H322M and Calu-3 cells. The ROS level in H322M cells was increased after PDTC treatment, and pretreatment with N-acetyl-cysteine (NAC) reversed PDTC-induced ACE2 suppression. Meanwhile, treatment with hydrogen peroxide augmented ACE2 suppression in H322M cells with p50 knockdown. Two repurposed NF-κB inhibitors, the anthelmintic drug triclabendazole and the antiprotozoal drug emetine, also reduced ACE2 mRNA and protein levels. Moreover, zinc supplementation augmented the suppressive effects of triclabendazole and emetine on ACE2 expression in H322M and Calu-3 cells. SIGNIFICANCE: These results suggest that ACE2 expression is modulated by ROS and NF-κB signaling in human lung cells, and the combination of zinc with triclabendazole or emetine shows promise for clinical treatment of ACE2-related disease.

Synthesis, Anticonvulsant, and Antinociceptive Activity of New 3-(2-Chlorophenyl)- and 3-(3-Chlorophenyl)-2,5-dioxo-pyrrolidin-1-yl-acetamides.

The new series of 3-(2-chlorophenyl)- and 3-(3-chlorophenyl)-pyrrolidine-2,5-dione-acetamide derivatives as potential anticonvulsant and analgesic agents was synthesized. The compounds obtained were evaluated in the following acute models of epilepsy: maximal electroshock (MES), psychomotor (6 Hz, 32 mA), and subcutaneous pentylenetetrazole (scPTZ) seizure tests. The most active substance-3-(2-chlorophenyl)-1-{2-[4-(4-fluorophenyl)piperazin-1-yl]-2-oxoethyl}-pyrrolidine-2,5-dione (6) showed more beneficial ED50 and protective index values than the reference drug-valproic acid (68.30 mg/kg vs. 252.74 mg/kg in the MES test and 28.20 mg/kg vs. 130.64 mg/kg in the 6 Hz (32 mA) test, respectively). Since anticonvulsant drugs are often effective in neuropathic pain management, the antinociceptive activity for two the promising compounds-namely, 6 and 19-was also investigated in the formalin model of tonic pain. Additionally, for the aforementioned compounds, the affinity for the voltage-gated sodium and calcium channels, as well as GABAA and TRPV1 receptors, was determined. As a result, the most probable molecular mechanism of action for the most active compound 6 relies on interaction with neuronal voltage-sensitive sodium (site 2) and L-type calcium channels. Compounds 6 and 19 were also tested for their neurotoxic and hepatotoxic properties and showed no significant cytotoxic effect.

Poria cocos polysaccharide attenuates damage of nervus in Alzheimer's disease rat model induced by D-galactose and aluminum trichloride.

Poria cocos polysaccharide (PCP) is a compound from Poria cocos, and which is used as a classical tonic agent. This article aims to investigate the effects of PCP on neuronal damage of hippocampus and cognitive function in a rat model of Alzheimer's disease induced by D-galactose and aluminum trichloride. Oxiracetam (ORC) was used as a positive drug in this experiment. The rats were treated with PCP at doses of 100, 200 and 300 mg/kg/day for 30 days and ORC at dose of 346 mg/kg/day after modeling. The results of behavioral test showed that PCP could prevent cognitive decline in Alzheimer's disease rats as assessed by Y-maze test and Morris water maze test. Results of hippocampus slices showed that neurons were integrated and regularly arranged in the groups, which were administered along with PCP. Moreover, PCP could reduce neuronal apoptosis in hippocampus of Alzheimer's disease rats. Furthermore, the activities of superoxide dismutase in the hippocampus were elevated by PCP administration, while acetyl cholinesterase, reactive oxygen, malondialdehyde and inflammatory factors levels were reduced. In addition, we found PCP could attenuate MAPK/NF-κB signal pathway in the hippocampus. All results illustrated that PCP could exert neuroprotective effects at least partly through alleviating oxidative stress, apoptosis, inflammation and inhibiting the MAPK/NF-κB pathway in Alzheimer's disease rats induced by D-galactose and aluminum trichloride.

Lead compounds for the development of SARS-CoV-2 3CL protease inhibitors.

We report the identification of three structurally diverse compounds - compound 4, GC376, and MAC-5576 - as inhibitors of the SARS-CoV-2 3CL protease. Structures of each of these compounds in complex with the protease revealed strategies for further development, as well as general principles for designing SARS-CoV-2 3CL protease inhibitors. These compounds may therefore serve as leads for the basis of building effective SARS-CoV-2 3CL protease inhibitors.

Discovery of Nanomolar Melanocortin-3 Receptor (MC3R)-Selective Small Molecule Pyrrolidine Bis-Cyclic Guanidine Agonist Compounds Via a High-Throughput "Unbiased" Screening Campaign.

The central melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are key regulators of body weight and energy homeostasis. Herein, the discovery and characterization of first-in-class small molecule melanocortin agonists with selectivity for the melanocortin-3 receptor over the melanocortin-4 receptor are reported. Identified via "unbiased" mixture-based high-throughput screening approaches, pharmacological evaluation of these pyrrolidine bis-cyclic guanidines resulted in nanomolar agonist activity at the melanocortin-3 receptor. The pharmacological profiles at the remaining melanocortin receptor subtypes tested indicated similar agonist potencies at both the melanocortin-1 and melanocortin-5 receptors and antagonist or micromolar agonist activities at the melanocortin-4 receptor. This group of small molecules represents a new area of chemical space for the melanocortin receptors with mixed receptor pharmacology profiles that may serve as novel lead compounds to modulate states of dysregulated energy balance.

Design and characterisation of an amorphous formulation of nifedipine for the treatment of autonomic dysreflexia.

OBJECTIVES: Current treatment for autonomic dysreflexia (AD) involves rupturing a liquid-filled soft capsule of nifedipine to aid rapid drug release and absorption, however, this application is not covered under the manufacturer's license. The objective of the current work was to design a rapidly dissolving solid dosage formulation for the treatment of AD as an alternative to the off-license "bite and swallow" use of currently available commercial products. METHODS: Amorphous solid dispersions (ASDs) of nifedipine were prepared by spray-drying using three different polymers: hydroxypropyl methyl cellulose (HPMC), polyvinyl pyrrolidone (PVP) and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol (Soluplus), at a 15% w/w drug loading and were formulated and compressed into tablets. Dissolution testing was performed in the paddle dissolution apparatus using either a monophasic or biphasic medium. KEY FINDINGS: The PVP-nifedipine ASD tablets exhibited rapid dissolution, with 35% of the total nifedipine dose dissolving within 15 min in the monophasic dissolution medium. The HPMC-nifedipine ASD exhibited a very slow dissolution, while the Solupus-nifedipine system exhibited no nifedipine release over 120 min. When tested in the biphasic dissolution medium, the PVP-nifedipine ASD tablets exhibited a release profile comparable to that of the pre-split/ruptured nifedipine soft capsule product. CONCLUSIONS: This study demonstrates that a nifedipine-PVP ASD is a promising formulation strategy in the treatment of AD.

A molecular modelling approach for identifying antiviral selenium-containing heterocyclic compounds that inhibit the main protease of SARS-CoV-2: an in silico investigation.

Coronavirus disease 2019 (COVID-19), an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been declared a global pandemic by the World Health Organization, and the situation worsens daily, associated with acute increases in case fatality rates. The main protease (Mpro) enzyme produced by SARS-CoV-2 was recently demonstrated to be responsible for not only viral reproduction but also impeding host immune responses. The element selenium (Se) plays a vital role in immune functions, both directly and indirectly. Thus, we hypothesised that Se-containing heterocyclic compounds might curb the activity of SARS-CoV-2 Mpro. We performed a molecular docking analysis and found that several of the selected selenocompounds showed potential binding affinities for SARS-CoV-2 Mpro, especially ethaselen (49), which exhibited a docking score of -6.7 kcal/mol compared with the -6.5 kcal/mol score for GC376 (positive control). Drug-likeness calculations suggested that these compounds are biologically active and possess the characteristics of ideal drug candidates. Based on the binding affinity and drug-likeness results, we selected the 16 most effective selenocompounds as potential anti-COVID-19 drug candidates. We also validated the structural integrity and stability of the drug candidate through molecular dynamics simulation. Using further in vitro and in vivo experiments, we believe that the targeted compound identified in this study (ethaselen) could pave the way for the development of prospective drugs to combat SARS-CoV-2 infections and trigger specific host immune responses.

Effects of the amide alkaloid piperyline on apoptosis, autophagy, and differentiation of pre-osteoblasts.

BACKGROUND: Amide alkaloidsare typical constituents in plants of the Piperaceae family. Most of the pharmacological properties of Piper nigrum L. are attributed to the major amide alkaloid, piperine. Piperyline (PIPE) is a further amide alkaloid that has been isolated from P. nigrum. HYPOTHESIS/PURPOSE: This study was performed to examine the biological effects of PIPE on pre-osteoblasts and elucidate the underlying mechanisms. STUDY DESIGN: We investigated the effects of PIPE in MC3T3E-1 cells, which are widely used for studying osteoblast behavior in in vitro cell systems. METHODS: We evaluated cell viability based on the MTT assay, apoptosis by TUNEL staining, adhesion and migration by cell adhesion and migration assays, and osteoblast differentiation by alkaline phosphatase activity and staining. Western blot and immunocytochemical analyses were used to investigate cell signaling pathways. RESULTS: We found that at concentrations ranging from 1 to 30 µM, PIPE inhibited cell growth and induced apoptosis in pre-osteoblasts, which was accompanied by the upregulation of apoptotic proteins but downregulation of anti-apoptotic proteins. In contrast, PIPE had no appreciable effect on the autophagy pathway. Nevertheless, PIPE reduced cell adhesion and migration via the inactivation of non-receptor tyrosine kinase (Src)/focal adhesion kinase (FAK) and mitogen-activated protein kinases, and also promoted the downregulation of matrix metalloproteinase 2 and 9 levels. Furthermore, at concentrations of 10 and 30 µM, PIPE suppressed osteoblast differentiation, as indicated by reductions in alkaline phosphatase staining and activity. In addition, PIPE reduced the protein levels of phospho-Smad1/5/8 and runt-related transcription factor 2, and the mRNA levels of osteopontin, alkaline phosphatase, and osteocalcin. CONCLUSION: The findings of this study indicate that PIPE has biological effects associated with cell adhesion, migration, proliferation, and osteoblast differentiation, and suggest a potential role for this alkaloid in the treatment of bone diseases.

Screening of a Custom-Designed Acid Fragment Library Identifies 1-Phenylpyrroles and 1-Phenylpyrrolidines as Inhibitors of Notum Carboxylesterase Activity.

The Wnt family of proteins are secreted signaling proteins that play key roles in regulating cellular functions. Recently, carboxylesterase Notum was shown to act as a negative regulator of Wnt signaling by mediating the removal of an essential palmitoleate. Here we disclose two new chemical scaffolds that inhibit Notum enzymatic activity. Our approach was to create a fragment library of 250 acids for screening against Notum in a biochemical assay followed by structure determination by X-ray crystallography. Twenty fragments were identified as hits for Notum inhibition, and 14 of these fragments were shown to bind in the palmitoleate pocket of Notum. Optimization of 1-phenylpyrrole 20, guided by structure-based drug design, identified 20z as the most potent compound from this series. Similarly, the optimization of 1-phenylpyrrolidine 8 gave acid 26. This work demonstrates that inhibition of Notum activity can be achieved by small, drug-like molecules possessing favorable in vitro ADME profiles.