Design of inhibitors of SARS-CoV-2 papain-like protease deriving from GRL0617: Structure-activity relationships.

The unique and complex structure of papain-like protease (PLpro) of the SARS-CoV-2 virus represents a difficult challenge for antiviral development, yet it offers a compelling validated target for effective therapy of COVID-19. The surge in scientific interest in inhibiting this cysteine protease emerged after its demonstrated connection to the cytokine storm in patients with COVID-19 disease. Furthermore, the development of new inhibitors against PLpro may also be beneficial for the treatment of respiratory infections caused by emerging coronavirus variants of concern. This review article provides a comprehensive overview of PLpro inhibitors, focusing on the structural framework of the known inhibitor GRL0617 and its analogs. We categorize PLpro inhibitors on the basis of their structures and binding site: Glu167 containing site, BL2 groove, Val70Ub site, and Cys111 containing catalytic site. We summarize and evaluate the majority of GRL0617-like inhibitors synthesized so far, highlighting their published biochemical parameters, which reflect their efficacy. Published research has shown that strategic modifications to GRL0617, such as decorating the naphthalene ring, extending the aromatic amino group or the orthomethyl group, can substantially decrease the IC50 from micromolar up to nanomolar concentration range. Some advantageous modifications significantly enhance inhibitory activity, paving the way for the development of new potent compounds. Our review places special emphasis on structures that involve direct modifications to the GRL0617 scaffold, including piperidine carboxamides and modified benzylmethylnaphthylethanamines (Jun9 scaffold). All these compounds are believed to inhibit the proteolytic, deubiquitination, and deISGylation activity of PLpro, biochemical processes linked to the severe progression of COVID-19. Finally, we summarize the development efforts for SARS-CoV-2 PLpro inhibitors, in detailed structure-activity relationships diagrams. This aims to inform and inspire future research in the search for potent antiviral agents against PLpro of current and emerging coronavirus threats.

The Gridlock transcriptional repressor impedes vertebrate heart regeneration by restricting expression of lysine methyltransferase.

Teleost zebrafish and neonatal mammalian hearts exhibit the remarkable capacity to regenerate through dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). Although many mitogenic signals that stimulate zebrafish heart regeneration have been identified, transcriptional programs that restrain injury-induced CM renewal are incompletely understood. Here, we report that mutations in gridlock (grl; also known as hey2), encoding a Hairy-related basic helix-loop-helix transcriptional repressor, enhance CM proliferation and reduce fibrosis following damage. In contrast, myocardial grl induction blunts CM dedifferentiation and regenerative responses to heart injury. RNA sequencing analyses uncover Smyd2 lysine methyltransferase (KMT) as a key transcriptional target repressed by Grl. Reduction in Grl protein levels triggered by injury induces smyd2 expression at the wound myocardium, enhancing CM proliferation. We show that Smyd2 functions as a methyltransferase and modulates the Stat3 methylation and phosphorylation activity. Inhibition of the KMT activity of Smyd2 reduces phosphorylated Stat3 at cardiac wounds, suppressing the elevated CM proliferation in injured grl mutant hearts. Our findings establish an injury-specific transcriptional repression program in governing CM renewal during heart regeneration, providing a potential strategy whereby silencing Grl repression at local regions might empower regeneration capacity to the injured mammalian heart.

Repurposing the antibacterial drugs for inhibition of SARS-CoV2-PLpro using molecular docking, MD simulation and binding energy calculation.

Papain-like protease (nsp-3; non-structural protein) of novel corona virus is an ideal target for developing drugs as it plays multiple important functions for viral growth and replication. For instance, role of nsp-3 has been recognized in cleavage of viral polyprotein; furthermore, in infected host it weakens the immune system via downregulating the production of type I interferon. This downregulation is promoted by removal of ubiquitin-like interferon-stimulated gene 15 protein (ISG15) from interferon-responsive factor 3 (IRF3) protein. Among known inhibitors of SARS-CoV-PLpro GRL0617 is by far the most effective inhibitor. As PLpro of SARS-CoV2 is having more than 80% similarity with SARS-CoV-PLpro, GRL0617 is reported to be effective even against SARS-CoV2. Owing to this similarity, certain key amino acids remain the same/conserved in both proteins. Among conserved amino acids Tyr268 for SARS-CoV2 and Tyr269 for SARS-CoV produce important hydrophobic interactions with aromatic rings of GRL0617. Here, in this study antibacterial compounds were collected from ZINC database, and they were filtered to select compounds that are having similar structural features as GRL0617. This filtered library of compound was then docked with SARS-CoV and CoV2-PLpro. Five hits were noted that were able to interact with Tyr268 (SARS-CoV2) and Tyr269 (SARS-CoV). Further, best hit 2-(2-((benzofuran-2-carboxamido)methyl)-5-methoxy-1H-indol-1-yl)acetic acid (ZINC44459905) was studied using molecular dynamic simulation where stability of protein-ligand complex as well as stability of produced interactions was noted.

Identifying structural-functional analogue of GRL0617, the only well-established inhibitor for papain-like protease (PLpro) of SARS-CoV2 from the pool of fungal metabolites using docking and molecular dynamics simulation.

The non-structural protein (nsp)-3 of SARS-CoV2 coronavirus is sought to be an essential target protein which is also named as papain-like protease (PLpro). This protease cleaves the viral polyprotein, but importantly in human host it also removes ubiquitin-like interferon-stimulated gene 15 protein (ISG15) from interferon responsive factor 3 (IRF3) protein which ultimately downregulates the production of type I interferon leading to weakening of immune response. GRL0617 is the most potent known inhibitor for PLpro that was initially developed for SARS outbreak of 2003. The PLpro of SARS-CoV and CoV2 share 83% sequence identity but interestingly have several identical conserved amino acids that suggests GRL0617 to be an effective inhibitor for PLpro of SARS-CoV2. GRL0617 is a naphthalene-based molecule and interacts with Tyr268 of SARS-CoV2-PLpro (and Tyr269 of SARS-CoV-PLpro). To identify PLpro inhibitors, we prepared a library of secondary metabolites from fungi with aromatic nature and docked them with PLpro of SARS-CoV and SARS-CoV2. We found six hits which interacts with Tyr268 of SARS-CoV2-PLpro (and Tyr269 of SARS-CoV-PLpro). More surprisingly the top hit, Fonsecin, has naphthalene moiety in its structure, which recruits Tyr268 of SARS-CoV2-PLpro (and Tyr269 of SARS-CoV-PLpro) and has binding energy at par with control (GRL0617). Molecular dynamics (MD) simulation showed Fonsecin to interact with Tyr268 of SARS-CoV2-PLpro more efficiently than control (GRL0617) and interacting with a greater number of amino acids in the binding cleft of PLpro.

Investigation of small molecule inhibitors of the SARS-CoV-2 papain-like protease by all-atom microsecond modelling, PELE Monte Carlo simulations, and in vitro activity inhibition.

The SARS-CoV-2 papain-like (PLpro) protease is essential for viral replication. We investigated potential antiviral effects of hypericin relative to the well-known noncovalent PLpro inhibitor GRL-0617. Molecular dynamics and PELE Monte Carlo simulations highlight favourable binding of hypericin and GRL-0617 to the naphthalene binding pocket of PLpro. Although not potent as GRL-0617 (45.8 vs 1.6 µM for protease activity, respectively), in vitro fluorogenic enzymatic assays with hypericin show concentration-dependent inhibition of both PLpro protease and deubiquitinating activities. Given its use in supplementations and the FDA conditional approval of a synthetic version, further evaluation of hypericin as a potential SARS-CoV-2 antiviral is warranted.

In vitro metabolic characterization of the SARS-CoV-2 papain-like protease inhibitors GRL0617 and HY-17542.

The SARS-CoV-2 pandemic requires a new therapeutic target for viral infection, and papain-like protease (Plpro) has been suggested as a druggable target. This in-vitro study was conducted to examine the drug metabolism of the GRL0617 and HY-17542, Plpro inhibitors. Metabolism of these inhibitors was studied to predict the pharmacokinetics in human liver microsomes. The hepatic cytochrome P450 (CYP) isoforms responsible for their metabolism were identified using recombinant enzymes. The drug-drug interaction potential mediated by cytochrome P450 inhibition was estimated. In human liver microsomes, the Plpro inhibitors had phase I and phase I + II metabolism with half-lives of 26.35 and 29.53 min, respectively. Hydroxylation (M1) and desaturation (-H2, M3) of the para-amino toluene side chain were the predominant reactions mediated with CYP3A4 and CYP3A5. CYP2D6 is responsible for the hydroxylation of the naphthalene side ring. GRL0617 inhibits major drug-metabolizing enzymes, including CYP2C9 and CYP3A4. HY-17542 is structural analog of GRL0617 and it is metabolized to GRL0617 through non-cytochrome P450 reactions in human liver microsomes without NADPH. Like GRL0617 and HY-17542 undergoes additional hepatic metabolism. The in-vitro hepatic metabolism of the Plpro inhibitors featured short half-lives; preclinical metabolism studies are needed to determine therapeutic doses for these inhibitors.

Efficacy of a feed additive consisting of 6-phytase produced by Komagataella phaffii CGMCC 12056 (APSA PHYTAFEED® 20,000 GR/L) for pigs for fattening (Andrés Pintaluba S.A).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the efficacy of 6-phytase produced by the genetically modified strain Komagataella phaffii CGMCC 12056 (APSA PHYTAFEED® 20,000 GR/L) as a zootechnical feed additive (functional group: digestibility enhancers) for pigs for fattening at a minimum recommended level of 1,000 U /kg feed. In a previous assessment, three trials were submitted; one of them showed significant improvements on zootechnical parameters supporting the efficacy of the additive. The other two trials evaluating apparent faecal phosphorus digestibility and bone mineralisation, showed no improvements on phosphorus retention in bone and, therefore, the FEEDAP Panel could not conclude on the efficacy of the additive. The applicant submitted two new efficacy studies in pigs for fattening evaluating the animal's zootechnical performance, apparent faecal phosphorus digestibility and bone mineralisation. One of the trials was not considered due to high rates of mortality, culling of animals and medical treatments applied. In the other trial, significant improvements on final body weight and apparent total track phosphorus digestibility were found in the animals that received the phytase at 1,000 U/kg complete feed. Owing to the lack of sufficient data, the FEEDAP Panel could not conclude on the efficacy of the additive in pigs for fattening.

Drug-Repurposing Screening Identified Tropifexor as a SARS-CoV-2 Papain-like Protease Inhibitor.

The global COVID-19 pandemic underscores the dire need for effective antivirals. Encouraging progress has been made in developing small-molecule inhibitors targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and main protease (Mpro). However, the development of papain-like protease (PLpro) inhibitors faces several obstacles. Nevertheless, PLpro represents a high-profile drug target given its multifaceted roles in viral replication. PLpro is involved in not only the cleavage of viral polyprotein but also the modulation of host immune response. In this study, we conducted a drug-repurposing screening of PLpro against the MedChemExpress bioactive compound library and identified three hits, EACC, KY-226, and tropifexor, as potent PLpro inhibitors with IC50 values ranging from 3.39 to 8.28 µM. The three hits showed dose-dependent binding to PLpro in the thermal shift assay. In addition, tropifexor inhibited the cellular PLpro activity in the FlipGFP assay with an IC50 of 10.6 µM. Gratifyingly, tropifexor showed antiviral activity against SARS-CoV-2 in Calu-3 cells at noncytotoxic concentrations. Overall, tropifexor represents a novel PLpro inhibitor that can be further developed as SARS-CoV-2 antivirals.

HIV-1 protease with 10 lopinavir and darunavir resistance mutations exhibits altered inhibition, structural rearrangements and extreme dynamics.

Antiretroviral drug resistance is a therapeutic obstacle for people with HIV. HIV protease inhibitors darunavir and lopinavir are recommended for resistant infections. We characterized a protease mutant (PR10x) derived from a highly resistant clinical isolate including 10 mutations associated with resistance to lopinavir and darunavir. Compared to the wild-type protease, PR10x exhibits ∼3-fold decrease in catalytic efficiency and Ki values of 2-3 orders of magnitude worse for darunavir, lopinavir, and potent investigational inhibitor GRL-519. Crystal structures of the mutant were solved in a ligand-free form and in complex with GRL-519. The structures show altered interactions in the active site, flap-core interface, hydrophobic core, hinge region, and 80s loop compared to the corresponding wild-type protease structures. The ligand-free crystal structure exhibits a highly curled flap conformation which may amplify drug resistance. Molecular dynamics simulations performed for 1 µs on ligand-free dimers showed extremely large fluctuations in the flaps for PR10x compared to equivalent simulations on PR with a single L76V mutation or wild-type protease. This analysis offers insight about the synergistic effects of mutations in highly resistant variants.

Multi-level attention pooling for graph neural networks: Unifying graph representations with multiple localities.

Graph neural networks (GNNs) have been widely used to learn vector representation of graph-structured data and achieved better task performance than conventional methods. The foundation of GNNs is the message passing procedure, which propagates the information in a node to its neighbors. Since this procedure proceeds one step per layer, the range of the information propagation among nodes is small in the lower layers, and it expands toward the higher layers. Therefore, a GNN model has to be deep enough to capture global structural information in a graph. On the other hand, it is known that deep GNN models suffer from performance degradation because they lose nodes' local information, which would be essential for good model performance, through many message passing steps. In this study, we propose multi-level attention pooling (MLAP) for graph-level classification tasks, which can adapt to both local and global structural information in a graph. It has an attention pooling layer for each message passing step and computes the final graph representation by unifying the layer-wise graph representations. The MLAP architecture allows models to utilize the structural information of graphs with multiple levels of localities because it preserves layer-wise information before losing them due to oversmoothing. Results of our experiments show that the MLAP architecture improves the graph classification performance compared to the baseline architectures. In addition, analyses on the layer-wise graph representations suggest that aggregating information from multiple levels of localities indeed has the potential to improve the discriminability of learned graph representations.

Inhibitors of SARS-CoV-2 PLpro.

The emergence of SARS-CoV-2 causing the COVID-19 pandemic, has highlighted how a combination of urgency, collaboration and building on existing research can enable rapid vaccine development to fight disease outbreaks. However, even countries with high vaccination rates still see surges in case numbers and high numbers of hospitalized patients. The development of antiviral treatments hence remains a top priority in preventing hospitalization and death of COVID-19 patients, and eventually bringing an end to the SARS-CoV-2 pandemic. The SARS-CoV-2 proteome contains several essential enzymatic activities embedded within its non-structural proteins (nsps). We here focus on nsp3, that harbours an essential papain-like protease (PLpro) domain responsible for cleaving the viral polyprotein as part of viral processing. Moreover, nsp3/PLpro also cleaves ubiquitin and ISG15 modifications within the host cell, derailing innate immune responses. Small molecule inhibition of the PLpro protease domain significantly reduces viral loads in SARS-CoV-2 infection models, suggesting that PLpro is an excellent drug target for next generation antivirals. In this review we discuss the conserved structure and function of PLpro and the ongoing efforts to design small molecule PLpro inhibitors that exploit this knowledge. We first discuss the many drug repurposing attempts, concluding that it is unlikely that PLpro-targeting drugs already exist. We next discuss the wealth of structural information on SARS-CoV-2 PLpro inhibition, for which there are now ∼30 distinct crystal structures with small molecule inhibitors bound in a surprising number of distinct crystallographic settings. We focus on optimisation of an existing compound class, based on SARS-CoV PLpro inhibitor GRL-0617, and recapitulate how new GRL-0617 derivatives exploit different features of PLpro, to overcome some compound liabilities.

Novel mutations in NSP-1 and PLPro of SARS-CoV-2 NIB-1 genome mount for effective therapeutics.

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), is rapidly acquiring new mutations. Analysis of these mutations is necessary for gaining knowledge regarding different aspects of therapeutic development. Previously, we have reported a Sanger method-based genome sequence of a viral isolate named SARS-CoV-2 NIB-1, circulating in Bangladesh. The genome has four novel non-synonymous mutations in V121D, V843F, A889V, and G1691C positions. RESULTS: Using different computational tools, we have found V121D substitution has the potential to destabilize the non-structural protein-1 (NSP-1). NSP-1 inactivates the type-1 interferon-induced antiviral system. Hence, this mutant could be a basis of attenuated vaccines against SARS-CoV-2. V843F, A889V, and G1691C are all located in nonstructural protein-3 (NSP-3). G1691C can decrease the flexibility of the protein. V843F and A889V might change the binding pattern and efficacy of SARS-CoV-2 papain-like protease (PLPro) inhibitor GRL0617. V843F substitution in PLPro was the most prevalent mutation in the clinical samples. This mutation showed a reduced affinity for interferon-stimulated gene-15 protein (ISG-15) and might have an impact on innate immunity and viral spread. However, V843F+A889V double mutant exhibited the same binding affinity as wild type PLPro. A possible reason behind this phenomenon can be that V843F is a conserved residue of PLPro which damaged the protease structure, but A889V, a less conserved residue, presumably neutralized that damage. CONCLUSIONS: Mutants of NSP-1 could provide attenuated vaccines against coronavirus. Also, these mutations of PLPro might be targeted to develop better anti-SARS therapeutics. We hope our study will help to get better insides during the development of attenuated vaccine and PLPro inhibitors.

Interaction of small molecules with the SARS-CoV-2 papain-like protease: In silico studies and in vitro validation of protease activity inhibition using an enzymatic inhibition assay.

The SARS-CoV-2 virus is causing COVID-19, an ongoing pandemic, with extraordinary global health, social, and political implications. Currently, extensive research and development efforts are aimed at producing a safe and effective vaccine. In the interim, small molecules are being widely investigated for antiviral effects. With respect to viral replication, the papain-like (PLpro) and main proteases (Mpro), are critical for processing viral replicase polypeptides. Further, the PLpro possesses deubiquitinating activity affecting key signalling pathways, including inhibition of interferon and innate immune antagonism. Therefore, inhibition of PLpro activity with small molecules is an important research direction. Our aim was to focus on identification of potential inhibitors of the protease activity of SARS-CoV-2 PLpro. We investigated 300 small compounds derived predominantly from our OliveNet™ library (222 phenolics) and supplemented with synthetic and dietary compounds with reported antiviral activities. An initial docking screen, using the potent and selective noncovalent PLpro inhibitor, GRL-0617 as a control, enabled a selection of 30 compounds for further analyses. From further in silico analyses, including docking to scenes derived from a publicly available molecular dynamics simulation trajectory (100 µs PDB 6WX4; DESRES-ANTON-11441075), we identified lead compounds for further in vitro evaluation using an enzymatic inhibition assay measuring SARS-CoV-2 PLpro protease activity. Our findings indicate that hypericin possessed inhibition activity, and both rutin and cyanidin-3-O-glucoside resulted in a concentration-dependent inhibition of the PLpro, with activity in the micromolar range. Overall, hypericin, rutin, and cyanidin-3-O-glucoside can be considered lead compounds requiring further characterisation for potential antiviral effects in appropriate model systems.

Role of Risk Assessment in Prevention of Work-Related Accidents and Diseases in Hospital Staff.

Risk assessment is an essential component of the occupational health and safety event prevention activity.The purpose of this study is to choose the most appropriate risk assessment method for hospitals. The main methods were compared. There are many assessment methods, each with its advantages and disadvantages, but none has been adapted to the specificities of hospital activity. We adapted the workplace assessment sheet from the INCDPM (National Research and Development Institute for Labor Protection Bucharest) method to the specific of the hospital units and used this method at the level of jobs, within the hospital's departments, calculating the global risk level per job position, workplace (department), and hospital. The clinical departments global risk level exceeds the average (3.00) for all jobs, but does not exceed, however, 3.50, representing an acceptable security level. For assess the psychosocial risks we used the ELVIE method. Looking ahead, the methods should be adapted to allow both numerical presentation of the results and graphic.

Grl1 Protein is a Candidate K Antigen in Tetrahymena thermophila.

In Tetrahymena, K antigens associate only with mature basal bodies and are expected to play important roles in the morphogenesis and function of the membrane skeleton around basal bodies, but these proteins have not been identified and their functions are unknown. Commercially available anti-human Rho GDP-dissociation inhibitor α (RhoGDIα) antibody (sc-33201) was accidentally found to show very similar immunofluorescence staining patterns to those of anti-K antigen antibodies, such as 424A8 and 10D12 mouse monoclonal antibodies, in Tetrahymena. A 40kDa protein recognized by this antibody was partially purified and identified as granule lattice protein 1 (Grl1p) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. In immunoblotting experiments this antibody was suggested to recognize endogenous Grl1p. The three-dimensional structure of proGrl1p protein predicted by I-TASSER was similar to a spectrin family protein. Grl1 may be a K antigen and a spectrin-like protein in Tetrahymena.

Anaerobic decolorization and detoxification of cationic red X-GRL by Shewanella oneidensis MR-1.

The ability of an electrochemically active bacterium, Shewanella oneidensis MR-1, to decolorize azo dye cationic red X-GRL (X-GRL) was investigated. S. oneidensis MR-1 showed a high decolorization capability for X-GRL under anaerobic conditions. The Mtr respiratory pathway was proved to be involved in the extracellular decolorization of X-GRL. The decolorization efficiency of S. oneidensis MR-1 was significantly inhibited when the initial X-GRL concentration was over 200 mg L-1. Increasing the inoculum volume of S. oneidensis MR-1 could obviously promote the X-GRL decolorization. The 100 mg L-1 X-GRL and 6% (v/v) inoculum volume were chosen as the optimal parameter. Under such a condition, almost all of X-GRL (100 mg L-1) could be completely reduced after 12-h incubation at the pH range of 5.5-8.0 and temperature range of 30-40°C. Salinity in the medium also affected X-GRL decolorization. Lactate and citric acid were found to be the suitable electron donors for X-GRL decolorization. Although the genotoxicity increased slightly, the phytotoxicity of X-GRL in the decolorization process was significantly reduced by S. oneidensis MR-1.

Effects of sludge thermal-alkaline pretreatment on cationic red X-GRL adsorption onto pyrolysis biochar of sewage sludge.

Thermal-alkaline pretreatment has traditionally been used to enhance anaerobic sludge digestion. In this study, after removing the supernatant, which could be used in anaerobic digestion, the Residual Solids of Thermal-Alkaline pretreated sewage Sludge (RSTAS) were used to prepare biochar via pyrolysis, which could then adsorb Cationic Red X-GRL(X-GRL). The experimental results showed that the RSTAS-biochar had a higher BET surface area and total pore volume than the biochar prepared from raw sludge (RS) (43.5% and 33.3%, respectively). The pretreatment enhanced the X-GRL adsorption capacity of the biochar by 1.5-49.2% at dosages between 12.5-100.0g/g, and the highest adsorption capacity increased from 39.1mg/g to 47.6mg/g. The biochar from RSTAS had a wider application pH range for X-GRL adsorption. The kinetics and isotherms for X-GRL adsorption onto the two biochars were well fitted to the pseudo-second-order and Langmuir isotherm models, respectively, which suggested that thermal-alkaline pretreatment had little effect on the adsorption mechanisms of X-GRL onto biochar.

Combined intra- and extracellular reduction involved in the anaerobic biodecolorization of cationic azo dye by Shewanella oneidensis MR-1.

Microbial reduction decolorization is a promising strategy for cationic azo dye pollution remediation, but the reduction mechanism is unclear yet. In this work, the anaerobic reduction decolorization mechanism of cationic red X-GRL (X-GRL) by Shewanella oneidensis MR-1 (MR-1) was investigated from both intracellular and extracellular aspects. The exogenous additional riboflavin treatment test was used to analyze the extracellular reduction mechanism of X-GRL, and the actual role of riboflavin during the reduction of X-GRL was identified by three-dimensional fluorescence analysis for the first time. The proteinase K and the electron competitor treatment tests were used to analyze the intracellular reduction mechanism of X-GRL. Moreover, the effect of external environment on the reduction mechanism of X-GRL was elucidated by the decolorization performance of MR-1 wild type and its mutants, ΔomcA/mtrC, ΔmtrA, ΔmtrB and ΔcymA, under different external pH conditions. The results indicated that X-GRL could be decolorized by MR-1 in both extracellular and intracellular spaces. The extracellular decolorization of X-GRL could be caused by Mtr respiratory pathway or the indirect reduction of riboflavin, while the intracellular decolorization might occur due to the intracellular reduction depending on CymA pathway and a NADH-dependent reduction catalyzed by intracellular azoreductases. Furthermore, the proportion of extracellular decolorization decreased, whereas that of intracellular decolorization increased as the environmental pH rose.

A novel central nervous system-penetrating protease inhibitor overcomes human immunodeficiency virus 1 resistance with unprecedented aM to pM potency.

Antiretroviral therapy for HIV-1 infection/AIDS has significantly extended the life expectancy of HIV-1-infected individuals and reduced HIV-1 transmission at very high rates. However, certain individuals who initially achieve viral suppression to undetectable levels may eventually suffer treatment failure mainly due to adverse effects and the emergence of drug-resistant HIV-1 variants. Here, we report GRL-142, a novel HIV-1 protease inhibitor containing an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran, which has extremely potent activity against all HIV-1 strains examined with IC50 values of attomolar-to-picomolar concentrations, virtually no effects on cellular growth, extremely high genetic barrier against the emergence of drug-resistant variants, and favorable intracellular and central nervous system penetration. GRL-142 forms optimum polar, van der Waals, and halogen bond interactions with HIV-1 protease and strongly blocks protease dimerization, demonstrating that combined multiple optimizing elements significantly enhance molecular and atomic interactions with a target protein and generate unprecedentedly potent and practically favorable agents.

Ultrasound assisted extraction of Maxilon Red GRL dye from water samples using cobalt ferrite nanoparticles loaded on activated carbon as sorbent: Optimization and modeling.

In this research, a selective, simple and rapid ultrasound assisted dispersive solid-phase micro-microextraction (UA-DSPME) was developed using cobalt ferrite nanoparticles loaded on activated carbon (CoFe2O4-NPs-AC) as an efficient sorbent for the preconcentration and determination of Maxilon Red GRL (MR-GRL) dye. The properties of sorbent are characterized by X-ray diffraction (XRD), Transmission Electron Microscopy (TEM), Vibrating sample magnetometers (VSM), Fourier transform infrared spectroscopy (FTIR), Particle size distribution (PSD) and Scanning Electron Microscope (SEM) techniques. The factors affecting on the determination of MR-GRL dye were investigated and optimized by central composite design (CCD) and artificial neural networks based on genetic algorithm (ANN-GA). CCD and ANN-GA were used for optimization. Using ANN-GA, optimum conditions were set at 6.70, 1.2mg, 5.5min and 174µL for pH, sorbent amount, sonication time and volume of eluent, respectively. Under the optimized conditions obtained from ANN-GA, the method exhibited a linear dynamic range of 30-3000ngmL-1 with a detection limit of 5.70ngmL-1. The preconcentration factor and enrichment factor were 57.47 and 93.54, respectively with relative standard deviations (RSDs) less than 4.0% (N=6). The interference effect of some ions and dyes was also investigated and the results show a good selectivity for this method. Finally, the method was successfully applied to the preconcentration and determination of Maxilon Red GRL in water and wastewater samples.