Site Of Target Protein Research Articles

A Method for the Examination of SDHI Fungicide Resistance Mechanisms in Phytopathogenic Fungi Using a Heterologous Expression System in Sclerotinia sclerotiorum.

Succinate dehydrogenase inhibitors (SDHIs) are a class of broad-spectrum fungicides used for management of diseases caused by phytopathogenic fungi. In many cases, reduced sensitivity to SDHI fungicides has been correlated with point mutations in the SdhB and SdhC target genes that encode components of the succinate dehydrogenase complex. However, the genetic basis of SDHI fungicide resistance mechanisms has been functionally characterized in very few fungi. Sclerotinia sclerotiorum is a fast-growing and SDHI fungicide-sensitive phytopathogenic fungus that can be conveniently transformed. Given the high amino acid sequence similarity and putative structural similarity of SDHI protein target sites between S. sclerotiorum and other common phytopathogenic ascomycete fungi, we developed an in vitro heterologous expression system that used S. sclerotiorum as a reporter strain. With this system, we were able to demonstrate the function of mutant SdhB or SdhC alleles from several ascomycete fungi in conferring resistance to multiple SDHI fungicides. In total, we successfully validated the function of Sdh alleles that had been previously identified in field isolates of Botrytis cinerea, Blumeriella jaapii, and Clarireedia jacksonii (formerly S. homoeocarpa) in conferring resistance to boscalid, fluopyram, or fluxapyroxad and used site-directed mutagenesis to construct and phenotype a mutant allele that is not yet known to exist in Monilinia fructicola populations. We also examined the functions of these alleles in conferring cross-resistance to more recently introduced SDHIs including inpyrfluxam, pydiflumetofen, and pyraziflumid. The approach developed in this study can be widely applied to interrogate SDHI fungicide resistance mechanisms in other phytopathogenic ascomycetes.

Risk Assessment of Hepatitis C Virus Resistance to Ns3/4a Protease Inhibitors (Pis)

Hepatitis C virus (HCV) persistently infects more than 175-185 million people globally. Drug resistance to anti-HCV DAAs therapeutics particularly NS3/4A has been a major concern for efficacy of HCV treatment due to generation of HCV quasispecies. Risk determination of the resistance of HCV to anti-NS3/4A inhibitors was accomplished using ViPR algorithm that computes and analyzes resistance-associated amino acid substitutions in HCV NS3 protein target site and reveal altered response to NS3/4A PIs. Spot comparison and data visualization was carried out using ncbi.nlm.nih.gov protein sequence graphics and R-package for Ubuntu version 12.04. Resistance to multiple NS3/4A PIs (telaprevir, simeprevir, faldaprevir and asunaprevir) was exhibited by HCV genotype 1 variant D1194A/E/T/G-NS3 (a substitution hot spot) while the risk of resistance to telaprevir (V1062 L NS3 variant) and simeprevir (S1148R variant) was demonstrated by HCV genotype 2. Genotype 3 & 4 revealed resistance against only telaprevir. Another almost pan-genotype (genotype 2,3,4,5 and unclassified) substitution hot spot was identified as anti-telaprevir variant V1062 L. Single substitution conferring multiple anti-NS3/4A PIs resistance was observed in D1194A NS3 variant (anti-simeprevir, anti-faldaprevir, anti-asunaprevir in genotype 1), Q1106K NS3 variant (anti-simeprevir, anti-faldaprevir in genotype 5 and 6) and T1080S NS3 variant (anti-faldaprevir, anti-telaprevir in genotype 5 and unclassified NS3 sequence). In addition to that, variant S1148G exhibited increased sensitivity to simeprevir in case of genotype 1. Because of a higher degree of HCV genomic variability, NS3 variants with decreased susceptibility to NS3/4A PIs exists, and therefore the resistance profile along with potency, adverse effect of PIs is noteworthy and should be well-considered while developing different protease inhibitors for effective treatment of HCV patients.

Epitopes of Protein Binders Are Related to the Structural Flexibility of a Target Protein Surface.

Protein binders including antibodies are known not to bind to random sites of target proteins, and their functional effectiveness mainly depends on the binding region, called the epitope. For the development of protein binders with desired functions, it is thus critical to understand which surface region protein binders prefer (or do not prefer) to bind. The current methods for epitope prediction focus on static indicators such as structural geometry or amino acid propensity, whereas protein binding events are in fact a consequence of dynamic interactions. Here, we demonstrate that the preference for a binding site by protein binders is strongly related to the structural flexibility of a target protein surface. Molecular dynamics simulations on unbound forms of antigen structures revealed that the antigen surface in direct contact with antibodies is less flexible than the rest of the surface. This tendency was shown to be similar in other non-antibody protein binders such as affibody, DARPin, monobody, and repebody. We also found that the relatedness of epitopes to the structural flexibility of a target protein surface is dependent on the secondary structure elements of paratopes. Monobody and repebody, whose binding sites are composed of β-strands, distinctively prefer to bind to a relatively more rigid region of a target protein. These observations enabled us to develop a simple epitope prediction method which shows a comparable performance to the commonly used ones.

Determining site occupancy of acetaminophen covalent binding to target proteins in vitro.

Acetaminophen (APAP)-related toxicity is caused by the formation of N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite able to covalently bind to protein thiols. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, using multiple reaction monitoring (MRM), was developed to measure APAP binding on selected target proteins, including glutathione S-transferases (GSTs). In vitro incubations with CYP3A4 were performed to form APAP in the presence of different proteins, including four purified GST isozymes. A custom alkylation agent was used to prepare heavy labeled modified protein containing a structural isomer of APAP on all cysteine residues for isotope dilution. APAP incubations were spiked with heavy labeled protein, digested with either trypsin or pepsin, followed by peptide fractionation by HPLC prior to LC-MRM analysis. Relative site occupancy on the protein-level was used for comparing levels of modification of different sites in target proteins, after validation of protein and peptide-level relative quantitation using human serum albumin as a model system. In total, seven modification sites were quantified, namely Cys115 and 174 in GSTM2, Cys15, 48 and 170 in GSTP1, and Cys50 in human MGST1 and rat MGST1. In addition, APAP site occupancies of three proteins from liver microsomes were also quantified by using heavily labeled microsomes spiked into APAP microsomal incubations. A novel approach employing an isotope-labeled alkylation reagent was used to determine site occupancies on multiple protein thiols.

PSC-db: A Structured and Searchable 3D-Database for Plant Secondary Compounds.

Plants synthesize a large number of natural products, many of which are bioactive and have practical values as well as commercial potential. To explore this vast structural diversity, we present PSC-db, a unique plant metabolite database aimed to categorize the diverse phytochemical space by providing 3D-structural information along with physicochemical and pharmaceutical properties of the most relevant natural products. PSC-db may be utilized, for example, in qualitative estimation of biological activities (Quantitative Structure-Activity Relationship, QSAR) or massive docking campaigns to identify new bioactive compounds, as well as potential binding sites in target proteins. PSC-db has been implemented using the open-source PostgreSQL database platform where all compounds with their complementary and calculated information (classification, redundant names, unique IDs, physicochemical properties, etc.) were hierarchically organized. The source organism for each compound, as well as its biological activities against protein targets, cell lines and different organism were also included. PSC-db is freely available for public use and is hosted at the Universidad de Talca.

Anticancer activities of phenolic compounds from Moringa oleifera leaves: in vitro and in silico mechanistic study

BackgroundMoringa oleifera is a common vegetable in many countries since ancient times, possesses numerous phenolic compounds having a wide array of biological activities. It possesses anticancer activity that can be used to develop new drugs for treatment of various types of cancers. The current study was conducted to evaluate the composition of phenolic compounds and in vitro and in silico anticancer activities of M. oleifera leaves extracts. The leaves of M. oleifera were subjected to extraction for solvent fraction using n-hexane, chloroform, ethyl acetate, butanol, and aqueous solvents. The solvent fractions were tested for anticancer activity in vitro against Hela cancer cell line and screened for phenolic compounds through reversed-phase high-performance liquid chromatography. The molecular docking approach was employed to check binding conformations of phytochemicals against the target protein.ResultThe result revealed that all the solvent fractions possess in vitro anticancer activity against Hela cancer cell line. The n-hexane fraction showed a 50% reduction in Hela cancer cell viability at 416 μg mL−1 as compared to control. The extracts of solvent-fraction contained 10 phenolic compounds viz. quercetin, gallic acid, sinapic acid, vanillic acid, 4-hydroxy-3-methoxy benzoic acid, p-coumaric acid, m-coumaric acid, 4-hydroxy-3-methoxy cinnamic acid, caffeic acid, and syringic acid. Molecular docking studies revealed that the ligands bind within the active site of target protein have good binding energy values.ConclusionThis study shows that M. oleifera leaves may have the potential to inhibit cancer cell growth and improving human health in addition to food ingredient innovations. Based on in vitro and in silico results, the phytochemicals from M. oleifera leaves can be used as leading drugs to treat cancer.Graphical abstract

Luteolin and abyssinone II as potential inhibitors of SARS-CoV-2: an in silico molecular modeling approach in battling the COVID-19 outbreak

BackgroundAt present, the entire world is in a war against COVID-19 pandemic which has gradually led us toward a more compromised “new normal” life. SARS-CoV-2, the pathogenic microorganism liable for the recent COVID-19 outbreak, is extremely contagious in nature resulting in an unusual number of infections and death globally. The lack of clinically proven therapeutic intervention for COVID-19 has dragged the world’s healthcare system into the biggest challenge. Therefore, development of an efficient treatment scheme is now in great demand. Screening of different biologically active plant-based natural compounds could be a useful strategy for combating this pandemic. In the present research, a collection of 43 flavonoids of 7 different classes with previously recorded antiviral activity was evaluated via computational and bioinformatics tools for their impeding capacity against SARS-CoV-2. In silico drug likeness, pharmacophore and Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) profile analysis of the finest ligands were carried out using DataWarrior, DruLiTo and admetSAR programs, respectively. Molecular docking was executed by AutoDock Vina, while molecular dynamics simulation of the target protein–ligand bound complexes was done using nanoscalable molecular dynamics and visual molecular dynamics software package. Finally, the molecular target analysis of the selected ligands within Homo sapiens was conducted with SwissTargetPredcition web server.ResultsOut of the forty-three flavonoids, luteolin and abyssinone II were found to develop successful docked complex within the binding sites of target proteins in terms of lowest binding free energy and inhibition constant. The root mean square deviation and root mean square fluctuation values of the docked complex displayed stable interaction and efficient binding between the ligands and target proteins. Both of the flavonoids were found to be safe for human use and possessed good drug likeness properties and target accuracy.ConclusionsConclusively, the current study proposes that luteolin and abyssinone II might act as potential therapeutic candidates for SARS-CoV-2 infection. In vivo and in vitro experiments, however, should be taken under consideration to determine the efficiency and to demonstrate the mechanism of action.

Genome-wide landscape of RNA-binding protein target site dysregulation reveals a major impact on psychiatric disorder risk.

Despite the strong genetic basis of psychiatric disorders, the underlying molecular mechanisms remain largely unmapped. RNA-binding proteins (RBPs) are responsible for most post-transcriptional regulation, from splicing to translational to localization. RBPs thus act as key gatekeepers of cellular homeostasis, especially in the brain. However, quantifying the pathogenic contribution of noncoding variants impacting RBP target sites is challenging. Here, we leverage a deep learning approach that can accurately predict RBP target site dysregulation effects of mutations, and discover that RBP dysregulation is a principal contributor to psychiatric disorder risk. RBP dysregulation explains a substantial amount of heritability not captured by large-scale molecular QTL studies, and has a stronger impact than common coding region variants. We share genome-wide profiles of RBP dysregulation, which we use to identify DDHD2 as a candidate schizophrenia risk gene. This resource provides a novel analytical framework to connect the full range of RNA regulation to complex disease.

In vitro and in silico evaluation of N-(alkyl/aryl)-2-chloro-4-nitro-5- [(4-nitrophenyl)sulfamoyl]benzamide derivatives for antidiabetic potential using docking and molecular dynamic simulations

A series of N-(alkyl/aryl)-2-chloro-4-nitro-5-[(4-nitrophenyl)sulfamoyl]benzamide derivatives were synthesized and evaluated for its in vitro antidiabetic potential against α-glucosidase and α-amylase enzymes and also for its antimicrobial potential. Compounds N-(2-methyl-4-nitrophenyl)-2-chloro-4-nitro-5-[(4-nitrophenyl)sulfamoyl]benzamide and N-(2-methyl-5-nitrophenyl)-2-chloro-4-nitro-5-[(4-nitrophenyl)sulfamoyl]benzamide were found to be the most potent α-glucosidase and α-amylase inhibitors with IC50 values of 10.13 and 1.52 µM, respectively. The docking results depicted reasonable dock score −10.2 to −8.0 kcal/mol (α-glucosidase), −11.1 to −8.3 kcal/mol (α-amylase) and binding interactions of synthesized molecules with respective targets with enzymes. During molecular dynamic simulations, analysis of RMSD of ligand protein complex suggested stability of the most active compound at binding site of target proteins. Compound N-(2-chloro-4-nitrophenyl)-2-chloro-4-nitro-5-[(4-nitrophenyl)sulfamoyl] benzamide showed antibacterial potential against Gram positive and Gram negative bacteria and compound N-(2-methyl-5-nitrophenyl)-2-chloro-4-nitro-5-[(4-nitrophenyl)sulfamoyl] benzamide showed excellent antifungal potential against Candida albicans and Aspergillus niger. The computational studies were also executed to predict the drug-likeness and ADMET properties of the title compounds. The N-(alkyl/aryl)-2-chloro-4-nitro-5-[(4-nitrophenyl)sulfamoyl]benzamide derivatives showed significant antidiabetic and antimicrobial potential which is equally supported by the molecular dynamic and docking studies. This study will prove useful in revealing the molecular structure and receptor target site details which can be further utilized for the development of newer active antidiabetic and antimicrobial agents.

Formulation and evaluation of rutin-loaded solid lipid nanoparticles for the treatment of brain tumor.

The primary requirement for curing cancer is the delivery of essential drug load at the cancer microenvironment with therapeutic efficacy. Considering this, the present study aims to formulate "Rutin"-encapsulated solid lipid nanoparticles (SLNs) for effective brain delivery across the blood-brain barrier (BBB). Rutin-loaded SLNs were fabricated by oil-in-water microemulsion technique and were characterized for their physicochemical properties. The in vivo biodistribution study of rutin-loaded SLNs was studied using Rattus norvegicus rats. Subsequently, in silico molecular docking and dynamic calculations were performed to examine the binding affinity as well as stability of rutin at the active site of target protein "epidermal growth factor receptor (EGFR)." Formulated rutin-loaded SLNs were predominantly spherical in shape with an average particle diameter of 100nm. Additionally, the biocompatibility and stability have been proved in vitro. The presence and biodistribution of rutin in vivo after 54h of injection were observed as 15.23 ± 0.32% in the brain, 8.68 ± 0.63% in the heart, 4.78 ± 0.28% in the kidney, 5.04 ± 0.37% in the liver, 0.92 ± 0.04% in the lung, and 11.52 ± 0.65% in the spleen, respectively. Molecular docking results revealed the higher binding energy of - 150.973kJ/mol of rutin with EGFR. Molecular dynamic simulation studies demonstrated that rutin with EGFR receptor complex was highly stable at 30ns. The observed results exemplified that the formulated rutin-loaded SLNs were stable in circulation for a period up to 5days. Thus, rutin-encapsulated SLN formulations can be used as a promising vector to target tumors across BBB. Graphical abstract.

A perspective on potential target proteins of COVID-19: Comparison with SARS-CoV for designing new small molecules

SARS-CoV-2 (COVID-19) epidemic has created an unprecedented medical and economic crisis all over the world. SARS-CoV-2 is found to have more contagious character as compared to MERS-CoV and is spreading in a very fast manner all around the globe. It has affected over 31 million people all over the world till date. This virus shares around 80% of genome similarity with SARS-CoV. In this perspective, we have explored three major targets namely; SARS-CoV-2 spike (S) protein, RNA dependent RNA polymerase, and 3CL or Mpro Protease for the inhibition of SARS-CoV-2. These targets have attracted attention of the medicinal chemists working on computer-aided drug design in developing new small molecules that might inhibit these targets for combating COVID-19 disease. Moreover, we have compared the similarity of these target proteins with earlier reported coronavirus (SARS-CoV). We have observed that both the coronaviruses share around 80% similarity in their amino acid sequence. The key amino acid interactions which can play a crucial role in designing new small molecule inhibitors against COVID-19 have been reported in this perspective. Authors believe that this study will help the medicinal chemists to understand the key amino acids essential for interactions at the active site of target proteins in SARS-CoV-2, based on their similarity with earlier reported viruses. In this review, we have also described the lead molecules under various clinical trials for their efficacy against COVID-19.

Synthesis, molecular docking and molecular dynamic simulation studies of 2-chloro-5-[(4-chlorophenyl)sulfamoyl]-N-(alkyl/aryl)-4-nitrobenzamide derivatives as antidiabetic agents

A series of 2-chloro-5-[(4-chlorophenyl)sulfamoyl]-N-(alkyl/aryl)-4-nitrobenzamide derivatives (5a–5v) has been synthesized and confirmed by physicochemical(Rf, melting point) and spectral means (IR, 1HNMR, 13CNMR). The results of in vitro antidiabetic study against α-glucosidase indicated that compound 5o bearing 2-CH3-5-NO2 substituent on phenyl ring was found to be the most active compound against both enzymes. The electron donating (CH3) group and electron withdrawing (NO2) group on a phenyl ring highly favoured the inhibitory activity against these enzymes. The docking simulations study revealed that these synthesized compounds displayed hydrogen bonding, electrostatic and hydrophobic interactions with active site residues. The structure activity relationship studies of these compounds were also corroborated with the help of molecular modeling studies. Molecular dynamic simulations have been done for top most active compound for validating its α-glucosidase and α-amylase inhibitory potential, RMSD analysis of ligand protein complex suggested the stability of top most active compound 5o in binding site of target proteins. In silico ADMET results showed that synthesized compounds were found to have negligible toxicity, good solubility and absorption profile as the synthesized compounds fulfilled Lipinski’s rule of 5 and Veber’s rule.

Investigation of bond topological and electrostatic properties of plumbagin molecule: An experimental and theoretical charge density study

Plumbagin is a naturally occurring derivative with several medicinal properties including antioxidants, antifungal, antimalarial, leprosy and antitumor properties; their structural and electrostatic properties are yet to be determined. The crystal structure of plumbagin has been solved; it shows that the compound crystallizes in P21/c space group with two molecules in the asymmetric unit. The electron density distribution of both molecules have determined from multipole model refinement. Among all the C–O bonds of both molecules (I & II), the OH group connected C–O bond and the methyl group connected C–C bond exhibits less electron density and the negative Laplacian of electron density as well. Further, the electrostatic potential (ESP) surface of two plumbagin molecules show some difference in their electronegative regions. The carbonyl O-atoms exhibit high electronegative ESP regions which are the key reactive locations of plumbagin when bind with the active site of target protein and in the ESP map, an aromatic π-cloud also observed in one of the molecule. The electron density distribution of O–H⋅⋅⋅O intermolecular interactions between the molecules I and II, reveals that these interactions are found very stronger than the other interactions in the crystal.

Design, synthesis, chemical characterization, biological evaluation, and docking study of new 1,3,4-oxadiazole homonucleoside analogs

Herein, we report the synthetic strategies and characterization of some novel 1,3,4-oxadiazole homonucleoside analogs that are relevant to potential antitumor and cytotoxic activities. The structure of all compounds is confirmed using various spectroscopic methods such as 1H-NMR, 13C-NMR, HRMS, and FTIR. These compounds were evaluated against three human cancer cell lines (MCF-7, SKBR3, and HL60 Cell Line). Preliminary investigations showed that the cytotoxic activity was markedly dependent on the nucleobase. Introduction of 5-Iodouracil 4g and theobromine 6b proved to be extremely beneficial even they were more potent than the reference drug (DOX). Also, the synthesized compounds were tested for their antiviral activities against the human varicella-zoster virus (VZV). The product 4h was (6-azauracil derivative) more potent to the reference (acyclovir) against the deficient TK − VZV strain by about 2-fold. Finally, molecular docking suggested that the anticancer activities of compounds 6b and 4g mediated by inhibiting dual proteins EGFR/HER2 with low micromolar inhibition constant Ki range. The 1,3,4-oxadiazole homonucleosides showed a strong affinity to binding sites of target proteins by forming H-bond, carbon–hydrogen bond, Pi-anion, Pi-sulfur, Pi-sigma, alkyl, and Pi-alkyl interactions.

RMAPS2: an update of the RNA map analysis and plotting server for alternative splicing regulation.

The rMAPS2 (RNA Map Analysis and Plotting Server 2) web server, freely available at http://rmaps.cecsresearch.org/, has provided the high-throughput sequencing data research community with curated tools for the identification of RNA binding protein sites. rMAPS2 analyzes differential alternative splicing or CLIP peak data obtained from high-throughput sequencing data analysis tools like MISO, rMATS, Piranha, PIPE-CLIP and PARalyzer, and then, graphically displays enriched RNA-binding protein target sites. The initial release of rMAPS focused only on the most common alternative splicing event, skipped exon or exon skipping. However, there was a high demand for the analysis of other major types of alternative splicing events, especially for retained intron events since this is the most common type of alternative splicing in plants, such as Arabidopsis thaliana. Here, we expanded the implementation of rMAPS2 to facilitate analyses for all five major types of alternative splicing events: skipped exon, mutually exclusive exons, alternative 5′ splice site, alternative 3′ splice site and retained intron. In addition, by employing multi-threading, rMAPS2 has vastly improved the user experience with significant reductions in running time, ∼3.5 min for the analysis of all five major alternative splicing types at once.

Indole-derived chalcones as anti-dermatophyte agents: In vitro evaluation and in silico study

A series of indole-derived methoxylated chalcones were described as anti-dermatophyte agents. The in vitro antifungal susceptibility testing against different dermatophytes revealed that most of compounds had potent activity against the dermatophyte strains. In particular, the 4-ethoxy derivative 4d with MIC values of 0.25−2 μg/ml was the most potent compound against Trichophyton interdigitale, Trichophyton veruccosum and Microsporum fulvum. Moreover, the 4-butoxy analog 4i displaying MIC values in the range of 1−16 μg/ml had the highest inhibitory activity against Trichophyton mentagrophytes, Microsporum canis, and Arthroderma benhamiae. To predict whether the synthesized compounds interact with tubulin binding site of dermatophytes, the 3D-structure of target protein was modeled by homology modeling and then used for molecular docking and molecular dynamics (MD) simulation studies. Docking simulation revealed that the promising compound 4d can properly bind with tubulin. The molecular dynamics analysis showed that interactions of compound 4d with the active site of target protein have binding stability throughout MD simulation. The results of this study could utilize in the design of more effective antifungal drugs with tubulin inhibition mechanism against keratinophilic fungi.

ORNAment: a database of putative RNA binding protein target sites in the transcriptomes of model species.

Protein–RNA interactions are essential for controlling most aspects of RNA metabolism, including synthesis, processing, trafficking, stability and degradation. In vitro selection methods, such as RNAcompete and RNA Bind-n-Seq, have defined the consensus target motifs of hundreds of RNA-binding proteins (RBPs). However, readily available information about the distribution features of these motifs across full transcriptomes was hitherto lacking. Here, we introduce oRNAment (o RNA motifs enrichment in transcriptomes), a database that catalogues the putative motif instances of 223 RBPs, encompassing 453 motifs, in a transcriptome-wide fashion. The database covers 525 718 complete coding and non-coding RNA species across the transcriptomes of human and four prominent model organisms: Caenorhabditis elegans, Danio rerio, Drosophila melanogaster and Mus musculus. The unique features of oRNAment include: (i) hosting of the most comprehensive mapping of RBP motif instances to date, with 421 133 612 putative binding sites described across five species; (ii) options for the user to filter the data according to a specific threshold; (iii) a user-friendly interface and efficient back-end allowing the rapid querying of the data through multiple angles (i.e. transcript, RBP, or sequence attributes) and (iv) generation of several interactive data visualization charts describing the results of user queries. oRNAment is freely available at http://rnabiology.ircm.qc.ca/oRNAment/.

Synthesis, characterization and Insilico molecular docking studies of novel chromene derivatives as Rab23 inhibitors

The Rab23 protein overexpression has a well-validated role in variety of human cancers. Therefore, the present research aimed to identify new Rab23 protein inhibitor candidates using computational based drug design methodology. A novel series of chromeno[2,3-b] pyridine derivatives has been synthesized by the cyclocondensation of 2-amino-3-cyano-4H-chromenes with cyclohexanone and cyclopentanone in ethanolicpiperidine solution. The 2-amino-4H-chromenes were obtained using one pot multicomponent condensation of resorcinol, malononitrile and aromatic aldehydes in the presence in ethanolicpiperidine solution. All newly synthetic compounds were characterized by spectral analysis IR, NMR and MS and elemental analysis techniques. The best binding modes of chromene derivatives were evaluated via molecular docking studies and binding energy calculations, using PyRx tool. The study has shown that the pyran and pyridine moieties interact favorably with the binding site of target protein, providing the mechanism of action against human sapiens Rab23 protein.

5‐Membered Mesoionic Insecticides: Synthesis and Evaluation of 1,3,4‐Thiadiazol‐4‐ium‐2‐olates with High Affinity for the Insect Nicotinic Acetylcholine Receptor

The vast majority of drugs and agrochemicals contain heterocyclic ring systems, which impact ease of synthesis, speed of compound derivatization, and protein target site binding, as well as uptake, transport and many other properties. Mesoionics are unique compact heterocyclic structures with an aromatic and highly polarized character. The design and synthesis of 5‐membered mesoionic thiadiazol‐4‐ium‐2‐olates with high insecticidal activity and potent in vitro affinity on insect nicotinic acetylcholine receptors (nAChRs) is described. Modelling experiments using a known acetylcholine‐binding protein as surrogate for the insect nAChR showed interactions with the thiadiazoliumolate that were consistent with that expected for related insecticides. Quantum chemical, photostability, greenhouse residual, as well as plant systemicity studies were conducted to understand the in vitro and in vivo behavior of this novel class of insecticides.

Engineering proteins for allosteric control by light or ligands.

Control of protein activity in living cells can reveal the role of spatiotemporal dynamics in signaling circuits. Protein analogs with engineered allosteric responses can be particularly effective in the interrogation of protein signaling, as they can replace endogenous proteins with minimal perturbation of native interactions. However, it has been a challenge to identify allosteric sites in target proteins where insertion of responsive domains produces an allosteric response comparable to the activity of native proteins. Here, we describe a detailed protocol to generate genetically encoded analogs of proteins that can be allosterically controlled by either rapamycin or blue light, as well as experimental procedures to produce and test these analogs in vitro and in mammalian cell lines. We describe computational methods, based on crystal structures or homology models, to identify effective sites for insertion of either an engineered rapamycin-responsive (uniRapR) domain or the light-responsive light-oxygen-voltage 2 (LOV2) domain. The inserted domains allosterically regulate the active site, responding to rapamycin with irreversible activation, or to light with reversible inactivation at higher spatial and temporal resolution. These strategies have been successfully applied to catalytic domains of protein kinases, Rho family GTPases, and guanine exchange factors (GEFs), as well as the binding domain of a GEF Vav2. Computational tasks can be completed within a few hours, followed by 1-2 weeks of experimental validation. We provide protocols for computational design, cloning, and experimental testing of the engineered proteins, using Src tyrosine kinase, GEF Vav2, and Rho GTPase Rac1 as examples.