Amino Acid Sites Research Articles

Pyrimidine-morpholine hybrids as potent druggable therapeutics for Alzheimer’s disease: Synthesis, biochemical and in silico analyses

The identification of effective and druggable cholinesterase inhibitors to treat progressive neurodegenerative Alzheimer’s disorder remains a continuous drug discovery hunt. In this perspective, the present study investigates the design and discovery of pyrimidine-morpholine hybrids (5a-l) as potent cholinesterase inhibitors. Palladium-catalyzed Suzuki-Miyaura cross-coupling reaction was employed to introduce the structural diversity on the pyrimidine heterocyclic core. A range of commercially available boronic acids was successfully coupled showing a high functional group tolerance. In vitro cholinesterase inhibitory potential using Ellman’s method revealed significantly strong potency. Compound 5h bearing a meta-tolyl substituent at 2-position of pyrimidine ring emerged as a lead candidate against AChE with an inhibitory potency of 0.43 ± 0.42 µM, ∼38-fold stronger value than neostigmine (IC50 = 16.3 ± 1.12 µM). Compound 5h also showed the lead inhibition against BuChE with an IC50 value of 2.5 ± 0.04 µM. The kinetics analysis of 5h revealed the non-competitive mode of inhibition against AChE whereas computational modelling results of potent leads depicted diverse contacts with the binding site amino acid residues. Molecular dynamics simulations revealed the stability of biomolecular system, while, ADME analysis demonstrated druglikeness behaviour of potent compounds. Overall, the investigated pyrimidine-morpholine scaffold presented a remarkable potential to be developed as efficacious anti-Alzheimer’s drugs.

Molecular modeling, synthesis, and in vitro acetylcholinesterase and butyrylcholinesterase inhibitory activities of novel benzimidazole-bearing thiadiazole derivatives

A new series of benzimidazole-based thiadiazole hybrids analogues (6a–p) as effective Alzheimer's inhibitors were synthesized and then evaluated for their inhibition profile against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes as compared to Donepezil as standard drug. All the synthesized benzimidazole-based thiadiazole analogues showed varied range of inhibitory potentials against targeted AChE & BuChE enzymes with IC50 values ranging from 1.32 ± 0.10 µM to 19.26 ± 0.60 µM (for AChE) and 1.94 ± 0.10 µM to 21.33 ± 0.70 µM (for BuChE) when compared to standard Donepezil (IC50 = 2.16 ± 0.050 µM for AChE) & 4.50 ± 0.10 µM for BuChE). As structure-activity relationship (SAR) studies revealed that the analogues 4d (bearing ortho‑hydroxy and para-NO2 on N-aryl ring along with ortho-NO2 substitution on another aryl ring) and 6k (that holds ortho‑hydroxy ¶-NO2 substitutions on N-aryl ring and 3,4-diCl moieties on another aryl ring) were emerged as the most potent analogues of AChE & BuChE enzymes having IC50 values of 1.32 ± 0.10 µM & 1.84 ± 0.20 µM (against AChE) and 1.94 ± 0.10 µM & 2.23 ± 0.20 µM (against BuChE) respectively. Furthermore, the active analogues were subjected to molecular docking studies in order to explore the binding interactions possess by potent analogues with the active sites of amino acids of targeted AChE & BuChE enzymes and result obtained shows that these active analogues furnished that several key interactions with targeted enzymes active sites. Additionally, all the synthesized analogues were elucidated structurally using variety of spectroscopic (1H NMR & 13C NMR) and spectrometric (HREI-MS) analysis.

Purifying selection leads to low protein diversity of the mitochondrial cyt b gene in avian malaria parasites

BackgroundMitochondrial respiration plays a central role in the survival of many eukaryotes, including apicomplexan parasites. A 479-bp fragment from the mitochondrial cytochrome b gene is widely used as a barcode to identify genetic lineages of avian malaria parasites Plasmodium and related haemosporidians. Here we looked for evidence of selection in the avian Plasmodium cyt b gene, using tests of selection and protein structure modeling. We also tested for the association between cyt b polymorphism and the host specificity of these parasites.ResultsBased on 1,089 lineages retrieved from the Malavi database, we found that the frequency of the most conserved amino acids in most sites was more than 90%, indicating that the protein diversity of the avian Plasmodium cyt b barcode was low. The exceptions were four amino acid sites that were highly polymorphic, though the substitutions had only slight functional impacts on the encoded proteins. The selection analyses revealed that avian Plasmodium cyt b was under strong purifying selection, and no positively selected sites were detected. Besides, lineages with a wide host range tend to share cyt b protein haplotypes.ConclusionsOur research indicates that purifying selection is the dominant force in the evolution of the avian Plasmodium cyt b lineages and leads to its low diversity at the protein level. Host specificity may also play a role in shaping the low mitochondrial diversity in the evolution of avian malaria parasites. Our results highlight the importance of considering selection pressure on the cyt b barcode region and lay a foundation for further understanding the evolutionary pattern of mitochondrial genes in avian malaria.

Monopolar spindle 1 contributes to tamoxifen resistance in breast cancer through phosphorylation of estrogen receptor α.

The overexpression of mitotic kinase monopolar spindle 1 (Mps1) has been identified in many tumor types, and targeting Mps1 for tumor therapy has shown great promise in multiple preclinical cancer models. However, the role played by Mps1 in tamoxifen (TAM) resistance in breast cancer has never been reported. The sensitivity of breast cancer cells to tamoxifen was analysed in colony formation assays and wound healing assays. Enhanced transactivational activity of estrogen receptor α (ERα) led by Mps1 overexpression was determined by luciferase assays. The interaction between Mps1 and ERα was verified by co-immunoprecipitation and proximity ligation assay. Phosphorylation of ERα by Mps1 was detected by in vitro kinase assay and such phosphorylation process in vivo was proven by co-immunoprecipitation. The potential phosphorylation site(s) of ERα were analyzed by mass spectrometry. Mps1 determines the sensitivity of breast cancer cells to tamoxifen treatment. Mps1 overexpression rendered breast cancer cells more resistant to tamoxifen, while an Mps1 inhibitor or siMps1 oligos enabled cancer cells to overcome tamoxifen resistance. Mechanistically, Mps1 interacted with estrogen receptor α and stimulated its transactivational activity in a kinase activity-dependent manner. Mps1 was critical for ERα phosphorylation at Thr224 amino acid site. Importantly, Mps1 failed to enhance the transactivational activity of the ERα-T224A mutant. Mps1 contributes to tamoxifen resistance in breast cancer and is a potential therapeutic that can overcome tamoxifen resistance in breast cancer.

Synthesis and Screening of Chemical Agents Targeting Viral Protein Genome-Linked Protein of Telosma Mosaic Virus.

The viral protein genome-linked protein (VPg) of telosma mosaic virus (TeMV) plays an important role in viral reproduction. In this study, the expression conditions of TeMV VPg were explored. A series of novel benzenesulfonamide derivatives were synthesized. The binding sites of the target compounds and TeMV VPg were studied by molecular docking, and the interaction was verified by microscale thermophoresis. The study revealed that the optimal expression conditions for TeMV VPg were in Escherichia coli Rosetta with IPTG concentration of 0.8 mM and induction temperature of 25 °C. Compounds A4, A6, A9, A16, and A17 exhibited excellent binding affinity to TeMV VPg, with Kd values of 0.23, 0.034, 0.19, 0.086, and 0.22 μM, respectively. LYS 121 is the key amino acid site. Compounds A9 inhibited the expression of TeMV VPg in Nicotiana benthamiana. The results suggested that TeMV VPg is a potential antiviral target to screen anti-TeMV compounds.

In-vitro and computational analysis of Urolithin-A for anti-inflammatory activity on Cyclooxygenase 2 (COX-2)

Cyclooxygenase 2 (COX-2) participates in the inflammation process by converting arachidonic acid into prostaglandin G2 which increases inflammation, pain and fever. COX-2 has an active site and a heme pocket and blocking these sites stops the inflammation. Urolithin A is metabolite of ellagitannin produced from humans and animals gut microbes. In the current study, Urolithin A showed good pharmacokinetic properties. Molecular docking of the complex of Urolithin A and COX-2 revealed the ligand affinity of −7.97 kcal/mol with the ligand binding sites at TYR355, PHE518, ILE517 and GLN192 with the 4-H bonds at a distance of 2.8 Å, 2.3 Å, 2.5 Å and 1.9 Å. The RMSD plot for Urolithin A and COX-2 complex was observed to be constant throughout the duration of dynamics. A total of 3 pair of hydrogen bonds was largely observed on average of 3 simulation positions for dynamics duration of 500 ns. The MMPBSA analysis showed that active site amino acids had a binding energy of –22.0368 kJ/mol indicating that throughout the simulation the protein of target was bounded by Urolithin A. In-silico results were validated by biological assays. Urolithin A strongly revealed to exhibit anti-inflammatory effect on COX-2 with an IC50 value of 44.04 µg/mL. The anti-inflammatory capability was also depicted through reduction of protein denaturation that showed 37.6 ± 0.1 % and 43.2 ± 0.07 % reduction of protein denaturation for BSA and egg albumin respectively at 500 µg/mL. The present study, suggests Urolithin A to be an effective anti-inflammatory compound for therapeutic use.

Mutation of aspartic acid 262 on estrogen receptor abrogates estradiol signaling pathway

Objective ERα (estrogen receptor alpha) exerts nuclear genomic actions and membrane-initiated non-genomic effects. The mutation of aspartic acid into alanine in vitro revealed the critical role of aspartic acid 258 (corresponding to mouse amino acid site 262) of ERα for non-nuclear function. Our previous in vitro study revealed that this mutation blocked estrogen’s non-genomic effects on vascular endothelial H2S release. Here, we studied the in vivo role of the aspartic acid 262 of ERα in the reproductive system and in the vascular tissue. Approach and Results We generated a mouse model harboring a point mutation of the murine counterpart of this aspartic acid into alanine (ERαD262A). Our results showed that the ERαD262A females are fertile with standard hormonal serum levels, but the uterine development and responded with estrogen and follicular development are disrupted. In line with our previous study, we found that the rapid dilation of the aorta was abrogated in ERαD262A mice. In contrast to the previously reported R264-ERα mice, the classical estrogen genomic effector SP1/NOS3/AP1 and the nongenomic effectors p-eNOs, p-AKT, and p-ERK were disturbed in the ERαD262A aorta. Besides, the serum H2S concentration was decreased in ERαD262A mice. Together, ERαD262A mice showed compromised both genomic and non-genomic actions in response to E2. Conclusions These data showed that aspartic acid 262 of ERα are important for both genomic and non-genomic effects of E2. Our data provide a theoretical basis for further selecting an effective non-genomic mouse model and provide a new direction for developing estrogen non-genomic effect inhibitors.

In silico prediction of mutation sites for anthranilate synthase from Serratia marcesens to deregulate tryptophan feedback inhibition

Allosteric feedback inhibition of the committed step in amino acid biosynthetic pathways is a major concern for production of amino acids at industrial scale. Anthranilate synthase (AS) catalyzes the first reaction of tryptophan biosynthetic pathway found in microorganisms and is feedback inhibited by its own product i.e. tryptophan. Here, we identified new mutant sites in AS using computational mutagenesis approach. MD simulations (20 ns) followed by MMPBSA and per residue decomposition energy analysis identified seven amino acid residues with best binding affinity for tryptophan. All 19 mutant structures were generated for each identified amino acid residue followed by simulation to evaluate effect of mutation on protein stability. Later, molecular docking studies were employed to generate mutant-tryptophan complex and structures with binding energies (kcal/mol) much higher than wild-type AS were selected. Finally, two mutants i.e., S37W and S37H were identified on the basis of positive binding scores and loss of tryptophan binding inside pocket. Further, MD simulations run for 200 ns were performed over these mutant-tryptophan complexes followed by RMSD, RMSF, radius of gyration , solvent accessible surface area , intra-protein hydrogen bond numbers, principal component analysis, free energy landscape (FEL) and secondary structure analysis to rationale effect of mutations on stability of protein. Cross correlation analysis of mutant site amino acids (S37W) with key residues of catalytic site (G325, T326, H395 and G482) was done to evaluate the effect of mutations on catalytic site conformation. Current computational mutagenesis approach predicted two mutants S37W and S37H with proposed deregulated feedback inhibition by tryptophan and retained catalytic activity. Communicated by Ramaswamy H. Sarma

Molecular Characteristics of Chicken Infectious Anemia Virus in Central and Eastern China from 2020 to 2022.

To evaluate the recent evolution of CIAV in China, 43 flocks of chickens from the provinces of Henan, Jiangsu, Hubei, and Anhui were screened via polymerase chain reaction during 2020-2022. Of these, 27 flocks tested positive for CIAV nucleic acids, including 12 which were positive for other immunosuppression viruses. Additionally, 27 CIAV strains were isolated, and their whole genomes were sequenced. The AH2001 and JS2002 strains shared the highest identity at 99.56%, and the HB2102 and HB2101 strains shared the lowest identity at 95.34%. Based on the genome sequences of these strains and reference strains, a phylogenetic tree was constructed and divided into eight main branches. Most of the strains were grouped with the East Asian strains, whereas the HB2101 strain belonged to the Brazil and Argentina cluster. A recombination event was detected in multiple strains, in which AH2002 recombined from KJ728827/China/2014 (from Taiwan Province) and HN2203, and AH2202 recombined from KX811526/China/2017 (from Shandong Province) and HN2203. All the obtained strains had a highly pathogenic Gln amino acid site at position 394 of the VP1. Overall, our findings demonstrate the importance of CIAV monitoring and provide data that aid in understanding the evolution of CIAV.

Genetic variation and recombination analysis of the GP5 (GP5a) gene of PRRSV-2 strains in China from 1996 to 2022.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been prevalent in China for more than 25 years and remains one of the most significant pathogens threatening the pig industry. The high rate of mutation and frequent recombination of PRRSV have exacerbated its prevalence, particularly with the emergence of highly pathogenic PRRSV (HP-PRRSV) has significantly increased the pathogenicity of PRRSV, posing a serious threat to the development of Chinese pig farming. To monitor the genetic variation of PRRSV-2 in China, the GP5 sequences of 517 PRRSV-2 strains from 1996 to 2022 were analyzed and phylogenetic trees were constructed. Furthermore, a total of 60 PRRSV strains, originating from various lineages, were carefully chosen for nucleotide and amino acid homologies analysis. The results showed that the nucleotide homologies of the PRRSV GP5 gene ranged from 81.4 to 100.0%, and the amino acid homologies ranged from 78.1 to 100.0%. Similarly, the PRRSV GP5a gene showed 78.0 ~ 100.0% nucleotide homologies and 70.2 ~ 100.0% amino acid homologies. Amino acid sequence comparisons of GP5 and GP5a showed that some mutations, such as substitutions, deletions, and insertions, were found in several amino acid sites in GP5, these mutations were primarily found in the signal peptide region, two highly variable regions (HVRs), and near two T-cell antigenic sites, while the mutation sites of GP5a were mainly concentrated in the transmembrane and intramembrane regions. Phylogenetic analysis showed that the prevalent PRRSV-2 strains in China were divided into lineages 1, 3, 5, and 8. Among these, strains from lineage 8 and lineage 1 are currently the main prevalent strains, lineage 5 and lineage 8 have a closer genetic distance. Recombination analysis revealed that one recombination event occurred in 517 PRRSV-2 strains, this event involved recombination between lineage 8 and lineage 1. In conclusion, this analysis enhances our understanding of the prevalence and genetic variation of PRRSV-2 in China. These findings provide significant insights for the development of effective prevention and control strategies for PRRS and serve as a foundation for future research in this field.

The interaction between apigenin and PKM2 restrains progression of colorectal cancer

Apigenin, a flavonoid that widely existed in vegetables and fruits, possesses anticarcinogenic, low toxicity, and no mutagenic properties, suggesting that apigenin is a potential therapeutic agent for tumors. However, the underlying anti-cancer molecular target of apigenin is still unclear. Therefore, to reveal the direct target and amino acid site of apigenin against colorectal cancer is the focus of this study. In the present study, the results proved that the anti-CRC activity of apigenin was positively correlated with pyruvate kinase M2 (PKM2) expression, characterized by the inhibition of cell proliferation and increase of apoptotic effects induced by apigenin in LS-174T cells of knock down PKM2. Next, pull-down and MALDI-TOF/TOF analysis determined that apigenin might interact directly with PKM2 in HCT-8 cells. Further, the study confirmed that lysine residue 433 (K433) was a key amino acid site for PKM2 binding to apigenin. Apigenin restricted the glycolysis of LS-174T and HCT-8 cells by targeting the K433 site of PKM2, thereby playing an anti-CRC role in vivo and in vitro. Meanwhile, apigenin markedly attenuated tumor growth without any adverse effects. Taken together, these findings reveal that apigenin is worthy of consideration as a promising PKM2 inhibitor for the prevention of CRC.

Petunia dihydroflavonol 4-reductase is only a few amino acids away from producing orange pelargonidin-based anthocyanins.

Anthocyanins are responsible for the color spectrum of both ornamental and natural flowers. However, not all plant species produce all colors. For example, roses are not blue because they do not naturally possess a hydroxylase that opens the pathway for delphinidin and its derivatives. It is more intriguing why some plants do not carry orange or scarlet red flowers with anthocyanins based on pelargonidin, because the precursor for these anthocyanins should be available if anthocyanins are made at all. The key to this is the substrate specificity of dihydroflavonol 4-reductase (DFR), an enzyme located at the branch point between flavonols and anthocyanins. The most common example is petunia, which does not bear orange flowers unless the enzyme is complemented by biotechnology. We changed a few amino acids in the active site of the enzyme and showed that the mutated petunia DFR started to favor dihydrokaempferol, the precursor to orange pelargonidin, in vitro. When transferred to petunia, it produced an orange hue and dramatically more pelargonidin-based anthocyanins in the flowers.

Evolution of drug resistance drives destabilization of flap region dynamics in HIV-1 protease

The HIV-1 protease is one of several common key targets of combination drug therapies for human immunodeficiency virus infection and acquired immunodeficiency syndrome. During the progression of the disease, some individual patients acquire drug resistance due to mutational hotspots on the viral proteins targeted by combination drug therapies. It has recently been discovered that drug-resistant mutations accumulate on the “flap region” of the HIV-1 protease, which is a critical dynamic region involved in nonspecific polypeptide binding during invasion and infection of the host cell. In this study, we utilize machine learning-assisted comparative molecular dynamics, conducted at single amino acid site resolution, to investigate the dynamic changes that occur during functional dimerization and drug binding of wild-type and common drug-resistant versions of the main protease. We also use a multiagent machine learning model to identify conserved dynamics of the HIV-1 main protease that are preserved across simian and feline protease orthologs. We find that a key conserved functional site in the flap region, a solvent-exposed isoleucine (Ile50) that controls flap dynamics is functionally targeted by drug resistance mutations, leading to amplified molecular dynamics affecting the functional ability of the flap region to hold the drugs. We conclude that better long-term patient outcomes may be achieved by designing drugs that target protease regions that are less dependent upon single sites with large functional binding effects.

Evolutionary analysis of actinins(ACTN) gene family for positive selective sites by finding the dn/ds (ω) value

Identifying positively selected amino acid sites is an important approach for making a conclusion about the function of proteins; an amino acid site that is undergoing positive selection is likely to play a key role in the function of the protein. Here, we used codon-based substitution models and maximum-likelihood (ML) methods to identify positively selected sites that are likely to influence the cohesiveness and mechanics of the cytoskeleton by cross-linking actinins filaments and other cytoskeleton components to create a scaffold that imparts stability and forms a bridge between the cytoskeleton and signaling pathways. We use MEGA 7 and PAML for polygenetic Analysis and positive selective Sites. Alpha-actinins from skeletal muscle as a protein factor promoting the super precipitation of actomyosin and inducing the gelation of F-actin solutions. The protein is located in Z-bands of sarcomeres in skeletal and cardiac muscles as well as near the fascia adherents of intercalated discs in cardiac muscle. In smooth muscle, it exists in cytoplasmic dense bodies and membrane-associated dense plaques. We used PAMLX to find out specific sites in these DNA sequences. These results show the location and identification of positively selected sites in the ACTININS gene family. ACTN1, ACTN2, ACTN3 and ACTN4 had (248G,250L), (234I,236N ,238P, 239K, 252F, 260E, 261Q, 314Q, 590N, 607L, 869S), (NO) and (248G, 250L) positive selective sites respectively. The aims of our work are the prediction of these sites to find the specific location of these sites in 3D structure and to replace it with suitable amino acids for the cure of obesity, muscles, cancer, and other chronic diseases

Synthesis, Biological Activity, and the Effect of Ionization Radiation on the Spectral, XRD, and TGA Analysis of Cu(I), Cu(II), Zn(II), and Cd(II) Complexes

New complexes of molecular formulae of [Cu(H2L1)I] (B1), [Cu(H2L)2(ClO4)2](B2), [Zn (H2L)2(H2O)]SO4(B3) and [Cd(H2L)Cl]Cl(B4) of ligand N-phenyl-2-(p-tolylglycyl)hydrazine -1-carbothioamide (B) have been prepared and characterized by 1H-NMR, Mass spectra, FT-IR, elemental analyses, molar conductance, UV–visible spectra, magnetic susceptibility measurements, thermogravimetric analysis (TGA/DTG) and X-ray powder diffraction before and after irradiation. Elemental analyses indicate that the metal complexes have 1:1 (metal/ ligand) (B1 and B4) or 1:2 stoichiometry for complexes (B2 and B3). The results confirmed that gamma ray enhanced the stability of irradiated compounds as compared to non-irradiated compounds. XRD patterns proved that increasing the crystallinity of the samples and the particles is in nanosize range before and after gamma irradiation. These studies revealed that Cu(II) and Zn(II) complexes adopted octahedral geometry, and Cu(I) and Cd(II) complexes were found to be tetrahedral. The theoretical conformational structure analyses were performed using density functional theory for ligand and complexes at B3LYP functional with 6–31G basis set for ligand and LANL2DZ basis set for complexes. The ligand (B, A) and complexes (B1–B4) and (A1–A4) before and after irradiated have been tested for their inhibitory effect on the growth of bacteria against gram-positive (Streptococcus pyogenes) and gram-negative (Escherichia coli). Molecular docking investigation proved that the Zn(II) complex had interesting interactions with active site amino acids of topoisomerase II DNA gyrase enzymes (code: 2XCT).

SUMOylation of AnxA6 facilitates EGFR-PKCα complex formation to suppress epithelial cancer growth

BackgroundThe Annexin A6 (AnxA6) protein is known to inhibit the epidermal growth factor receptor (EGFR)-extracellular signal regulated kinase (ERK)1/2 signaling upon EGF stimulation. While the biochemical mechanism of AnxA6 inactivating phosphorylation of EGFR and ERK1/2 is not completely explored in cancer cells.MethodsCells were transiently co-transfected with pFlag-AnxA6, pHA-UBC9 and pHis-SUMO1 plasmids to enrich the SUMOylated AnxA6 by immunoprecipitation, and the modification level of AnxA6 by SUMO1 was detected by Western blot against SUMO1 antibody. The SUMOylation level of AnxA6 was compared in response to chemical SUMOylation inhibitor treatment. AnxA6 SUMOylation sites were further identified by LC–MS/MS and amino acid site mutation validation. AnxA6 gene was silenced through AnxA6 targeting shRNA-containing pLKO.1 lentiviral transfection in HeLa cells, while AnxA6 gene was over-expressed within the Lenti-Vector carrying AnxA6 or mutant AnxA6K299R plasmid in A431 cells using lentiviral infections. Moreover, the mutant plasmid pGFP-EGFRT790M/L858R was constructed to test AnxA6 regulation on EGFR mutation-induced signal transduction. Moreover, cell proliferation, migration, and gefitinib chemotherapy sensitivity were evaluated in HeLa and A431 cells under AnxA6 konckdown or AnxA6 overexpression by CCK8, colony form and wound healing assays. And tumorigenicity in vivo was measured in epithelial cancer cells-xenografted nude mouse model.ResultsAnxA6 was obviously modified by SUMO1 conjugation within Lys (K) residues, and the K299 was one key SUMOylation site of AnxA6 in epithelial cancer cells. Compared to the wild type AnxA6, AnxA6 knockdown and its SUMO site mutant AnxA6K299R showed less suppression of dephosphorylation of EGFR-ERK1/2 under EGF stimulation. The SUMOylated AnxA6 was prone to bind EGFR in response to EGF inducement, which facilitated EGFR-PKCα complex formation to decrease the EGF-induced phosphorylation of EGFR-ERK1/2 and cyclin D1 expression. Similarly, AnxA6 SUMOylation inhibited dephosphorylation of the mutant EGFR, thereby impeding EGFR mutation-involved signal transduction. Moreover, AnxA6 knockdown or the K299 mutant AnxA6K299R conferred AnxA6 inability to suppress tumor progression, resulting in drug resistance to gefitinib in epithelial cancer cells. And in epithelial cancer cells-xenografted nude mouse model, both the weight and size of tumors derived from AnxA6 knockdown or AnxA6K299R mutation-expressing cells were much greater than that of AnxA6-expressing cells.ConclusionsBesides EGFR gene mutation, protein SUMOylation modification of EGFR-binding protein AnxA6 also functions pivotal roles in mediating epithelial cancer cell growth and gefitinib drug effect.4V8MbH9D2-V-dxkGbe_YjXVideo

DeCOIL: Optimization of Degenerate Codon Libraries for Machine Learning-Assisted Protein Engineering.

With advances in machine learning (ML)-assisted protein engineering, models based on data, biophysics, and natural evolution are being used to propose informed libraries of protein variants to explore. Synthesizing these libraries for experimental screens is a major bottleneck, as the cost of obtaining large numbers of exact gene sequences is often prohibitive. Degenerate codon (DC) libraries are a cost-effective alternative for generating combinatorial mutagenesis libraries where mutations are targeted to a handful of amino acid sites. However, existing computational methods to optimize DC libraries to include desired protein variants are not well suited to design libraries for ML-assisted protein engineering. To address these drawbacks, we present DEgenerate Codon Optimization for Informed Libraries (DeCOIL), a generalized method that directly optimizes DC libraries to be useful for protein engineering: to sample protein variants that are likely to have both high fitness and high diversity in the sequence search space. Using computational simulations and wet-lab experiments, we demonstrate that DeCOIL is effective across two specific case studies, with the potential to be applied to many other use cases. DeCOIL offers several advantages over existing methods, as it is direct, easy to use, generalizable, and scalable. With accompanying software (https://github.com/jsunn-y/DeCOIL), DeCOIL can be readily implemented to generate desired informed libraries.

Identification of Zika virus NS2B-NS3 protease and NS5 polymerase inhibitors by structure-based virtual screening of FDA-approved drugs

Zika virus (ZIKV) is a mosquito-borne human flavivirus responsible that causing emergency outbreaks in Brazil. ZIKV is suspected of causing Guillain-Barre syndrome in adults and microcephaly. The NS2B-NS3 protease and NS5 RNA-dependent RNA polymerase (RdRp), central to ZIKV multiplication, have been identified as attractive molecular targets for drugs. We performed a structure-based virtual screening of 2,659 FDA-approved small molecule drugs in the DrugBank database using AutoDock Vina in PyRx v0.8. Accordingly, 15 potential drugs were selected as ZIKV inhibitors because of their high values (binding affinity - binding energy) and we analyzed the molecular interactions between the active site amino acids and the compounds. Among these drugs, tamsulosin was found to interact most efficiently with NS2B/NS3 protease, as indicated by the lowest binding energy value (−8.27 kJ/mol), the highest binding affinity (−5.7 Kcal/mol), and formed H-bonds with amino acid residues TYRB130, SERB135, TYRB150. Furthermore, biotin was found to interact most efficiently with NS5 RdRp with a binding energy of −150.624 kJ/mol, a binding affinity of −5.6 Kcal/mol, and formed H-bonds with the amino acid residues ASPA665 and ASPA540. In vitro, in vivo, and clinical studies are needed to demonstrate anti-ZIKV safety and the efficacy of these FDA-approved drug candidates. Communicated by Ramaswamy H. Sarma

Exploration of Remarkably Potential Multitarget-Directed N-Alkylated-2-(substituted phenyl)-1H-benzimidazole Derivatives as Antiproliferative, Antifungal, and Antibacterial Agents.

Improving lipophilicity for drugs to penetrate the lipid membrane and decreasing bacterial and fungal coinfections for patients with cancer pose challenges in the drug development process. Here, a series of new N-alkylated-2-(substituted phenyl)-1H-benzimidazole derivatives were synthesized and characterized by 1H and 13C NMR, FTIR, and HRMS spectrum analyses to address these difficulties. All the compounds were evaluated for their antiproliferative, antibacterial, and antifungal activities. Results indicated that compound 2g exhibited the best antiproliferative activity against the MDA-MB-231 cell line and also displayed significant inhibition at minimal inhibitory concentration (MIC) values of 8, 4, and 4 μg mL-1 against Streptococcus faecalis, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus compared with amikacin. The antifungal data of compounds 1b, 1c, 2e, and 2g revealed their moderate activities toward Candida albicans and Aspergillus niger, with MIC values of 64 μg mL-1 for both strains. Finally, the molecular docking study found that 2g interacted with crucial amino acids in the binding site of complex dihydrofolate reductase with nicotinamide adenine dinucleotide phosphate.

Relationship between Sugarcane eIF4E Gene and Resistance against Sugarcane Streak Mosaic Virus.

Sugarcane mosaic disease, mainly caused by Sugarcane streak mosaic virus (SCSMV), has serious adverse effects on the yield and quality of sugarcane. Eukaryotic translation initiation factor 4E (eIF4E) is a natural resistance gene in plants. The eIF4E-mediated natural recessive resistance results from non-synonymous mutations of the eIF4E protein. In this study, two sugarcane varieties, CP94-1100 and ROC22, were selected for analysis of their differences in resistance to SCSMV. Four-base missense mutations in the ORF region of eIF4E resulted in different conserved domains. Therefore, the differences in resistance to SCSMV are due to the inherent differences in eIF4E of the sugarcane varieties. The coding regions of eIF4E included 28 SNP loci and no InDel loci, which were affected by negative selection and were relatively conserved. A total of 11 haploids encoded 11 protein sequences. Prediction of the protein spatial structure revealed three non-synonymous mutation sites for amino acids located in the cap pocket of eIF4E; one of these sites existed only in a resistant material (Yuetang 55), whereas the other site existed only in a susceptible material (ROC22), suggesting that these two sites might be related to the resistance to SCSMV. The results provide a strong basis for further analysis of the functional role of eIF4E in regulating mosaic resistance in sugarcane.