Human Enzyme Research Articles

Generation of SARS-CoV-2 spike receptor binding domain mutants and functional screening for immune evaders using a novel lentivirus-based system.

The emergence of rapid and continuous mutations of severe acute respiratory syndrome 2 (SARS-CoV-2) spike glycoprotein that increased with the Omicron variant points out the necessity to anticipate such mutations for conceiving specific and adaptable therapies to avoid another pandemic. The crucial target for the antibody treatment and vaccine design is the receptor binding domain (RBD) of the SARS-CoV-2 spike. It is also the site where the virus has shown its high ability to mutate and consequently escape immune response. We developed a robust and simple method for generating a large number of functional SARS-CoV-2 spike RBD mutants by error-prone PCR and a novel nonreplicative lentivirus-based system. We prepared anti-RBD wild type (WT) polyclonal antibodies and used them to screen and select for mutant libraries that escape inhibition of virion entry into recipient cells expressing human angiotensin-converting enzyme 2 and transmembrane serine protease 2. We isolated, cloned, and sequenced six mutants totally bearing nine mutation sites. Eight mutations were found in successive WT variants, including Omicron and other recombinants, whereas one is novel. These results, together with the detailed functional analyses of two mutants provided the proof of concept for our approach.

SUBJECT REVIEW PHASES OF SUGAR METABOLISM AND THE EFFECT OF HORMONES ON IT

Sugar metabolism is the biochemical processes that imply formation, breakdown, and conversion of sugar in human body. Several biological processes are involved during Sugar metabolism. This report discusses comprehensively both sugar metabolism phases and digestion pathways. The biological activities related to sugar metabolism in every relevant human body organs, chemical reactions, hormones and enzymes effects, transport routes and absorption mechanisms have been highlighted.

Novel Mitochondrial and DNA Damaging Fluorescent Calix[4]arenes Bearing Isatin Groups as Aromatase Inhibitors: Design, Synthesis and Anticancer Activity

Breast cancer causes a high rate of mortality all over the world. Therefore, the present study focuses on the anticancer activity of new lower rim-functionalized calix[4]arenes integrated with isatin and the p-position of calixarenes with 1,4-dimethylpyridinium iodine against various human cancer cells such as MCF-7 and MDA-MB-231 breast cancer cell lines, as well as the PNT1A healthy epithelial cell line. It was observed that compound 6c had the lowest values in MCF-7 (8.83 µM) and MDA-MB-231 (3.32 µM). Cell imaging and apoptotic activity studies were performed using confocal microscopy and flow cytometry, respectively. The confocal imaging studies with 6c showed that the compound easily entered the cell, and it was observed that 6c accumulated in the mitochondria.The Comet assay test was used to detect DNA damage of compounds in cells. It was found that treated cells had abnormal tail nuclei and damaged DNA structures compared with untreated cells. In vitro human aromatase enzyme inhibition profiles showed that compound 6c had a remarkable inhibitory effect on aromatase. Compound 6c displayed a significant inhibition capacity on aromatase enzyme with the IC50 value of 0.104 ± 0.004 µM. Thus, not only the anticancer activity of the new fluorescent derivatives, which are the subject of this study, but the aromatase inhibitory profiles have also been proven.

In silico approaches to study the human asparagine synthetase: An insight of the interaction between the enzyme active sites and its substrates.

Cancer is a leading concern and important cause of death worldwide. Cancer is a non-communicable illness defined as uncontrolled division of cells. It can develop into metastatic cancer when tumor cells migrate to other organs. In recent years evidence has emerged that the bioavailability of Asn play a crucial role in cancer metastasis. Asn is a non-essential amino acid formed from an ATP dependent catalyzed reaction by the enzyme asparagine synthetase (ASNS), where Asp and Gln are converted to Asn and Glu, respectively. The human ASNS enzyme consist of 561 amino acids, with a molecular weight of 64 KDa. ASNS governs the activation of transcriptional factors that regulate the process of metastasis. In this work the 3D model of ASNS in E. coli (AS-B) and the human ASNS docked with its different ligands have been used to study the 3D mechanism of the conversion of Asp and Gln to Asn and Glu, in human ASNS. The stability evaluation of the docked complexes was checked by molecular dynamic simulation through the bioinformatic tool Desmond. The binding residues and their interactions can be exploited for the development of inhibitors, as well as for finding new drug molecules against ASNS and prevention of metastatic cancer.

Metabolic characterization of the new benzimidazole synthetic opioids - nitazenes.

The recent re-emergence and the increasing popularity of nitazenes, a group of new synthetic opioids (NSO) that belong to the benzimidazole chemical class, has raised public health concerns. As a class of potential opioid analgesic agents whose development was discontinued in the 1960s due to their high potential for abuse, very little is known about their metabolism and physiologic disposition. In the current study, three nitazenes-butonitazene, isotonitazene and protonitaze were incubated in human liver microsomes (HLM), human S9 (HS9) fractions and recombinant cytochrome P450 enzymes. All three nitazenes were rapidly metabolized in both HLM and HS9 with over 95% depletion within 60min. In HLM, butonitazene, isotonitazene and protonitazene had in vitro intrinsic clearance (CLint) (µL/min/mg protein) values of 309, 221 and 216 respectively compared to 150 of verapamil, the positive control. In HS9, CLint values were 217, 139, and 150 for butonitazene, isotonitazene and protonitazene respectively compared to only 35 for testosterone, the control probe substrate. Putative metabolite identified from this study include products of hydroxylation, desethylation, dealkylation, desethylation followed by dealkylation, and desethylation followed by hydroxylation. The metabolic phenotyping showed CYP2D6, CYP2B6 and CYP2C8 and the major hepatic enzymes responsible for the metabolism of nitazenes. Within 30min of incubation, CYP2D6 depleted butonitazene (99%), isotonitazene (72%) and butonitazene (100%) significantly. The rapid metabolism of nitazenes may be an important factor in accurate and timely detections and quantitation of the unchanged drugs in human matrices following intoxication or in forensic analysis. The involvement of multiple polymorphic CYPs in their metabolism may play important roles in the susceptibility to intoxication and/or addiction, depending on the activity of the metabolites.

Prevalence of Glucose-6-Phosphate Dehydrogenase deficiency among Hausa and Fulani ethnics in Kano, North-West Nigeria

Background: Glucose-6-phosphate dehydrogenase catalyzes the rate-determining step in the pentose phosphate pathway. Its activity is a key determinant of the reduced nicotinamide adenine dinucleotide phosphate to oxidized nicotinamide adenine dinucleotide phosphate (NADPH-to-NADP+) ratio in the cytoplasm and thus contributes to the replenishment of the antioxidant glutathione system. Glucose-6-phosphate dehydrogenase deficiency is a common X-linked human enzyme defect of red blood cells. Individuals with this gene defect appear normal until exposed to oxidative stress which induces haemolysis but it is infrequently taken into consideration in health practices. Aim: The present work was designed to determine the frequency of occurrence of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency among Hausa and Fulani ethnics. Material and methods: Cross-sectional comparative study in which 5 mL of venous blood samples were collected from 1000 individuals (596 males and 404 females) all of whom gave informed consent were screened for G-6-PD-deficiency by qualitative methaemoglobin reduction and quantitative enzyme activity measurement techniques. Results: Glucose-6-phoshate dehydrogenase deficiency was found in 194 (19.4%) of participants studied. Of these 10.3% were Fulani and 22.2% were Hausa with mean G-6-PD enzyme activity of 2.5 IU/gHb in a range from 1.4 to 2.4 IU/gHb. G-6-PD deficiency was found in 121 of 596 (20.3%) males and 73 of 404 (18.1%) females screened. Conclusions: These results suggest high occurrence of phenotypic G-6-PD deficiency in Hausa ethnics of Kano with lower enzyme status among males living with G-6-PD deficiency.

Omicron variants bind to human angiotensin-converting enzyme 2 (ACE2) much stronger due to higher number of charged-charged interactions

Since the start of COVID-19 pandemic, several mutant variants of SARS-CoV-2 have emerged with different virulence and transmissibility patterns. Some of these variants have been labeled as variants of concern (VOC). There are mainly five strain clades with VOC status: Alpha, Beta, Gamma, Delta, and Omicron. Omicron sub-variants have been currently in circulation around the world, and they show faster transmissibility and lower virulence compared to others. Receptor binding domain (RBD) of SARS-CoV-2 spike protein is the region where it binds to human angiotensin-converting enzyme 2 (hACE2) on the host cell. Mutations on RBD might have direct or indirect effects on differential disease patterns of these variants. In this study, we analyzed sequence and structures of SARS-CoV-2 variants’ RBD domains and documented their predicted affinities and contact interactions with hACE2. We found that Omicron sub-variants have much higher hACE2 affinities compared to other VOC strains. To understand reasons behind this, we checked biophysical characteristics of RBD-hACE2 contacts. Surprisingly, number of charged-charged interactions of Omicron sub-variants were on average 4-fold higher. These higher charged residue mutations on epitope region of Omicron sub-variants leading to stronger affinity for hACE2 might shed light onto why Omicron has less severe disease symptoms.

Exploration of Anti-Diabetic Potential of Rubus ellipticus smith through Molecular Docking, Molecular Dynamics Simulation, and MMPBSA Calculation

Diabetes is a chronic metabolic disorder affecting a majority of the population worldwide. Hyperglycemia leading to diabetes mellitus could be managed through the inhibition of human pancreatic α-amylase enzyme. Phytochemicals are frequently reported to possess anti-diabetic activity through inhibition of normal functioning of α-amylase. This study aims to find potential α-amylase inhibitors from Rubus ellipticus Smith with molecular-level understanding using different computational tools. From the molecular docking calculations, rubuside F and rubuside D possessed good binding affinity of -10.0 kcal/mol and -9.9 kcal/mol, respectively better than the reference drugs (acarbose, miglitol, voglibose and metformin). Both the compounds showed good geometrical stability from molecular dynamics simulation accessed in terms of RMSD, hydrogen bond count, SASA, Rg and RMSF. Binding free energy changes of -27.92±4.15 kcal/mol and -28.75±4.15 kcal/mol, respectively for the two ligands indicated sustained thermodynamic spontaneity present in the adducts. The two phytochemicals could be proposed as potential inhibitors of human pancreatic α-amylase for the treatment of diabetes. Further, in vitro and in vivo experiments are recommended for the verification of computational insights in the course of drug design and discovery process.

Prevalence Of Glucose-6-Phosphate Dehydrogenase Deficiency in People Living in Katsina Metropolis

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most prevalent human enzyme defects observed in malaria endemic areas. The study included 212 participants, with 142 (67%) male and 70 (33%) female, aged between14 to 59 with a mean age of 24.20±9.35 years. The study was granted ethical approval by the Katsina State Ministry of Health. Each subject gave his/her written informed permission after counseling. G6PD deficiency, malaria parasite, reduced glutathione (GSH), glutathione peroxidase (GPx), packed cell volume (PCV) and hemoglobin (Hb) were assayed using standard procedures. Among the 212 participants tested in the study, 40 (19%) were found to be G6PD deficient and 172 (81%) participants were normal. Among 212 participants tested, 75 (35.4%) were infected with malaria parasite out of which 13 (17.3%) were G6PD deficient and 62 (82.7%) were normal. Among the 40 G6PD deficient subjects, 33 (82.5%) were male while 7 (17.5%) were females. GSH concentration was lowered significantly in G6PD-deficient subjects compared to the normal subjects. GPx, Hb and PCV showed no significant difference (p>0.05) in the deficient subjects compared to the normal subjects. The results of this study suggest that G6PD deficiency is quite common with a prevalence of 19% in the population of Katsina metropolis. The data also shows that males are more likely to be affected by G6PD deficiency than females.

Nutritional evaluation of probiotics enriched rabadi beverage (PERB) and molecular mapping of digestive enzyme with dietary fibre for exploring the therapeutic potential

Advent of the silent pandemic of obesity, which is associated with onset of various non-communicable and communicable disorders; led for the necessity to develop a functional food product with good nutritional profile to improve the overall health. Therefore, the current study was aimed to develop a probiotic enriched rabadi beverage. The beverage was prepared by using pearl millet, buttermilk and fortified with L. rhamnosus, Lactobacillus sp and S. faecalis. Subsequently, analysed for different parameters. Molecular mapping was also performed to show the inhibition profiling between cellulose and the human digestive enzymes. There was a significant difference for pH, acidity, viscosity, dry matter, and organic matter of PERB for autoclaved and non-autoclaved batches in all the used strains. However, a non-significant difference was observed in terms of different fermentation temperatures. The ash content varied significantly (0.32 ± 0.02%) in beverages fortified with S. faecalis fermented at 350 C, whereas moisture content had a non-significant effect on all the strains at different fermentation temperatures. Molecular mapping revealed the inhibition profiling in terms of binding affinity (kcal/mol) for various human digestive enzymes (Recombinant Acetylcholinesterase: −8.1, lysosomal acid-alpha-glucosidase: −6.6, and pancreatic alpha-amylase: −6.4) with cellulose. The study concluded that the PERB has a good amount of ADF, NDF, and hemicellulose content as well as indicated the significance of dietary fibre (cellulose) in regulating the digestibility through the interaction of gastrointestinal enzymes in order to explore the therapeutic potential of the PERB. Therefore, the developed PERB would serve as a potential source for better nutrition and overall health.

Laxilignans A-C from the leaves of Terminalia laxiflora Engl. and their α-glucosidase inhibitory activity

Three previously undescribed dibenzylbutane lignans, laxilignans A−C, together with fifteen known compounds were isolated and characterized from the leaves of Terminalia laxiflora Engl. The structures of laxilignans A−C were elucidated by extensive 1D and 2D NMR analyses, together with mass spectrometry. The inhibitory activity of the isolated compounds against α-glucosidase enzyme, obtained from Saccharomyces cerevisiae, was evaluated in comparison to acarbose (IC50 = 16.42 ± 0.14 µM). The results revealed that corilagin, chebulagic acid, laxilignan A, termilignan B, termitomenin C, arjunglucoside II, and laxilignan B possess α-glucosidase inhibitory potential, with IC50 values in the range of 1.61 ± 0.05 – 403.5 ± 29.5 μM, respectively. The results of the molecular docking revealed that the two laxilignans A and B showed comparable binding affinities to those of acarbose with the binding sites of the human α-glucosidase enzyme. These findings suggest that dibenzylbutane lignans may act as promising lead compounds for the development of α-glucosidase inhibitors.

Differential Outcomes of Infection by Wild-Type SARS-CoV-2 and the B.1.617.2 and B.1.1.529 Variants of Concern in K18-hACE2 Transgenic Mice.

SARS-CoV-2 is a respiratory virus with neurological complications including the loss of smell and taste, headache, and confusion that can persist for months or longer. Severe neuronal cell damage has also been reported in some cases. The objective of this study was to compare the infectivity of the wild-type virus, Delta (B.1.617.2) and Omicron (B.1.1.529) variants in transgenic mice that express the human angiotensin-converting enzyme 2 (hACE2) receptor under the control of the keratin 18 promoter (K18) and characterize the progression of infection and inflammatory response in the lungs, brain, medulla oblongata, and olfactory bulbs of these animals. We hypothesized that wild type, Delta and Omicron differentially infect K18-hACE2 mice, thereby inducing distinct cellular responses. K18-hACE2 female mice were intranasally infected with wild-type, Delta, or Omicron variants and euthanized either at 3 days post-infection (dpi) or at the humane endpoint. None of the animals infected with the Omicron variant reached the humane endpoint and were euthanized at day 8 dpi. Virological and immunological analyses were performed in the lungs, brains, medulla oblongata and olfactory bulbs isolated from infected mice. At 3 dpi, mice infected with wild type and Delta displayed significantly higher levels of viral RNA in the lungs than mice infected with Omicron, while in the brain, Delta and Omicron resulted in higher levels of viral RNA than with the wild type. Viral RNA was also detected in the medulla oblongata of mice infected by all these virus strains. At this time point, the mice infected with wild type and Delta displayed a marked upregulation of many inflammatory markers in the lungs. On the other hand, the upregulation of inflammatory markers was observed only in the brains of mice infected with Delta and Omicron. At the humane endpoint, we observed a significant increase in the levels of viral RNA in the lungs and brains of mice infected with wild type and Delta, which was accompanied by the elevated expression of many inflammatory markers. In contrast, mice which survived infection with the Omicron variant showed high levels of viral RNA and the upregulation of cytokine and chemokine expression only in the lungs at 8 dpi, suggesting that infection and inflammatory response by this variant is attenuated in the brain. Reduced RNA levels and the downregulation of inflammatory markers was also observed in the medulla oblongata and olfactory bulbs of mice infected with Omicron at 8 dpi as compared with mice infected with wild-type and Delta at the humane end point. Collectively, these data demonstrate that wild-type, Delta, and Omicron SARS-CoV-2 induce distinct levels of infection and inflammatory responses in K18-hACE2 mice. Notably, sustained brain infection accompanied by the upregulation of inflammatory markers is a critical outcome in mice infected with wild type and Delta but not Omicron.

Mechanisms of SARS-CoV-2 Inactivation Using UVC Laser Radiation.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has had a tremendous impact on humanity. Prevention of transmission by disinfection of surfaces and aerosols through a chemical-free method is highly desirable. Ultraviolet C (UVC) light is uniquely positioned to achieve inactivation of pathogens. We report the inactivation of SARS-CoV-2 virus by UVC radiation and explore its mechanisms. A dose of 50 mJ/cm2 using a UVC laser at 266 nm achieved an inactivation efficiency of 99.89%, while infectious virions were undetectable at 75 mJ/cm2 indicating >99.99% inactivation. Infection by SARS-CoV-2 involves viral entry mediated by the spike glycoprotein (S), and viral reproduction, reliant on translation of its genome. We demonstrate that UVC radiation damages ribonucleic acid (RNA) and provide in-depth characterization of UVC-induced damage of the S protein. We find that UVC severely impacts SARS-CoV- 2 spike protein's ability to bind human angiotensin-converting enzyme 2 (hACE2) and this correlates with loss of native protein conformation and aromatic amino acid integrity. This report has important implications for the design and development of rapid and effective disinfection systems against the SARS-CoV-2 virus and other pathogens.

Structure-based drug discovery to identify SARS-CoV2 spike protein–ACE2 interaction inhibitors

After the emergence of the COVID-19 pandemic in late 2019, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has undergone a dynamic evolution driven by the acquisition of genetic modifications, resulting in several variants that are further classified as variants of interest (VOIs), variants under monitoring (VUM) and variants of concern (VOC) by World Health Organization (WHO). Currently, there are five SARS-CoV-2 VOCs (Alpha, Beta, Delta, Gamma and Omicron), two VOIs (Lambda and Mu) and several other VOIs that have been reported globally. In this study, we report a natural compound, Curcumin, as the potential inhibitor to the interactions between receptor binding domain (RBD(S1)) and human angiotensin-converting enzyme 2 (hACE2) domains and showcased its inhibitory potential for the Delta and Omicron variants through a computational approach by implementing state of the art methods. The study for the first time revealed a higher efficiency of Curcumin, especially for hindering the interaction between RBD(S1) and hACE-2 domains of Delta and Omicron variants as compared to other lead compounds. We investigated that the mutations in the RBD(S1) of VOC especially Delta and Omicron variants affect its structure compared to that of the wild type and other variants and therefore altered its binding to the hACE2 receptor. Molecular docking and molecular dynamics (MD) simulation analyses substantially supported the findings in terms of the stability of the docked complexes. This study offers compelling evidence, warranting a more in-depth exploration into the impact of these alterations on the binding of identified drug molecules with the Spike protein. Further investigation into their potential therapeutic effects in vivo is highly recommended. Communicated by Ramaswamy H. Sarma

Optimization of sequence and chiral content enhances therapeutic potential of tilapia piscidin peptides

Because antimicrobial peptides (AMPs) often exhibit broad-spectrum bactericidal potency, we sought to develop peptide-based antimicrobials for potential clinical use against drug-resistant pathogens. To accomplish this goal, we first optimized the amino acid sequence of a broad-spectrum AMP known as Tilapia Piscidin 4 (TP4). Then, we used the optimized sequence to create a pair of heterochiral variants (TP4-α and TP4-β) with different percentages of D-enantiomers, as poly-L peptides often exhibit poor pharmacokinetic profiles. The conformations of the peptide pair exhibited inverted chirality according to CD and NMR spectroscopic analyses. Both heterochiral peptides displayed enhanced stability and low hemolysis activities. Irrespective of their different d-enantiomer contents, both heterochiral peptides exhibited bactericidal activities in the presence of human serum or physiological enzymes. However, the peptide with higher d-amino acid content (TP4-β) caused better bacterial clearance when tested in mice infected with NDM-1 K. pneumoniae. In addition, we observed a relatively higher hydrogen bonding affinity in a simulation of the interaction between TP4-β and a model bacterial membrane. In sum, our results demonstrate that the current design strategy may be applicable for development of new molecules with enhanced stability and in vivo antimicrobial activity.

Evidence of a Sjögren's disease-like phenotype following COVID-19 in mice and humans.

Sjögren's Disease (SjD) is a systemic autoimmune disease characterized by lymphocytic inflammation of the lacrimal and salivary glands (SG), dry eyes and mouth, and systemic symptoms. SARS-CoV-2 may trigger the development or progression of autoimmune diseases. To test this, we used a mouse model of SARS-CoV-2 infection and convalescent patients' blood and SG in order to understand the development of SjD-like autoimmunity after infection. First, SARS-CoV-2-infected human angiotensin-converting enzyme 2 (ACE2) transgenic mice exhibited decreased salivation, elevated antinuclear antibodies (ANA), and lymphocytic infiltration in the lacrimal and SG. The sera from patients with COVID-19 sera showed increased ANA (i.e., anti-SSA [Sjögren's-syndrome-related antigen A]/anti-Ro52 and anti-SSB [SS-antigen B]/anti-La). Male patients showed elevated anti-SSA compared with female patients, and female patients exhibited diverse ANA patterns. SG biopsies from convalescent COVID-19 patients were microscopically similar to SjD SG with focal lymphocytic infiltrates in 4 of 6 patients and 2 of 6 patients exhibiting focus scores of at least 2. Lastly, monoclonal antibodies produced in recovered patients blocked ACE2/spike interaction and cross-reacted with nuclear antigens. Our study shows a direct association between SARS-CoV-2 and SjD. Hallmark features of SjD-affected SGs were histologically indistinguishable from convalescent COVID-19 patients. The results implicate that SARS-CoV-2 could be an environmental trigger for SjD.

Venomous gland transcriptome and venom proteomic analysis of the scorpion Androctonus amoreuxi reveal new peptides with anti-SARS-CoV-2 activity

The recent COVID-19 pandemic shows the critical need for novel broad spectrum antiviral agents. Scorpion venoms are known to contain highly bioactive peptides, several of which have demonstrated strong antiviral activity against a range of viruses. We have generated the first annotated reference transcriptome for the Androctonus amoreuxi venom gland and used high performance liquid chromatography, transcriptome mining, circular dichroism and mass spectrometric analysis to purify and characterize twelve previously undescribed venom peptides. Selected peptides were tested for binding to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and inhibition of the spike RBD – human angiotensin-converting enzyme 2 (hACE2) interaction using surface plasmon resonance-based assays. Seven peptides showed dose-dependent inhibitory effects, albeit with IC50 in the high micromolar range (117–1202 µM). The most active peptide was synthesized using solid phase peptide synthesis and tested for its antiviral activity against SARS-CoV-2 (Lineage B.1.1.7). On exposure to the synthetic peptide of a human lung cell line infected with replication-competent SARS-CoV-2, we observed an IC50 of 200 nM, which was nearly 600-fold lower than that observed in the RBD – hACE2 binding inhibition assay. Our results show that scorpion venom peptides can inhibit the SARS-CoV-2 replication although unlikely through inhibition of spike RBD – hACE2 interaction as the primary mode of action. Scorpion venom peptides represent excellent scaffolds for design of novel anti-SARS-CoV-2 constrained peptides. Future studies should fully explore their antiviral mode of action as well as the structural dynamics of inhibition of target virus-host interactions.

Protein-protein interfaces as druggable targets: a common motif of the pyridoxal-5′-phosphate-dependent enzymes to receive the coenzyme from its producer

Pyridoxal-5′-phosphate (PLP), a phosphorylated form of vitamin B6, acts as a coenzyme for numerous reactions, including those changed in cancer and/or associated with the disease prognosis. Since highly reactive PLP may modify cellular proteins, it is hypothesized to be directly transferred from its donors to acceptors. Our goal is to validate the hypothesis by finding common motif(s) in a multitude of the PLP-dependent enzymes for binding the limited number of the PLP donors, namely pyridoxal kinase (PdxK), pyridox(am)in-5′-phosphate oxidase (PNPO) and the PLP-binding protein (PLPBP). Experimentally confirmed interactions between the PLP donors and acceptors reveal that PdxK and PNPO interact with the PLP acceptors of folds I and II, while PLPBP - with those of folds III and V. Aligning the sequences and 3D structures of the identified interactors of PdxK and PNPO, we have found a common motif in the PLP-dependent enzymes of folds I and II. The motif extends from the enzyme surface to the neighborhood of the PLP binding site, represented by an exposed alfa-helix, a partially buried beta-strand and residual loops. Pathogenicity of mutations in human PLP-dependent enzymes within or in the vicinity of the motif, but outside of the active sites, supports functional significance of the motif that may provide an interface for the direct transfer of PLP from the sites of its synthesis to those of the coenzyme binding. The enzyme-specific amino acid residues of the common motif may be used to develop selective inhibitors blocking PLP delivery to the PLP-dependent enzymes critical for proliferation of malignant cells.

Protective Efficacy of Novel Engineered Human ACE2-Fc Fusion Protein Against Pan-SARS-CoV-2 Infection In Vitro and in Vivo.

Enduring occurrence of severe COVID-19 for unvaccinated, aged, or immunocompromised individuals remains an urgent need. Soluble human angiotensin-converting enzyme 2 (ACE2) has been used as a decoy receptor to inhibit SARS-CoV-2 infection, which is limited by moderate affinity. We describe an engineered, high-affinity ACE2 that is consistently effective in tissue cultures in neutralizing all strains tested, including Delta and Omicron. We also found that treatment of AC70 hACE2 transgenic mice with hACE2-Fc receptor decoys effectively reduced viral infection, attenuated tissue histopathology, and delayed the onset of morbidity and mortality caused by SARS-CoV-2 infection. We believe that using this ACE2-Fc protein would be less likely to promote the escape mutants of SARS-CoV-2 as frequently as did those neutralizing antibody therapies. Together, our results emphasize the suitability of our newly engineered hACE2-Fc fusion protein for further development as a potent antiviral agent against Pan-SARS-CoV-2 infection.

In vitro inhibition of six active sesquiterpenoids in zedoary turmeric oil on human liver cytochrome P450 enzymes

In vitro inhibition of six active sesquiterpenoids in zedoary turmeric oil on human liver cytochrome P450 enzymes