Human Enzyme Research Articles

In Vivo and In Vitro Induction of Cytochrome P450 3A4 by Thalidomide in Humanized-Liver Mice and Experimental Human Hepatocyte HepaSH cells.

Autoinduction of cytochrome P450 (P450) 3A4-mediated metabolism of thalidomide was investigated in humanized-liver mice and human hepatocyte-derived HepaSH cells. The mean plasma ratios of 5-hydroxythalidomide and glutathione adducts to thalidomide were significantly induced (3.5- and 6.0-fold, respectively) by thalidomide treatment daily at 1000 mg/kg for 3 days and measured at 2 h after the fourth administration (on day 4). 5-Hydroxythalidomide was metabolically activated by P450 3A4 in HepaSH cells pretreated with 300 and 1000 μM thalidomide, and 5,6-dihydroxythalidomide was detected. Significant induction of P450 3A4 mRNA expression (4.1-fold) in the livers of thalidomide-treated mice occurred. Thalidomide exerts a variety of actions through multiple mechanisms following bioactivation by induced human P450 3A enzymes.

Characterization of the metabolic contributions of cytochrome P450 isoforms to bicyclol using the relative activity factor method

Bicyclol is a hepatoprotective agent widely used for treating chronic hepatitis and drug-induced liver injuries in clinics. The purpose of the study was to elucidate the contribution of CYP450 enzymes to the metabolism of bicyclol using the relative activity factor approach. After incubation with human liver microsomes and recombinant human liver CYP450 enzymes, the calculated contribution of CYP3A4 and 2C19 to the metabolism of bicyclol was 85.6–90.3% and 9.2–9.7%, respectively. The metabolism was interrupted in the presence of CYP3A4 and 2C19 selective inhibitors. These findings help to predict or avoid metabolic drug–drug interactions or toxicity in clinical applications of bicyclol.

Greener Synthesis, Molecular Docking and Anticancer Studies of A New Series of 6‐Hydroxy‐5‐(3‐(3‐hydroxy‐1,4‐dioxo‐1,4‐dihydronaphthalen‐2‐yl)‐2‐oxoindolin‐3‐yl) pyrimidine‐2,4(1H,3H)‐diones

AbstractGreener solvent mediated simple and efficient synthesis of 6‐hydroxy‐5‐(3‐(3‐hydroxy‐1,4‐dioxo‐1,4‐dihydronaphthalen‐2‐yl)‐2‐oxoindolin‐3‐yl)pyrimidine‐2,4(1H,3H)‐diones has been achieved through the one‐pot multicomponent condensation reaction between 2‐hydroxy‐1,4‐naphthoquinone, substituted isatin and barbituric acid/thiobarbituric acid/1,3‐dimethylbarbituric acid using ethanol‐water as a solvent without any catalyst at room temperature has been described. Molecular docking studies have been performed with Akt kinase enzyme protein which helped to rationalize the binding energy of all the synthesized compounds. The docking of compound 4d, with human Akt kinase enzyme showed a good binding interaction with lowest binding energy. Based on the results from the molecular docking studies, the compound 4d was further studied for its in‐vitro anticancer activity against human breast cancer cells (MCF‐7) and human normal breast cells (HBL 100) by MTT assay which also proved the potential anticancer activity of 4d.

Sites and Zones of Maximum Reactivity of the most Stable Structure of the Receptor-binding Domain of Wild-type SARS-CoV-2 Spike Protein: A Quantum Density Functional Theory Study

Today, it is well known that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has four types of proteins within its structure, between them the spike protein (S). The infection mechanism is carried out by the entry of the virus into the human host cell through the S protein, which strongly interacts with the human cell receptor angiotensin-converting enzyme 2 (ACE2). In this work, we propose an atomic model of the Receptor Binding Domain (RBD) of the S spike protein of the wild-type SARS-CoV-2 virus. The molecular structure of the model was composed of 50 amino acids that were chemically bonded, starting with Leucine and ending with one amino acid Tyrosine. The novelty of our work lies in the importance of knowing the sites and zones of maximum reactivity of the RBD from the fundamental levels of quantum mechanics considering the atomic structure of matter. For this, the local and global reactivity indices of the RBD were calculated, such as frontier orbitals, Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO), Fukui indices, chemical potential, chemical hardness, electrophilicity index; with this, it will be possible to know what type of molecules are more likely to interact with the RBD structure, and in this way, new knowledge will be generated at the quantum, atomic and molecular level to inhibit the virulent effects of wild-type SARS-CoV-2. Finally, in order to identify the functional groups within the most stable structure and thereby verify the future reactions that can be carried out between the RBD structure and biomolecules, the Infrared (IR) absorption spectrum was calculated. For this work, we used Material Studio v6.0 which uses the density functional theory (DFT) implemented in its DMol3 computational code. The IR spectrum was obtained using the Spartan ‘94 computer code. One novelty would be that we found nine amino acids more that could make the RBD and ACE2 binding further the already known. Thus, the Mulliken charge distribution indicates that the highest concentrations of positive and negative charge are found in the zones 477S, 478T, 484E, and 501N amino acids letting ionic or Van der Waals possible interactions with other structures.

Sex toys for pleasure, but there are risks: In silico toxicity studies of leached Micro/Nanoplastics and phthalates

The widespread use of sex toys has sparked concerns about potential health risks associated with the leaching of micro/nanoplastics (M/NPs) and phthalates. In this in silico toxicity analysis, we investigated the immunotoxicity potential of compounds mechanically produced from sex toys. The research focuses on understanding their interactions with critical human cytochrome P450 enzymes (CYP34A and CYP2D6), which are central to drug metabolism and immune function. Four types of M/NPs (polyethylene terephthalate - PET, polyvinyl chloride - PVC, polydimethylsiloxane - PDMS, polyisoprene) and eight phthalates (Diethyl phthalate - DEP, Bis(4-methyl-2-pentyl) phthalate, Bis(2-methoxyethyl) phthalate, Benzyl butyl phthalate - BBP, Dibutyl Phthalate - DBP, Diethylhexyl phthalate - DEHP, Dinonyl phthalate - DNP, Di-n-octyl phthalate - DnOP) from sex toys were analyzed. Molecular dynamics and docking simulations were conducted to assess the binding affinity of these compounds to the enzymes. PET exhibited the highest toxicity, with a binding affinity of -8.3 kcal/mol and -8.9 kcal/mol for CYP3A4 and CYP2D6, respectively, surpassing control values (-7.8 kcal/mol and -6.3 kcal/mol). BBP showed considerable toxicity, binding strongly to CYP3A4 (-7.8 kcal/mol) and CYP2D6 (-9.1 kcal/mol). PDMS and DEP were the lowest toxic compounds in both groups. Molecular interactions revealed hydrogen bonds, pi interactions, and unfavorable donor-donor effects between compounds and specific amino acid residues within the enzymes. While these in silico findings provide insights, they underscore the need for comprehensive chemical analyses and absorption studies to validate health risks. This research emphasizes the necessity of evaluating the toxicity of M/NPs and phthalates from sex toys, promoting further investigations to protect individuals' health during product use.

Discovery of a Novel Potent Tetrazole Antifungal Candidate with High Selectivity and Broad Spectrum.

Thirty-one novel albaconazole derivatives were designed and synthesized based on our previous work. All compounds exhibited potent in vitro antifungal activities against seven pathogenic fungi. Among them, tetrazole compound D2 was the most potent antifungal with MIC values of <0.008, <0.008, and 2 μg/mL against Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus, respectively, the three most common and critical priority pathogenic fungi. In addition, compound D2 also exhibited potent activity against fluconazole-resistant C. auris isolates. Notably, compound D2 showed a lower inhibitory activity in vitro against human CYP450 enzymes as well as a lower inhibitory effect on the hERG K+ channel, indicating a low risk of drug-drug interactions and QT prolongation. Moreover, with improved pharmacokinetic profiles, compound D2 showed better in vivo efficacy than albaconazole at reducing fungal burden and extending the survival of C. albicans-infected mice. Taken together, compound D2 will be further investigated as a promising candidate.

Forecasting SARS-CoV-2 spike protein evolution from small data by deep learning and regression

The emergence of SARS-CoV-2 variants during the COVID-19 pandemic caused frequent global outbreaks that confounded public health efforts across many jurisdictions, highlighting the need for better understanding and prediction of viral evolution. Predictive models have been shown to support disease prevention efforts, such as with the seasonal influenza vaccine, but they require abundant data. For emerging viruses of concern, such models should ideally function with relatively sparse data typically encountered at the early stages of a viral outbreak. Conventional discrete approaches have proven difficult to develop due to the spurious and reversible nature of amino acid mutations and the overwhelming number of possible protein sequences adding computational complexity. We hypothesized that these challenges could be addressed by encoding discrete protein sequences into continuous numbers, effectively reducing the data size while enhancing the resolution of evolutionarily relevant differences. To this end, we developed a viral protein evolution prediction model (VPRE), which reduces amino acid sequences into continuous numbers by using an artificial neural network called a variational autoencoder (VAE) and models their most statistically likely evolutionary trajectories over time using Gaussian process (GP) regression. To demonstrate VPRE, we used a small amount of early SARS-CoV-2 spike protein sequences. We show that the VAE can be trained on a synthetic dataset based on this data. To recapitulate evolution along a phylogenetic path, we used only 104 spike protein sequences and trained the GP regression with the numerical variables to project evolution up to 5 months into the future. Our predictions contained novel variants and the most frequent prediction mapped primarily to a sequence that differed by only a single amino acid from the most reported spike protein within the prediction timeframe. Novel variants in the spike receptor binding domain (RBD) were capable of binding human angiotensin-converting enzyme 2 (ACE2) in silico, with comparable or better binding than previously resolved RBD-ACE2 complexes. Together, these results indicate the utility and tractability of combining deep learning and regression to model viral protein evolution with relatively sparse datasets, toward developing more effective medical interventions.

Antibiotics daptomycin interacts with S protein of SARS-CoV-2 to promote cell invasion of Omicron (B1.1.529) pseudovirus

ABSTRACT The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has posed enormous challenges to global public health. The use of antibiotics has greatly increased during the SARS-CoV-2 epidemic owing to the presence of bacterial co-infection and secondary bacterial infections. The antibiotics daptomycin (DAP) is widely used in the treatment of infectious diseases caused by gram-positive bacteria owing to its highly efficient antibacterial activity. It is pivotal to study the antibiotics usage options for patients of coronavirus infectious disease (COVID-19) with pneumonia those need admission to receive antibiotics treatment for bacterial co-infection in managing COVID-19 disease. Herein, we have revealed the interactions of DAP with the S protein of SARS-CoV-2 and the variant Omicron (B1.1.529) using the molecular docking approach and Omicron (B1.1.529) pseudovirus (PsV) mimic invasion. Molecular docking analysis shows that DAP has a certain degree of binding ability to the S protein of SARS-CoV-2 and several derived virus variants, and co-incubation of 1–100 μM DAP with cells promotes the entry of the PsV into human angiotensin-converting enzyme 2 (hACE2)-expressing HEK-293T cells (HEK-293T-hACE2), and this effect is related to the concentration of extracellular calcium ions (Ca2+). The PsV invasion rate in the HEK-293T-hACE2 cells concurrently with DAP incubation was 1.7 times of PsV infection alone. In general, our findings demonstrate that DAP promotes the infection of PsV into cells, which provides certain reference of antibiotics selection and usage optimization for clinicians to treat bacterial coinfection or secondary infection during SARS-CoV-2 infection.

Evaluation of Lippia scaberrima Sond. and Aspalathus linearis (Burm.f.) R. Dahlgren extracts on human CYP enzymes and gold nanoparticle synthesis: implications for drug metabolism and cytotoxicity

BackgroundMetabolism is an important component of the kinetic characteristics of herbal constituents, and it often determines the internal dose and concentration of these effective constituents at the target site. The metabolic profile of plant extracts and pure compounds need to be determined for any possible herb-drug metabolic interactions that might occur.MethodsVarious concentrations of the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with fermented and unfermented Aspalathus linearis extract were used to determine the inhibitory potential on placental, microsomal and recombinant human hepatic Cytochrome P450 enzymes. Furthermore, the study investigated the synthesis and characterization of gold nanoparticles from the ethanolic extract of Lippia scaberrima as a lead sample. Confirmation and characterization of the synthesized gold nanoparticles were conducted through various methods. Additionally, the cytotoxic properties of the ethanolic extract of Lippia scaberrima were compared with the gold nanoparticles synthesized from Lippia scaberrima using gum arabic as a capping agent.ResultsAll the samples showed varying levels of CYP inhibition. The most potent inhibition took place for CYP2C19 and CYP1B1 with 50% inhibitory concentration (IC50) values of less than 0.05 µg/L for the essential oil tested and IC50-values between 0.05 µg/L-1 µg/L for all the other combinations and extracts tested, respectively. For both CYP1A2 and CYP2D6 the IC50-values for the essential oil, the extracts and combinations were found in the range of 1 – 10 µg/L. The majority of the IC50 values found were higher than 10 µg/L and, therefore, were found to have no inhibition against the CYP enzymes tested.ConclusionTherefore, the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with Aspalathus linearis do not possess any clinically significant CYP interaction potential and may be further investigated for their adjuvant potential for use in the tuberculosis treatment regimen. Furthermore, it was shown that the cytotoxic potential of the Lippia scaberrima gold nanoparticles was reduced by twofold when compared to the ethanolic extract of Lippia scaberrima.

Nutriose, a Dextrin-based Natural Polysaccharide Polymer with Beneficial Activities as a Candidate for Future Drug Delivery: A Review.

Nutriose is a dextrin-based soluble fiber prepared from starch. Cereals such as maize, wheat, and barley are the primary sources of nutrients for commercial production. Nutriose is resistant to digestion by human enzymes in the stomach and small intestine. It is mostly undamaged when it enters the colon after traveling through the digestive tract, where it generates shortchain fatty acids (SCFAs) as byproducts. These SCFAs, which include butyrate, propionate, and acetate, have a number of health advantages. They foster an environment in the colon that is advantageous for gut health-promoting bacteria like lactobacilli and bifidobacteria. Nutriose fermentation leads to a more balanced composition of the gut microbiota, which may have advantages for the immune system, better digestion, and increased nutrient absorption. As a result, nutriose is currently being utilized as a prebiotic. Several publications have previously demonstrated the impact of nutriose on stimulating gut mucosal immunity and boosting colonic fermentation and excretion in rats. Nanoformulations and nutrisomes have already been prepared and evaluated in recent years. A novel nutriose-based polymeric coating mix has already been tested as a potential colon-targeting material. As a natural polysaccharide, nutriose's possible uses in pharmaceuticals may increase in the near future. The purpose of this study is to critically analyze existing data to determine the potential of nutriose as a natural polymer for various drug delivery systems.

Abstract SY11-02: Small molecule inhibitors of RNA modifying enzymes as precision cancer therapeutics

Abstract Over 100 modifications to RNA are known to occur in human cells, where they influence many aspects of RNA biology, including protein and nucleic acid interactions. Our survey of human RNA-modifying enzymes with selectively essential phenotypes in specific cancers, suggests that these cancer-essential enzymes fall into four mechanistic categories: group transfer enzymes; base editors; nucleases; and helicases. Our team has discovered nanomolar or picomolar inhibitors of cancer-essential enzymes within each of these four categories. DHX9, a novel cancer target, is a multifunctional DEAH-box RNA helicase which can unwind regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops), circular RNA and DNA/RNA G-quadruplexes. DHX9 interacts with and regulate a large variety of proteins, including key proteins in DNA damage repair pathways such as BRCA1, ATR, Ku86, and WRN. We previously demonstrated that DHX9 inhibition is selectively efficacious in microsatellite high (MSI-H)/defective mismatch repair (dMMR) tumor models, in vitro and in vivo. Further profiling of DHX9 inhibitors across a broad panel of cancer cell lines reveals that tumor cells with Loss-of-Function (LOF) mutations in the DNA damage repair genes BRCA1 and/or BRCA2 (as defined by somatic mutations including single-nucleotide variants and/or copy number loss), are also selectively responsive to DHX9 inhibitor treatment. Selective dependence on DHX9 was observed in both ovarian and breast cancer cell lines that exhibit BRCA1 and/or BRCA2 LOF. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress. Cell lines that exhibit BRCA1 and/or BRCA2 LOF appear unable to resolve this replication stress and showed S-G2 phase cell cycle arrest prior to onset of apoptosis. An orally bioavailable DHX9 inhibitor was dosed in vivo to assess DHX9 dependency within multiple human xenografts representing triple negative breast cancer and high-grade serous ovarian cancer with BRCA1 and/or BRCA2 LOF. In all models, DHX9 inhibition was well tolerated for a period of up to 28 days, with robust and significant tumor growth inhibition - including tumor regression - observed in multiple BRCA1 and/or BRCA2 LOF models; in contrast, minimal tumor growth inhibition was observed in BRCA1 and BRCA2 wild type models. These results portend DHX9 inhibition as a novel treatment modality for patients with BRCA1 and/or BRCA2 LOF across multiple tumor types, including breast and ovarian cancer. Citation Format: Robert A. Copeland, Jennifer Castro, Matthew H. Daniels, Deepali Gotur, Young-Tae Lee, Shihua Yao, David Brennan, Brian T. Johnston, Monique Laidlaw, Sunaina Pai, Jie Wu, Rishabh Bansal, Anugraha Raman, Shane M. Buker, Julie Liu, E. Allen Sickmier, Kevin Knockenhauer, Chuang Lu, Stephen J. Blakemore, Serena J. Silver, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager. Small molecule inhibitors of RNA modifying enzymes as precision cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY11-02.

The thiol methyltransferase activity of TMT1A (METTL7A) is conserved across species

Although few resistance mechanisms for histone deacetylase inhibitors (HDACis) have been described, we recently demonstrated that TMT1A (formerly METTL7A) and TMT1B (formerly METTL7B) can mediate resistance to HDACis with a thiol as the zinc-binding group by methylating and inactivating the drug. TMT1A and TMT1B are poorly characterized, and their normal physiological role has yet to be determined. As animal model systems are often used to determine the physiological function of proteins, we investigated whether the ability of these methyltransferases to methylate thiol-based HDACis is conserved across different species. We found that TMT1A was conserved across rats, mice, chickens, and zebrafish, displaying 85.7%, 84.8%, 60.7%, and 51.0% amino acid sequence identity, respectively, with human TMT1A. Because TMT1B was not found in the chicken or zebrafish, we focused our studies on the TMT1A homologs. HEK-293 cells were transfected to express mouse, rat, chicken, or zebrafish homologs of TMT1A and all conferred resistance to the thiol-based HDACIs NCH-51, KD-5170, and romidepsin compared to empty vector-transfected cells. Additionally, all homologs blunted the downstream effects of HDACi treatment such as increased p21 expression, increased acetylated histone H3, and cell cycle arrest. Increased levels of dimethylated romidepsin were also found in the culture medium of cells transfected to express any of the TMT1A homologs after a 24 h incubation with romidepsin compared to empty-vector transfected cells. Our results indicate that the ability of TMT1A to methylate molecules is conserved across species. Animal models may therefore be useful in elucidating the role of these enzymes in humans.

Determination, expression and characterization of an UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) from the Pacific oyster, Crassostrea gigas

Molluscs are intermediate hosts for several parasites. The recognition processes, required to evade the host’s immune response, depend on carbohydrates. Therefore, the investigation of mollusc glycosylation capacities is of high relevance to understand the interaction of parasites with their host. UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT-I) is the key enzyme for the biosynthesis of hybrid and complex type N-glycans catalysing the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the α-1,3 Man antenna of Man5GlcNAc2. Thereby, the enzyme produces a suitable substrate for further enzymes, such as α-mannosidase II, GlcNAc-transferase II, galactosyltransferases or fucosyltransferases. The sequence of GnT- I from the Pacific oyster, Crassostrea gigas, was obtained by homology search using the corresponding human enzyme as the template. The obtained gene codes for a 445 amino acids long type II transmembrane glycoprotein and shared typical structural elements with enzymes from other species. The enzyme was expressed in insect cells and purified by immunoprecipitation using protein A/G-plus agarose beads linked to monoclonal His-tag antibodies. GnT-I activity was determined towards the substrates Man5-PA, MM-PA and GnM-PA. The enzyme displayed highest activity at pH 7.0 and 30 °C, using Man5-PA as the substrate. Divalent cations were indispensable for the enzyme, with highest activity at 40 mM Mn2+, while the addition of EDTA or Cu2+ abolished the activity completely. The activity was also reduced by the addition of UDP, UTP or galactose. In this study we present the identification, expression and biochemical characterization of the first molluscan UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I, GnT-I, from the Pacific oyster Crassostrea gigas.Graphical abstractIllustration of GnT-I activity. (a) Transfer of GlcNAc to Man5-PA, creating Man5GlcNAc3-PA. (b) Transfer of GlcNAc to MM-PA, creating MGn-PA. (c) Transfer of GlcNAc to GnM-PA, creating GnGn-PA. Blue squares represent N-acetylglucosamine, green cycles depict mannose. Graphic illustration of N-glycans were created using bioRENDER.

Exploring immune evasion of SARS-CoV-2 variants using a pseudotyped system

In the United States, coronavirus disease 2019 (COVID-19) cases have consistently been linked to the prevailing variant XBB.1.5 of SARS-CoV-2 since late 2022. A system has been developed for producing and infecting cells with a pseudovirus (PsV) of SARS-CoV-2 to investigate the infection in a Biosafety Level 2 (BSL-2) laboratory. This system utilizes a lentiviral vector carrying ZsGreen1 and Firefly luciferase (Fluc) dual reporter genes, facilitating the analysis of experimental results. In addition, we have created a panel of PsV variants that depict both previous and presently circulating mutations found in circulating SARS-CoV-2 strains. A series of PsVs includes the prototype SARS-CoV-2, Delta B.1.617.2, BA.5, XBB.1, and XBB.1.5. To facilitate the study of infections caused by different variants of SARS-CoV-2 PsV, we have developed a HEK-293T cell line expressing mCherry and human angiotensin converting enzyme 2 (ACE2). To validate whether different SARS-CoV-2 PsV variants can be used for neutralization assays, we employed serum from rats immunized with the PF-D-Trimer protein vaccine to investigate its inhibitory effect on the infectivity of various SARS-CoV-2 PsV variants. According to our observations, the XBB variant, particularly XBB.1.5, exhibits stronger immune evasion capabilities than the prototype SARS-CoV-2, Delta B.1.617.2, and BA.5 PsV variants. Hence, utilizing the neutralization test, this study has the capability to forecast the effectiveness in preventing future SARS-CoV-2 variants infections.

Investigating the effect of polyphenols from nuts on human carbohydrate digestion in vitro

Recent studies have documented the importance of postprandial hyperglycaemia in the incidence of chronic diseases, including type 2 diabetes. Inhibition of digestive enzymes, including membrane-bound brush-border α-glucosidases, leads to slowed carbohydrate digestion and absorption, and reduced postprandial glycemia. Nuts are widely eaten around the world and have the potential to inhibit α-glucosidases through their content of polyphenols and other bioactive compounds. According to our recent systematic review(1), no study has investigated the inhibitory effects of nut extracts on human α-glucosidase activities. Almost all studies in this area have been conducted on yeast α-glucosidase, with only a few using rat α-glucosidase. While there is no sequence homology between yeast and human α-glucosidase, there is 74% to 78% sequence homology between rat and human α-glucosidases(1). The lack of studies on the effect of bioactive compounds from nuts on human α-glucosidases, along with the growing attention to nuts as an important component of a healthy diet with the potential to reduce the risk of chronic diseases(2), highlights the need for research to evaluate the inhibitory effect of nut extracts on human α-glucosidases. The aim of the current study is to explore the inhibitory effect of extracts from nuts on human carbohydrate digestive enzymes. Walnuts and almonds were ground and defatted with hexane, extracted in 80% (v/v) acetone, and further purified using solid-phase extraction to obtain phenolic-rich extracts. The Folin–Ciocalteu assay was used to approximate the polyphenol content of the samples. Following our recently published detailed protocol(3), cell-free extracts from human intestinal Caco-2/TC7 cells were used as a source of α-glucosidase in enzyme inhibition assays, with sucrose, maltose and isomaltose as substrates and appropriate controls. The assay products were quantified using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Glucose production in the presence of various concentrations of phenol-rich nut extracts was compared using a one-way ANOVA and half-maximal inhibitory concentration (IC50) values were calculated. The Folin–Ciocalteu data demonstrate that walnut extracts comprise a relatively high polyphenol content, with 18.1 ± 0.23 mg (epigallocatechin gallate [EGCG] equivalent) per gram of fresh weight, while almond extracts contain 0.87 ± 0.03 mg EGCG equivalent/g of fresh weight. The walnut phenolic-rich extract dose-dependently inhibited human intestinal sucrase and maltase activities (both p&lt;0.01), with IC50 values of 1.67 mg/mL and 2.84 mg/mL, respectively. We demonstrate that phenolic-rich walnut extracts can inhibit human α-glucosidases in vitro and therefore walnuts may contribute to slowing carbohydrate digestion in humans. As such, we plan to assess the effects of walnuts on postprandial glycaemia in vivo.

Design and engineering of logic genetic-enzymatic gates based on the activity of the human CYP2C9 enzyme in permeabilized Saccharomyces cerevisiae cells

Gene circuits allow cells to carry out complex functions such as the precise regulation of biological metabolic processes. In this study, we combined, in the yeast S. cerevisiae, genetic regulatory elements with the enzymatic reactions of the human CYP2C9 and its redox partner CPR on luciferin substrates and diclofenac. S. cerevisiae cells were permeabilized and used as enzyme bags in order to host these metabolic reactions. We engineered three different (genetic)-enzymatic basic Boolean gates (YES, NOT, and N-IMPLY). In the YES and N-IMPLY gates, human CYP2C9 was expressed under the galactose-inducible GAL1 promoter. The carbon monoxide releasing molecule CORM-401 was used as an input in the NOT and N-IMPLY gates to impair CYP2C9 activity through inhibition of the Fe+2- heme prosthetic group in the active site of the human enzyme. Our study provides a new approach in designing synthetic bio-circuits and optimizing experimental conditions to favor the heterologous expression of human drug metabolic enzymes over their endogenous counterparts. This new approach will help study precise metabolic attributes of human P450s.

Safety and pharmacokinetics of antifungal agent VT-1598 and its primary metabolite, VT-11134, in healthy adult subjects: phase 1, first-in-human, randomized, double-blind, placebo-controlled study of single-ascending oral doses of VT-1598.

VT-1598 is a novel fungal CYP51 inhibitor and 1-tetrazole-based antifungal drug candidate with improved selectivity minimizing off-target binding to and inhibition of human CYP450 enzymes. Data are presented from this first clinical study in the evaluation of the safety and pharmacokinetic (PK) of single ascending doses of 40, 80, 160, 320, and 640mg VT-1598, comprising a 160mg cohort in both fasting and fed states. Eight healthy adults per dose were randomized to receive either oral VT-1598 or placebo (3:1). Over the dose range, exposures were with relatively high variation. The maximum plasma concentrations (Cmax) for VT-1598 were 31.00-279.4ng/ml and for its primary metabolite, VT-11134, were 27.80-108.8ng/ml. The plasma area under the concentration-time curve to the last measurable concentration (AUC0-last) for VT-1598 were 116.1-4507 ng*h/ml, and for VT-11134 were 1140-7156 ng*h/ml. The dose proportionality was inconclusive based on the results of the power model. The peak concentration time (Tmax) was 4-5 h for VT-1598 and for VT-11134. Half-life was 103-126 h for VT-11134. After food intake, Cmax of VT-1598 increased by 44% (geometric mean ratio (GMR), 1.44; 90%CI [0.691, 2.19]) and AUC0-last by 126% (GMR, 2.26; 90%CI [1.09, 3.44]), while exposure of VT-11134 was decreased 23% for Cmax (GMR, 0.77; 90%CI [0.239, 1.31]) and unchanged for AUC0-last (GMR, 1.02; 90%CI [0.701, 1.33]). Neither VT-1598 nor VT-11134 were detected in urine. No serious adverse events (AEs) or AEs leading to early termination were observed. The safety and PK profiles of VT-1598 support its further clinical development.

Protein Interaction Map of APOBEC3 Enzyme Family Reveals Deamination-Independent Role in Cellular Function

Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination-independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence are not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and mapped a diverse set of protein–protein and protein–RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein-folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein–protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology. Data are available via ProteomeXchange with the identifier PXD044275.

Human surfactant protein A inhibits SARS-CoV-2 infectivity and alleviates lung injury in a mouse infection model.

SARS coronavirus 2 (SARS-CoV-2) infects human angiotensin-converting enzyme 2 (hACE2)-expressing lung epithelial cells through its spike (S) protein. The S protein is highly glycosylated and could be a target for lectins. Surfactant protein A (SP-A) is a collagen-containing C-type lectin, expressed by mucosal epithelial cells and mediates its antiviral activities by binding to viral glycoproteins. This study examined the mechanistic role of human SP-A in SARS-CoV-2 infectivity and lung injury in vitro and in vivo. Human SP-A can bind both SARS-CoV-2 S protein and hACE2 in a dose-dependent manner (p<0.01). Pre-incubation of SARS-CoV-2 (Delta) with human SP-A inhibited virus binding and entry and reduced viral load in human lung epithelial cells, evidenced by the dose-dependent decrease in viral RNA, nucleocapsid protein (NP), and titer (p<0.01). We observed significant weight loss, increased viral burden, and mortality rate, and more severe lung injury in SARS-CoV-2 infected hACE2/SP-A KO mice (SP-A deficient mice with hACE2 transgene) compared to infected hACE2/mSP-A (K18) and hACE2/hSP-A1 (6A2) mice (with both hACE2 and human SP-A1 transgenes) 6 Days Post-infection (DPI). Furthermore, increased SP-A level was observed in the saliva of COVID-19 patients compared to healthy controls (p<0.05), but severe COVID-19 patients had relatively lower SP-A levels than moderate COVID-19 patients (p<0.05). Collectively, human SP-A attenuates SARS-CoV-2-induced acute lung injury (ALI) by directly binding to the S protein and hACE2, and inhibiting its infectivity; and SP-A level in the saliva of COVID-19 patients might serve as a biomarker for COVID-19 severity.

Mechanistic insight into biotransformation of novel triazine-based flame retardant 1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazinane-2,4,6-trione by human cytochrome P450s

The escalating focus on the environmental occurrence and toxicology of emerging pollutants underscores the imperative need for a profound exploration of their metabolic transformations mediated by human CYP450 enzymes. Such investigations have the potential to unravel the intricate metabolite profiles, substantially altering the toxicological outcomes. In this study, we integrated the computational simulations with in vitro metabolism experiments to investigate the metabolic activity and mechanism of an emerging pollutant, 1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazinane-2,4,6-trione (TDBP-TAZTO), catalyzed by human CYP450s. The results highlight the important contributions of CYP2E1, 3A4 and 2C9 to the biotransformation of TDBP-TAZTO, leading to the identification of four distinct metabolites. The effective binding conformations governing biotransformation reactions of TDBP-TAZTO within active CYP450s are unveiled. Structural instability of primary hydroxyTDBP-TAZTO products suggests three potential outcomes: (1) generation of an alcohol metabolite through successive debromination and reduction reactions, (2) formation of a dihydroxylated metabolite through secondary hydroxylation by CYP450, and (3) production of an N-dealkylated metabolite via decomposition and isomerization reactions in the aqueous environment. The formation of a desaturated debrominated metabolite may arise from H-abstraction and barrier-free Br release during the primary oxidation, potentially competing with the generation of hydroxyTDBP-TAZTO. These findings provide detailed mechanistic insight into TDBP-TAZTO biotransformation by CYP450s, which can enrich our understanding of the metabolic fate and associated health risk of this chemical.