Enzyme Assay Research Articles

Characterization and synergistic activity of heterologously expressed microbial-derived endoglucanase and bifunctional cellulase on wheat straw

Cellulases are divided into endoglucanase, exoglucanase, and β-glucosidase based on their catalytic activity. Eight cellulases were recombinantly expressed in Escherichia coli BL21 to investigate their effects on the enzymatic hydrolysis of wheat straw. Among them, cellulase 2006 exhibited the highest endoglucanase activity (432.25 U/mg), while Bf1 displayed superior exoglucanase and β-glucosidase activities (577.46 and 1991.63 U/mg respectively). Bioinformatic and enzymatic analyses revealed that both cellulases displayed notable thermal and pH stability. The enzyme kinetics parameters revealed that Km values for cellulases 2006 and Bf1 were 15.98 and 14.19 mg/mL, respectively, with Vmax values of 20.78 and 16.38 μmol/min/mg. In a prokaryotic co-expression system, the mixed cellulase Bf2006 exhibited endoglucanase, exoglucanase, and β-glucosidase activities (130.78, 1406.36, and 1119.25 U/mg, respectively). The enzymatic hydrolysis assay revealed that these three cellulases acted on the cellulose macromolecules in wheat straw, increasing reducing sugar content and decreasing crystallinity. Endoglucanase 2006 acted on various organic compounds rich in phenols and aromatic heterocycles, while Bf1 primarily acted on compounds containing glucose units. Bf2006 significantly affected the content of lignin, neutral detergent fiber, acid detergent fiber, and the microstructure of wheat straw, with degradation products primarily consisting of disaccharides, oligosaccharides, polysaccharides, glycosides, and other carbohydrates. This study provides theoretical guidance for the production and application of mixed cellulase Bf2006, serving as a reference for the industrial use of different cellulase types.

Exploring the potential use of Caenorhabditis elegans as an animal model for evaluating chemical-induced intestinal dysfunction

Evaluating intestinal toxicity is crucial for identifying and preventing the harmful effects of environmental chemicals. Owing to the limitations of existing models in evaluating intestinal toxicity, the development of alternative models is urgently needed. This study explored the potential use of the nematode Caenorhabditis elegans as a model animal for assessing chemical-induced intestinal dysfunction. Changes in intestinal permeability and nutrient absorption in C. elegans individuals exposed to four intestine-disrupting chemicals (sodium dodecyl sulfate (SDS), dextran sulfate sodium (DSS), lipopolysaccharide (LPS) and ethanol) were examined using dye stain assays, an enzymatic photometric assay, and fluorescent probe uptake assays. Additionally, epigallocatechin-3-gallate (EGCG), an intestine-protecting phytochemical, was chosen to prevent ethanol-induced intestinal damage. The results indicated that SDS, DSS, LPS, and ethanol compromised the intestinal barrier in C. elegans. SDS had no effect on glucose absorption, but LPS, DSS, and ethanol inhibited or tended to inhibit glucose absorption. SDS, DSS, LPS, and ethanol reduced fatty acid absorption. LPS increased peptide absorption at a low dose but decreased it at a high dose; SDS, DSS, and ethanol attenuated peptide absorption. EGCG protected against the disruption of the intestinal barrier that was induced by ethanol treatment. These results suggest that C. elegans is a suitable surrogate model animal for evaluating chemical-induced intestinal dysfunction. These findings also provide new insights into the effects of SDS, DSS, LPS, and ethanol on intestinal function and highlight the potential of EGCG as a natural dietary intervention to protect individuals who use excess alcohol from intestinal injury.

Transformative nanobioplasmonic effects: Toxicological implications of plasmonic silver nanoparticles in aquatic biological models

Silver nanoparticles (AgNPs) present unique properties, such as the induced localized surface plasmon resonance (LSPR) provoked under illumination with a proper wavelength, allowing these nanomaterials to be applied in fields such as catalysis and biomedicine. The study of AgNPs is also highly relevant from the environmental pollution viewpoint due to their high production and application in commercial products. Consequently, AgNPs reach aquatic environments and can be plasmonically stimulated under natural light conditions. This study investigates the toxic effects promoted by AgNPs under plasmonic excitation on the survival and physiology of the crustacean Daphnia similis. Two AgNP shapes (spherical and triangular) with plasmon bands absorbing in different spectral regions in the visible range were studied. The organisms were exposed to different AgNP concentrations under five different light conditions. Survival and changes in enzymatic biomarkers of oxidative stress and lipid storage were evaluated. Under LSPR conditions, we observed increased lethality for both AgNP shapes. LSPR effects of AgNPs showed mortality 2.6 and 1.7 times higher than the treatment under dark conditions for spherical and triangular morphologies respectively. The enzymatic assays demonstrated that plasmonic treatments triggered physiological responses. Significantly decreased activities were observed exclusively under LSPR conditions for both AgNP shapes. Considering all treatments, spherical AgNPs showed lower LC50 values than triangular ones, indicating their higher toxic potential. Our results demonstrate that LSPR AgNPs can induce biological responses associated with oxidative stress and survival. Therefore, this study highlights the potential risks of environmental contamination by plasmonically active metallic nanomaterials. These materials can enhance their toxicity when light-excited, yet the results also indicate promising opportunities for light-based therapies.

Omeprazole and its analogs exhibit insecticidal potencies as inhibitors of insect choline acetyltransferase

Choline acetyltransferase (ChAT) is crucial for acetylcholine synthesis and regulates diverse functions in numerous biological processes. Omeprazole, an inhibitor on human ChAT, was evaluated here on insect ChAT as a potential inhibitor, as well as its insecticidal potency on Nilaparvata lugens, a major insect pest on rice. The evaluation also included omeprazole analogs and α-NETA, in order to explore a superior leading compound targeting on insect ChAT. In toxicity test, α-NETA and omeprazole exhibited insecticidal activity, among which omeprazole exhibited activity with a mortality of around 50 % on N. lugens nymphs at 0.4 mg/mL. In vitro crude enzyme assays showed that omeprazole acted as an inhibitor on insect ChAT with a high selectivity and exciting potency compared with α-NETA and control. Three residues (Tyr84, Val95, Tyr589) was critical in N. lugens ChAT for interacting with its substrate choline through molecular docking, and it also revealed that omeprazole exhibited a higher binding affinity toward ChAT catalytic tunnel compared with α-NETA. Based on this, we screened omeprazole analogs for their affinity to N. lugens ChAT, and two compounds stood out. The 5-hydroxy omeprazole had the highest binding affinity by prediction, and 5-O-desmethyl omeprazole was with the lowest binding affinity. The toxicity bioassay and enzyme activity test were then performed on these two compounds. Aligned with the docking results, 5-hydroxy omeprazole showed a strong inhibitory effect and insecticidal activity. In summary, omeprazole and 5-hydroxy omeprazole could serve as lead compounds for insecticides targeting on insect ChAT, a novel target.

Chaga Mushroom Triterpenoids Inhibit Dihydrofolate Reductase and Act Synergistically with Conventional Therapies in Breast Cancer

Inonotus obliquus (Chaga) is a medicinal mushroom with several pharmacological properties that is used as a tea in traditional Chinese medicine. In this study, Chaga water extract was digested in vitro to mimic the natural processing and absorption of its biocomponents when it is consumed as functional beverage, and its anticancer activities were evaluated in breast cancer (BC) cell lines, representing HER2-positive and triple-negative subtypes. After chemical characterization by liquid chromatography/mass spectrometry (HR-QTOF) analysis, the effect of Chaga biocomponents on cell viability and cell cycle progression was assessed by MTT assay, FACS analysis, and Western blot. Dihydrofolate reductase (DHFR) activity was measured by an enzymatic assay. Four highly bioactive triterpenoids (inotodiol, trametenolic acid, 3-hydroxy-lanosta-8,24-dien-21-al, and betulin) were identified as the main components, able to decrease BC cell viability and block the cell cycle in G0/G1 by inducing the downregulation of cyclin D1, CDK4, cyclin E, and phosphorylated retinoblastoma protein. DHFR was identified as their crucial target. Moreover, bioactive Chaga components exerted a synergistic action with cisplatin and with trastuzumab in SK-BR-3 cells by inhibiting both HER2 and HER1 activation and displayed an immunomodulatory effect. Thus, Inonotus obliquus represents a source of triterpenoids that are effective against aggressive BC subtypes and display properties of targeted drugs.

A defensive pathway from NAC and TCP transcription factors activates a BAHD acyltransferase for (Z)-3-hexenyl acetate biosynthesis to resist herbivore in tea plant (Camellia sinensis).

Numerous herbivore-induced plant volatiles (HIPVs) play important roles in plant defense. In tea plants (Camellia sinensis), (Z)-3-hexenyl acetate (3-HAC) has been characterized as associated with resistance to herbivores. To date, how tea plants biosynthesize and regulate 3-HAC to resist herbivores remain unclear. Based on transcriptomes assembled from Ectropis obliqua-fed leaves, a cDNA encoding BAHD acyltransferase, namely CsCHAT1, was highly induced in leaves fed with E. obliqua. Enzymatic assays showed that CsCHAT1 converted (Z)-3-hexenol into 3-HAC. Further suppression of CsCHAT1 expression reduced the accumulation of 3-HAC and lowered the resistance of tea plants to E. obliqua, while 3-HAC replenishment rescued the reduced resistance of CsCHAT1-silenced tea plants against E. obliqua. Two transcription factors (TFs), CsNAC30 and CsTCP11, were co-expressed with CsCHAT1. An integrative approach of biochemistry, DNA-protein interaction, gene silencing, and metabolic profiling revealed that the two TFs positively regulated the expression of CsCHAT1. The suppression of either one decreased the production of 3-HAC and eliminated the resistance of tea plants to E. obliqua. Notably, the suppression of either one considerably impaired JA-induced 3-HAC biosynthesis in tea plant. The proposed pathway can be targeted for innovative agro-biotechnologies protecting tea plants from damage by E. obliqua.

Simultaneous and Rapid Detection of Glucose and Insulin: Coupling Enzymatic and Aptamer-Based Assays.

Diabetes management demands precise monitoring of key biomarkers, particularly insulin (I) and glucose (G). Herein, we present a bioelectronic chip device that enables the simultaneous detection of I and G in biofluids within 2 min. This dual biosensor chip integrates aptamer-based insulin sensing with enzymatic glucose detection on a single platform, employing a four-electrode sensor chip. The insulin voltammetric sensor employs a G-quadraplex methylene-blue-modified aptamer, while the amperometric biocatalytic glucose sensor utilizes a second-generation mediator-based approach. Simultaneous reagent-less sensing of I and G has been achieved by addressing key challenges. These include combining different surface chemistries, assay formats, and detection principles at closely spaced working electrodes and the substantially different concentration levels of the I and G targets. An attractive analytical performance, with no apparent crosstalk, is demonstrated for the simultaneous detection of millimolar G concentrations and picomolar I concentrations in single microliter serum or saliva sample droplets. This dual biosensor offers rapid, cost-effective, and reliable monitoring, addressing the unmet need for integrated multiplexed diabetes biomarker detection in decentralized settings. Such integration of enzymatic and aptamer-based bioassays could greatly expand the scope of decentralized testing in healthcare beyond diabetes care.

Development of an Antiviral Medicinal Plant and Natural Product Database (avMpNp Database) from Biodiversity.

The construction of compound databases (DB) is a strategy for the rational search of bioactive compounds and drugs for new and old diseases. In order to bring greater impact to drug discovery, we propose the development of a DB of bioactive antiviral compounds. Several research groups have presented evidence of the antiviral activity of medicinal plants and compounds isolated from these plants. We believe that compiling these discoveries in a DB would benefit the scientific research community and increase the speed to discover new potential drugs and medicines. Thus, we present the Antiviral Medicinal Plant and Natural Product DB (avMpNp DB) as an important source for acquiring, organizing, and distributing knowledge related to natural products and antiviral drug discovery. The avMpNp DB contains a series of chemically diverse compounds with drug-like profiles. To test the potential of this DB, SARS-CoV-2 Mpro and PLpro enzymatic inhibition assays were performed for available compounds resulting in IC50 values ranging from 6.308±0.296 to 15.795±0.155 μM. As a perspective, artificial intelligence tools will be added to implement computational predictions, as well as other chemical functionalities that allow data validation.

Small Molecule Inhibitors of Mycobacterium tuberculosis Topoisomerase I Identified by Machine Learning and In Vitro Assays

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a leading infectious cause of death globally. The treatment of patients becomes much more difficult for the increasingly common multi-drug resistant TB. Topoisomerase I is essential for the viability of M. tuberculosis and has been validated as a new target for the discovery of novel treatment against TB resistant to the currently available drugs. Virtual high-throughput screening based on machine learning was used in this study to identify small molecules that target the binding site of divalent ion near the catalytic tyrosine of M. tuberculosis topoisomerase I. From the virtual screening of more than 2 million commercially available compounds, 96 compounds were selected for testing in topoisomerase I relaxation activity assay. The top hit that has IC50 of 7 µM was further investigated. Commercially available analogs of the top hit were purchased and tested with the in vitro enzyme assay to gain further insights into the molecular scaffold required for topoisomerase inhibition. Results from this project demonstrated that novel small molecule inhibitors of bacterial topoisomerase I can be identified starting with the machine-learning-based virtual screening approach.

Benzocarbazoledinones as SARS-CoV-2 Replication Inhibitors: Synthesis, Cell-Based Studies, Enzyme Inhibition, Molecular Modeling, and Pharmacokinetics Insights

Endemic and pandemic viruses represent significant public health challenges, leading to substantial morbidity and mortality over time. The COVID-19 pandemic has underscored the urgent need for the development and discovery of new, potent antiviral agents. In this study, we present the synthesis and anti-SARS-CoV-2 activity of a series of benzocarbazoledinones, assessed using cell-based screening assays. Our results indicate that four compounds (4a, 4b, 4d, and 4i) exhibit EC50 values below 4 μM without cytotoxic effects in Calu-3 cells. Mechanistic investigations focused on the inhibition of the SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) have used enzymatic assays. Notably, compounds 4a and 4b showed Mpro inhibition activity with IC50 values of 0.11 ± 0.05 and 0.37 ± 0.05 µM, respectively. Furthermore, in silico molecular docking, physicochemical, and pharmacokinetic studies were conducted to validate the mechanism and assess bioavailability. Compound 4a was selected for preliminary drug-likeness analysis and in vivo pharmacokinetics investigations, which yielded promising results and corroborated the in vitro and in silico findings, reinforcing its potential as an anti-SARS-CoV-2 lead compound.

Effects of a novel HDAC6-selective inhibitor's radiosensitization on cancer cells.

The radiation sensitivity of tumor cells is a critical determinant of their therapeutic response to radiotherapy. Histone deacetylase 6 (HDAC6), beyond its known role in modulating tubulin acetylation and influencing cell motility, is also involved in the DNA damage response, potentially enhancing tumor cell radiosensitivity. Targeted HDAC6 inhibitors have shown substantial promise in preclinical studies aimed at increasing radiosensitivity and inhibiting cellular migration. A new HDAC inhibitor, named OXHA, was designed by substituting the phenyl cap of SAHA with an N,5-diphenyloxazole-2-carboxamide group. The inhibitory activity of OXHA was evaluated via in vitro enzymatic assays. Its effects on tumor cell migration and radiosensitization potential were assessed using scratch wound healing assays, micronucleus formation, and clonogenic survival assays. Enzymatic assays confirmed OXHA's selective inhibition of HDAC6. Compared to SAHA, OXHA significantly increased α-tubulin acetylation while minimally impacting histone H3 acetylation, indicating a high selectivity for HDAC6. In combination with X-ray irradiation, OXHA markedly impaired wound healing in A549 and HepG2 cells, enhanced micronucleus formation, and reduced clonogenic survival across multiple tumor lines. OXHA exhibits potent and selective HDAC6 inhibition, effectively impeding tumor cell migration and enhancing radiosensitivity across multiple cell lines. These findings suggest that OXHA has strong potential as a therapeutic strategy to improve radiotherapy efficacy.

Chemical Heating for Minimally Instrumented Point-of-Care (POC) Molecular Diagnostics

The minimal instrumentation of portable medical diagnostic devices for point-of-care applications is facilitated by using chemical heating in place of temperature-regulated electrical heaters. The main applications are for isothermal nucleic acid amplification tests (NAATs) and other enzymatic assays that require elevated, controlled temperatures. In the most common implementation, heat is generated by the exothermic reaction of a metal (e.g., magnesium, calcium, or lithium) with water or air, buffered by a phase-change material that maintains a near-constant temperature to heat the assay reactions. The ability to incubate NAATs electricity-free and to further to detect amplification with minimal instrumentation opens the door for fully disposable, inexpensive molecular diagnostic devices that can be used for pathogen detection as needed in resource-limited areas and during natural disasters, wars, and civil disturbances when access to electricity may be interrupted. Several design approaches are reviewed, including more elaborate schemes for multiple stages of incubation at different temperatures.

Molecular Interactions of the Plant Steroid Hormone Epibrassinolide on Human Drug-Sensitive and Drug-Resistant Small-Cell Lung Carcinoma Cells

Background: Small-cell lung cancer (SCLC) has a poor prognosis because it is often diagnosed after it has spread and develops multi-drug resistance. Epibrassinolide (EB) is a plant steroid hormone with widespread distribution and physiological effects. In plants, EB-activated gene expression occurs via a GSK-mediated signaling pathway, similar to Wnt-β-catenin signaling in animal cells that is elevated in cancer cells. Methods: This mechanistic parallel prompted investigations of the molecular interactions of EB on drug-sensitive (H69) and multi-drug-resistant (VPA) SCLC cells. Cellular and molecular investigations were performed. Results: Pharmacologic interactions between EB and the Wnt signaling inhibitors IGC-011 and PRI-724 were determined by the combination index method and showed antagonism, indicating that EB acts on the same pathway as these inhibitors. Following incubation of drug-sensitive and drug-resistant SCLC cells with EB, there was a reduction in β-catenin (e.g., 3.8 to 0.7 pg/µg protein), accompanied by a reduction in β-catenin promoter activity, measured by firefly luciferase-coupled promoter element transfection. Cellular β-catenin concentration is regulated by the active form of GSK3β. In Wnt signaling, active GSK3β is converted to inactive pGSK3β, thereby increasing the concentration of β-catenin. After incubation of SCLC cells with EB, there was a reduction in the inactive form (pGSK3β) and a relative increase in the active form (GSK3β). In vitro enzyme assays showed that EB did not inhibit purified GSK3β, but there was non-competitive inhibition when SCLC cell extracts were used as the source of enzyme. This indirect inhibition by EB indicates that it may act on the Wnt pathway by blocking the phosphorylation of GSK3β. The protein levels of three SCLC tumor markers, namely, NSE, CAV1, and MYCL1, were elevated in drug-resistant SCLC cells. EB incubation led to a significant reduction in the levels of the three markers. Two major effects of EB on SCLC cells are the promotion of apoptosis and the reversal of drug resistance. Transcriptional analyses showed that after exposure of SCLC cells to EB, there were increases in the expression of genes encoding apoptotic inducers (e.g., BAX and FAS) and effectors (e.g., CASP3) and reductions in the expression of genes encoding apoptosis inhibitors (e.g., survivin). PGP1 and MRP1, two membrane efflux pumps expressed in SCLC cells, were elevated in drug-resistant cells, but EB incubation did not affect these protein levels. Cellular assays of drug efflux by PGP1 showed an increase in drug-resistant cells, but EB did not alter efflux activity. Following exposure to human liver microsomes, EB was metabolized by NADPH-dependent oxidation and UDPG-dependent glucuronidation, as evidenced by the elimination of EB cytotoxicity against SCLC cells. Conclusions: Taken together, these data indicate that EB, a steroid hormone in plants consumed in the human diet, is pharmacologically active in drug-sensitive and drug-resistant SCLC cells in the Wnt signaling pathway, alters apoptotic gene expression, and is a substrate for microsomal modifications.

Ultradeep O-GlcNAc proteomics reveals widespread O-GlcNAcylation on tyrosine residues of proteins

As a unique type of glycosylation, O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) on Ser/Thr residues of proteins was discovered 40 y ago. O-GlcNAcylation is catalyzed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation is an essential glycosylation that regulates the functions of many proteins in virtually all cellular processes. However, deep and site-specific characterization of O-GlcNAcylated proteins remains a challenge. We developed an ultradeep O-GlcNAc proteomics workflow by integrating digestion with multiple proteases, two mass spectrometric approaches (i.e., electron-transfer/higher-energy collision dissociation [EThcD] and HCD product-dependent electron-transfer/higher-energy collision dissociation [HCD-pd-EThcD]), and two data analysis tools (i.e., MaxQuant and Proteome Discoverer). The performance of this strategy was benchmarked by the analysis of whole lysates from PANC-1 (a pancreatic cancer cell line). In total, 2,831 O-GlcNAc sites were unambiguously identified, representing the largest O-GlcNAc dataset of an individual study reported so far. Unexpectedly, in addition to confirming known sites and identifying many other sites of Ser/Thr modification, O-GlcNAcylation was found on 121 tyrosine (Tyr) residues of 93 proteins. In vitro enzymatic assays showed that OGT catalyzes the transfer of O-GlcNAc onto Tyr residues of peptides and OGA catalyzes its removal. Taken together, our work reveals widespread O-GlcNAcylation on Tyr residues of proteins and that Tyr O-GlcNAcylation is mediated by OGT and OGA. As another form of glycosylation, Tyr O-GlcNAcylation is likely to have important regulatory roles.

Functional identification of soluble uric acid as an endogenous inhibitor of CD38

Excessive elevation or reduction of soluble uric acid (sUA) levels has been linked to some of pathological states, raising another subject that sUA at physiological levels may be essential for the maintenance of health. Yet, the fundamental physiological functions and molecular targets of sUA remain largely unknown. Using enzyme assays and in vitro and in vivo metabolic assays, we demonstrate that sUA directly inhibits the hydrolase and cyclase activities of CD38 via a reversible non-competitive mechanism, thereby limiting nicotinamide adenine dinucleotide (NAD+) degradation. CD38 inhibition is restricted to sUA in purine metabolism, and a structural comparison using methyl analogs of sUA such as caffeine metabolites shows that 1,3-dihydroimidazol-2-one is the main functional group. Moreover, sUA at physiological levels prevents crude lipopolysaccharide (cLPS)-induced systemic inflammation and monosodium urate (MSU) crystal-induced peritonitis in mice by interacting with CD38. Together, this study unveils an unexpected physiological role for sUA in controlling NAD+ availability and innate immunity through CD38 inhibition, providing a new perspective on sUA homeostasis and purine metabolism.

Functional identification of soluble uric acid as an endogenous inhibitor of CD38.

Excessive elevation or reduction of soluble uric acid (sUA) levels has been linked to some of pathological states, raising another subject that sUA at physiological levels may be essential for the maintenance of health. Yet, the fundamental physiological functions and molecular targets of sUA remain largely unknown. Using enzyme assays and in vitro and in vivo metabolic assays, we demonstrate that sUA directly inhibits the hydrolase and cyclase activities of CD38 via a reversible non-competitive mechanism, thereby limiting nicotinamide adenine dinucleotide (NAD+) degradation. CD38 inhibition is restricted to sUA in purine metabolism, and a structural comparison using methyl analogs of sUA such as caffeine metabolites shows that 1,3-dihydroimidazol-2-one is the main functional group. Moreover, sUA at physiological levels prevents crude lipopolysaccharide (cLPS)-induced systemic inflammation and monosodium urate (MSU) crystal-induced peritonitis in mice by interacting with CD38. Together, this study unveils an unexpected physiological role for sUA in controlling NAD+ availability and innate immunity through CD38 inhibition, providing a new perspective on sUA homeostasis and purine metabolism.

The Kinetics of Carbon-Carbon-Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity.

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the b-ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon-carbon-bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here, we report enzyme kinetics of the KS-mediated carbon-carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS is likely rate-limiting to the fatty acid biosynthesis, its kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS-mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. We identify positive cooperativity of the KS dimer, which we suggest to affect the conformational variability of the multienzyme. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

The Kinetics of Carbon‐Carbon‐Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the b‐ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon‐carbon‐bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here, we report enzyme kinetics of the KS‐mediated carbon‐carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS is likely rate‐limiting to the fatty acid biosynthesis, its kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS‐mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. We identify positive cooperativity of the KS dimer, which we suggest to affect the conformational variability of the multienzyme. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

CNSC-31. INVESTIGATING THE THERAPEUTIC POTENTIAL OF STK17A INHIBITION IN GLIOBLASTOMA

Abstract For decades, the survival rate for glioblastoma (GBM) has remained nearly stagnant, with a 5-year survival rate of only 5%. There is an unmet medical need for therapeutic development, because while GBM is the most common primary brain tumor, only four FDA approved drugs have been developed for GBM over the last century. By taking advantage of RNA sequencing, a potential target for GBM has been identified: the kinase STK17A (Serine/Threonine Kinase17A). We confirmed that STK17A is overexpressed in gliomas by analyzing public databases and single-cell RNA sequencing from our own GBM patients, and further associated it with poor patient outcome. Little is known about STK17A, but it has been found to be involved in cell proliferation and tumorigenesis. The role of STK17A in GBM pathophysiology and its therapeutic potential as a drug target remains a critical knowledge gap. To better understand STK17A’s role in GBM pathophysiology, we first knocked down STK17A in GBM cell lines and determined that STK17A had functional roles in proliferation and morphology. Similar conclusions were drawn after removing STK17A in GBM xenograft mouse models. These findings support STK17A as a viable therapeutic target. We then designed and optimized novel STK17A inhibitors for evaluations through kinase enzymatic and biological assays, in vitro proliferation and toxicity assays, and in vivo Drug Metabolism and Pharmacokinetics (DMPK) assays. We then used these potent, selective, and highly brain penetrant STK17A inhibitors to investigate the anti-tumor effects of STK17A inhibition in GBM and demonstrate the efficacy of our novel STK17A inhibitors in vivo. In conclusion, this work seeks to contribute to the field of targeted therapies for GBM by optimizing and examining a novel brain penetrant STK17A inhibitor, initiating studies for the treatment of GBM in vitro and in vivo, and providing proof of efficacy for further studies in GBM patients.

Protein phosphatase-1 regulates the binding of filamin C to FILIP1 in cultured skeletal muscle cells under mechanical stress

The actin-binding protein filamin c (FLNc) is a key mediator in the response of skeletal muscle cells to mechanical stress. In addition to its function as a structural scaffold, FLNc acts as a signaling adaptor which is phosphorylated at S2234 in its mechanosensitive domain 20 (d20) through AKT. Here, we discovered a strong dephosphorylation of FLNc-pS2234 in cultured skeletal myotubes under acute mechanical stress, despite high AKT activity. We found that all three protein phosphatase 1 (PP1) isoforms are part of the FLNc d18-21 interactome. Enzymatic assays demonstrate that PP1 efficiently dephosphorylates FLNc-pS2234 and in vitro and in cells upon PP1 activation using specific modulators. FLNc-pS2234 dephosphorylation promotes the interaction with FILIP1, a mediator for filamin degradation. Altogether, we present a model in which dephosphorylation of FLNc d20 by the dominant action of PP1c prevails over AKT activity to promote the binding of the filamin degradation-inducing factor FILIP1 during acute mechanical stress.