Enzyme Kinetics Research Articles

Enzyme variants in biosynthesis and biological assessment of different molecular weight hyaluronan

In the present study, low- and high-molecular-weight hyaluronic acids (LMW-HA and HMW-HA) were synthesized in vitro by truncated Streptococcus equisimilis hyaluronan synthases (SeHAS). The enzyme kinetic parameters were determined for each enzyme variant. The MW, structure, dispersity, and biological activity of polymers were determined by electrophoresis, FTIR spectroscopy, carbazole, cell proliferation, and cell migration assay, respectively. The specific activities were calculated as 7.5, 6.8, 4.9, and 2.8 µgHA µgenzyme−1 min−1 for SeHAS, HAS123, HAS23, and HASIntra, respectively. The results revealed SeHAS produced a polydisperse HMW-HA (268 kDa), while HAS123 and HAS23 produced a polydisperse LMW-HA (< 30 kDa). Interestingly, HASIntra produced a low-disperse LMW-HA. Kinetics studies revealed the truncated variants displayed increased Km values for two substrates when compared to the wild-type enzyme. Biological assessments indicated all LMW-HAs showed a dose-dependent proliferation activity on endothelial cells (ECs), whereas HMW-HAs exhibited an inhibitory effect. Also, LMW-HAs had the highest cell migration effect at 10 µg/mL, while at 200 µg/mL, both LMW- and HMW-HAs postponed the healing recovery rate. The study elucidated that the transmembrane domains (TMDs) of SeHAS affect the enzyme kinetics, HA-titer, HA-size, and HA-dispersity. These findings open new insight into the rational engineering of SeHAS to produce size-defined HA.Graphical abstract

Structural insight into the binding mode of cefotaxime and meropenem to TEM-1, SHV-1, KPC-2, and Amp-C type beta-lactamases.

Antimicrobial resistance is an emerging threat to public health around the world. The study employs computational and biophysical methods to investigate the properties of cefotaxime and meropenem's binding to various beta-lactamases like TEM-1, SHV-1, KPC-2, and Amp-C. The enzyme kinetics of purified proteins revealed an increase in Michaelis constant (Km) value in the presence of meropenem and cefotaxime, indicating a decrease in enzyme affinity for nitrocefin. Proteins interact with meropenem/cefotaxime, causing quenching through complex formation. All proteins have one binding site, and binding constant (Kb) values are 104, indicating strong interaction. The study found that meropenem and cefotaxime had high fitness scores for Amp-C, KPC-2,TEM-1 and SHV-1, with binding energy ranging from -7.4 to -7.8, and hydrogen bonds between them. Molecular Dynamic simulation of protein-ligand complexes revealed cefotaxime-binding proteins have slightly lower Root Mean Square Deviation(RMSD) than meropenem-binding proteins, indicating stable association antibiotics with these proteins.

Size- and Shape-controlled Biodegradable Polymer Brushes Based on l-Amino Acid for Intracellular Drug Delivery and Deep-Tissue Penetration.

We report size- and shape-controlled polymer brushes based on l-amino acid bioresource and study the role of polymer topology on the enzymatic biodegradation and deep-tissue penetration under in vitro and in vivo. For this purpose, l-tyrosine-based propargyl-functionalized monomer is tailor-made and polymerized via solvent-free melt polycondensation strategy to yield hydrophobic and clickable biodegradable poly(ester-urethane)s. Postpolymerization click chemistry strategy is applied to make well-defined amphiphilic one-dimensional rodlike and three-dimensional spherical polymer brushes by merely varying the lengths of PEG-azides in the reaction. These core-shell polymer brushes are found to be nontoxic and nonhemolytic and capable of loading clinical anticancer drug doxorubicin and deep-tissue penetrable near-infrared biomarker IR-780. In vitro enzymatic drug-release kinetics and lysotracker-assisted real-time live-cell confocal bioimaging revealed that the rodlike polymer brush is superior than its spherical counterparts for faster cellular uptake and enzymatic biodegradation at the endolysosomal compartments to release DOX at the nucleus. Further, in vivo live-animal bioimaging by IVIS technique established that the IR-780-loaded rodlike polymer brush exhibited efficient deep-tissue penetration ability and emphasized the importance of polymer brush topology control for biological activity. Polymer brushes exhibit good stability in the blood plasma for more than 72 h, they predominately accumulate in the digestive organs like liver and kidney, and they are less toxic to heart and brain tissues. IVIS imaging of cryotome tissue slices of organs confirmed the deep-penetrating ability of the polymer brushes. The present investigation opens opportunity for bioderived and biodegradable polymer brushes as next-generation smart drug-delivery scaffolds.

Taking advantage of the interaction between the sulfoxide and heme cofactor to develop indoleamine 2, 3-dioxygenase 1 inhibitors

Taking advantage of key interactions between sulfoxide and heme cofactor, we used the sulfoxide as the anchor functional group to develop two series of indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitors: 2-benzylsulfinylbenzoxazoles (series 1) and 2-phenylsulfinylbenzoxazoles (series 2). In vitro enzymatic screening shows that both series can inhibit the activity of IDO1 in low micromolar (series 1) or nanomolar (series 2) levels. They also show inhibitory selectivity between IDO1 and tryptophan 2, 3-dioxygenase 2. Interestingly, although series 1 is less potent IDO1 inhibitors of these two series, it exhibited stronger inhibitory activity toward kynurenine production in interferon-γ stimulated BxPC-3 cells. Enzyme kinetics and binding studies demonstrated that 2-sulfinylbenzoxazoles are non-competitive inhibitors of tryptophan, and they interact with the ferrous form of heme. These results demonstrated 2-sulfinylbenzoxazoles as type II IDO1 inhibitors. Furthermore, molecular docking studies supports the sulfoxide being of the key functional group that interacts with the heme cofactor. Compound 22 (series 1) can inhibit NO production in a concentration dependent manner in lipopolysaccharides (LPS) stimulated RAW264.7 cells, and can relieve pulmonary edema and lung injury in LPS induced mouse acute lung injury models.

Exploring the interdependence of calcium and chloride activation of O2 evolution in photosystem II.

Calcium and chloride are activators of oxygen evolution in photosystem II (PSII), the light-absorbing water oxidase of higher plants, algae, and cyanobacteria. Calcium is an essential part of the catalytic Mn4CaO5 cluster that carries out water oxidation and chloride has two nearby binding sites, one of which is associated with a major water channel. The co-activation of oxygen evolution by the two ions is examined in higher plant PSII lacking the extrinsic PsbP and PsbQ subunits using a bisubstrate enzyme kinetics approach. Analysis of three different preparations at pH 6.3 indicates that the Michaelis constant, KM, for each ion is less than the dissociation constant, KS, and that the affinity of PSII for Ca2+ is about ten-fold greater than for Cl-, in agreement with previous studies. Results are consistent with a sequential binding model in which either ion can bind first and each promotes the activation by the second ion. At pH 5.5, similar results are found, except with a higher affinity for Cl- and lower affinity for Ca2+. Observation of the slow-decaying Tyr Z radical, YZ•, at 77 K and the coupled S2YZ• radical at 10 K, which are both associated with Ca2+ depletion, shows that Cl- is necessary for their observation. Given the order of electron and proton transfer events, this indicates that chloride is required to reach the S3 state preceding Ca2+ loss and possibly for stabilization of YZ• after it forms. Interdependence through hydrogen bonding is considered in the context of the water environment that intervenes between Cl- at the Cl-1 site and the Ca2+/Tyr Z region.

Synthesis and Evaluation of diaryl ether modulators of the leukotriene A4 hydrolase aminopeptidase activity

Activation of the aminopeptidase (AP) activity of leukotriene A4 hydrolase (LTA4H) presents a potential therapeutic strategy for resolving chronic inflammation. Previously, ARM1 and derivatives were found to activate the AP activity using the alanine-p-nitroanilide (Ala-pNA) as a reporter group in an enzyme kinetics assay. As an extension of this previous work, novel ARM1 derivatives were synthesized using a palladium-catalyzed Ullmann coupling reaction and screened using the same assay. Analogue 5, an aminopyrazole (AMP) analogue of ARM1, was found to be a potent AP activator with an AC50 of 0.12 μM. An X-ray crystal structure of LTA4H in complex with AMP was refined at 2.7 Å. Despite its AP activity with Ala-pNA substrate, AMP did not affect hydrolysis of the previously proposed natural ligand of LTA4H, Pro-Gly-Pro (PGP). This result highlights a discrepancy between the hydrolysis of more conveniently monitored chromogenic synthetic peptides typically employed in assays and endogenous peptides. The epoxide hydrolase (EH) activity of AMP was measured in vivo and the compound significantly reduced leukotriene B4 (LTB4) levels in a murine bacterial pneumonia model. However, AMP did not enhance survival in the murine pneumonia model over a 14-day period. A liver microsome stability assay showed metabolic stability of AMP. The results suggested that accelerated Ala-pNA cleavage is not sufficient for predicting therapeutic potential, even when the full mechanism of activation is known.

Genistein and metformin regulate glycerol kinase and the enzymes of glycerol 3-phosphate shuttle in a differential manner in myocytes, hepatocytes and adipocytes

Glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (GPDH) are critical in glucose homeostasis. The role of genistein and metformin on these enzymes and glucose production was investigated in C2C12, HepG2, and 3 T3-L1 cells. Enzyme kinetics, Real-Time PCR and western blots were performed to determine enzyme activities and expressions of mRNAs and proteins. Glucose production and uptake were also measured in these cells. siRNAs were used to assess their impact on the enzymes and glucose production. Ki values for the compounds were determined using purified GK and GPDH. Genistein decreased GK activity by ~45 %, while metformin reduced cGPDH and mGPDH activities by ~32 % and ~ 43 %, respectively. Insignificant changes in expressions (mRNAs and proteins) of the enzymes were observed. The compounds showed dose-dependent alterations in glucose production and uptake in these cells. Genistein non-competitively inhibited GK activity (Ki 19.12 μM), while metformin non-competitively inhibited cGPDH (Ki 75.52 μM) and mGPDH (Ki 54.70 μM) activities. siRNAs transfection showed ~50 % and ~ 35 % decrease in activities of GK and mGPDH and a decrease in glucose production (0.38-fold and 0.42-fold) in 3 T3-L1 cells. Considering the differential effects of the compounds, this study may provide insights into the potential therapeutic strategies for type II diabetes mellitus.

Optimizing the conversion of phosphoenolpyruvate to lactate by enzymatic channeling with mixed nanoparticle display.

Co-assembling enzymes with nanoparticles (NPs) into nanoclusters allows them to access channeling, a highly efficient form of multienzyme catalysis. Using pyruvate kinase (PykA) and lactate dehydrogenase (LDH) to convert phosphoenolpyruvic acid to lactic acid with semiconductor quantum dots (QDs) confirms how enzyme cluster formation dictates the rate of coupled catalytic flux (kflux) across a series of differentially sized/shaped QDs and 2D nanoplatelets (NPLs). Enzyme kinetics and coupled flux were used to demonstrate that by mixing different NP systems into clusters, a >10× improvement in kflux is observed relative to free enzymes, which is also ≥2× greater than enhancement on individual NPs. Cluster formation was characterized with gel electrophoresis and transmission electron microscopy (TEM) imaging. The generalizability of this mixed-NP approach to improving flux is confirmed by application to a seven-enzyme system. This represents a powerful approach for accessing channeling with almost any choice of enzymes constituting a multienzyme cascade.

Sequence similarity network analysis of drug- and dye-modifying azoreductase enzymes found in the human gut microbiome

Drug metabolism by human gut microbes is often exemplified by azo bond reduction in the anticolitic prodrug sulfasalazine. Azoreductase activity is often found in incubations with cell cultures or ex vivo gut microbiome samples and contributes to the xenobiotic metabolism of drugs and food additives. Applying metagenomic studies to personalized medicine requires knowledge of the genes responsible for sulfasalazine and other drug metabolism, and candidate genes and proteins for drug modifications are understudied. A representative gut-abundant azoreductase from Anaerotignum lactatifermentan DSM 14214 efficiently reduces sulfasalazine and another drug, phenazopyridine, but could not reduce all azo-bonded drugs in this class. We used enzyme kinetics to characterize this enzyme for its NADH-dependent reduction of these drugs and food additives and performed computational docking to provide the groundwork for understanding substrate specificity in this family. We performed an analysis of the Flavodoxin-like fold InterPro family (IPR003680) by computing a sequence similarity network to classify distinct subgroups of the family and then performed chemically-guided functional profiling to identify proteins that are abundant in the NIH Human Microbiome Project dataset. This strategy aims to reduce the number of unique azoreductases needed to characterize one protein family in the diverse set of potential drug- and dye-modifying activities found in the human gut microbiome.

Enzymatic degradability of diclofenac ozonation products: A mechanistic analysis

The treatment of waterborne micropollutants, such as diclofenac, presents a significant challenge to wastewater treatment plants due to their incomplete removal by conventional methods. Ozonation is an effective technique for the degradation of micropollutants. However, incomplete oxidation can lead to the formation of ecotoxic by-products that require a subsequent post-treatment step. In this study, we analyze the susceptibility of micropollutant ozonation products to enzymatic digestion with laccase from Trametes versicolor to evaluate the potential of enzymatic treatment as a post-ozonation step. The omnipresent micropollutant diclofenac is used as an example, and the enzymatic degradation kinetics of all 14 detected ozonation products are analyzed by high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) and tandem mass spectrometry (MS2). The analysis shows that most of the ozonation products are responsive to chemo-enzymatic treatment but show considerable variation in enzymatic degradation kinetics and efficiencies. Mechanistic investigation of representative transformation products reveals that the hydroxylated aromatic nature of the ozonation products matches the substrate spectrum, facilitating their rapid recognition as substrates by laccase. However, after initiation by laccase, the subsequent chemical pathway of the enzymatically formed radicals determines the global degradability observed in the enzymatic process. Substrates capable of forming stable molecular oxidation products inhibit complete detoxification by oligomerization. This emphasizes that it is not the enzymatic uptake of the substrates but the channelling of the reaction of the substrate radicals towards the oligomerization of the substrate radicals that is the key step in the further development of an enzymatic treatment step for wastewater applications.

Electrochemical Impedance Spectroscopy for the Sensing of the Kinetic Parameters of Engineered Enzymes.

The study presents a promising approach to enzymatic kinetics using Electrochemical Impedance Spectroscopy (EIS) to assess fundamental parameters of modified enteropeptidases. Traditional methods for determining these parameters, while effective, often lack versatility and convenience, especially under varying environmental conditions. The use of EIS provides a novel approach that overcomes these limitations. The enteropeptidase underwent genetic modification through the introduction of single amino acid modifications to assess their effect on enzyme kinetics. However, according to the one-sample t-test results, the difference between the engineered enzymes and hEKL was not statistically significant by conventional criteria. The kinetic parameters were analyzed using fluorescence spectroscopy and EIS, which was found to be an effective tool for the real-time measurement of enzyme kinetics. The results obtained through EIS were not significantly different from those obtained through traditional fluorescence spectroscopy methods (p value >> 0.05). The study validates the use of EIS for measuring enzyme kinetics and provides insight into the effects of specific amino acid changes on enteropeptidase function. These findings have potential applications in biotechnology and biochemical research, suggesting a new method for rapidly assessing enzymatic activity.

Screening of α-Glucosidase Inhibitors in Cichorium glandulosum Boiss. et Huet Extracts and Study of Interaction Mechanisms.

Cichorium glandulosum Boiss. et Huet (CGB) extract has an α-glucosidase inhibitory effect (IC50 = 59.34 ± 0.07 μg/mL, positive control drug acarbose IC50 = 126.1 ± 0.02 μg/mL), but the precise enzyme inhibitors implicated in this process are not known. The screening of α-glucosidase inhibitors in CGB extracts was conducted by bioaffinity ultrafiltration, and six potential inhibitors (quercetin, lactucin, 3-O-methylquercetin, hyperoside, lactucopicrin, and isochlorogenic acid B) were screened as the precise inhibitors. The binding rate calculations and evaluation of enzyme inhibitory effects showed that lactucin and lactucopicrin exhibited the greatest inhibitory activities. Next, the inhibiting effects of the active components of CGB, lactucin and lactucopicrin, on α-glucosidase and their mechanisms were investigated through α-glucosidase activity assay, enzyme kinetics, multispectral analysis, and molecular docking simulation. The findings demonstrated that lactucin (IC50 = 52.76 ± 0.21 μM) and lactucopicrin (IC50 = 17.71 ± 0.64 μM) exhibited more inhibitory effects on α-glucosidase in comparison to acarbose (positive drug, IC50 = 195.2 ± 0.30 μM). Enzyme kinetic research revealed that lactucin inhibits α-glucosidase through a noncompetitive inhibition mechanism, while lactucopicrin inhibits it through a competitive inhibition mechanism. The fluorescence results suggested that lactucin and lactucopicrin effectively reduce the fluorescence of α-glucosidase by creating lactucin-α-glucosidase and lactucopicrin-α-glucosidase complexes through static quenching. Furthermore, the circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR) analyses revealed that the interaction between lactucin or lactucopicrin and α-glucosidase resulted in a modification of the α-glucosidase's conformation. The findings from molecular docking and molecular dynamics simulations offer further confirmation that lactucopicrin has a robust binding affinity for certain residues located within the active cavity of α-glucosidase. Furthermore, it has a greater affinity for α-glucosidase compared to lactucin. The results validate the suppressive impact of lactucin and lactucopicrin on α-glucosidase and elucidate their underlying processes. Additionally, they serve as a foundation for the structural alteration of sesquiterpene derived from CGB, with the intention of using it for the management of diabetic mellitus.

A flexible self-supported electrochemical sensor Co-NC/PS@CC for real-time detection of cell-released H2O2

BackgroundHydrogen peroxide (H2O2) is an important reactive oxygen species (ROS) molecule involved in cell metabolism regulation, transcriptional regulation, and cytoskeleton remodeling. Real-time monitoring of H2O2 levels in live cells is of great significance for disease prevention and diagnosis. ResultsWe utilized carbon cloth (CC) as the substrate material and employed a single-atom catalysis strategy to prepare a flexible self-supported sensing platform for the real-time detection of H2O2 secreted by live cells. By adjusting the coordination structure of single-atom sites through P and S doping, a cobalt single-atom nanoenzyme Co-NC/PS with excellent peroxidase-like activity was obtained. Furthermore, we explored the enzyme kinetics and possible catalytic mechanism of Co-NC/PS. Due to the excellent flexibility, high conductivity, strong adsorption performance of carbon cloth, and the introduction of non-metallic atom-doped active sites, the developed Co-NC/PS@CC exhibited ideal sensing performance. Experimental results showed that the linear response range for H2O2 was 1–17328 μM, with a detection limit (LOD) of 0.1687 μM. Additionally, the sensor demonstrated good reproducibility, repeatability, anti-interference, and stability. SignificanceThe Co-NC/PS@CC prepared in this study has been successfully applied for detecting H2O2 secreted by MCF-7 live cells, expanding the application of single-atom nanoenzymes in live cell biosensing, with significant implications for health monitoring and clinical diagnostics.

Development of Improved Spectrophotometric Assays for Biocatalytic Silyl Ether Hydrolysis.

Reported herein is the development of assays for the spectrophotometric quantification of biocatalytic silicon-oxygen bond hydrolysis. Central to these assays are a series of chromogenic substrates that release highly absorbing phenoxy anions upon cleavage of the sessile bond. These substrates were tested with silicatein, an enzyme from a marine sponge that is known to catalyse the hydrolysis and condensation of silyl ethers. It was found that, of the substrates tested, tert-butyldimethyl(2-methyl-4-nitrophenoxy)silane provided the best assay performance, as evidenced by the highest ratio of enzyme catalysed reaction rate compared with the background (uncatalysed) reaction. These substrates were also found to be suitable for detailed enzyme kinetics measurements, as demonstrated by their use to determine the Michaelis-Menten kinetic parameters for silicatein.

HLM chip – A microfluidic approach to study the mechanistic basis of cytochrome P450 inhibition using immobilized human liver microsomes

Cytochrome P450 (CYP) system is a critical elimination route to most pharmaceuticals in human, but also prone to drug-drug interactions arising from the fact that concomitantly administered pharmaceuticals inhibit one another's CYP metabolism. The most severe form of CYP interactions is irreversible inhibition, which results in permanent inactivation of the critical CYP pathway and is only restored by de novo synthesis of new functional enzymes. In this study, we conceptualize a microfluidic approach to mechanistic CYP inhibition studies using human liver microsomes (HLMs) immobilized onto the walls of a polymer micropillar array. We evaluated the feasibility of these HLM chips for CYP inhibition studies by establishing the stability and the enzyme kinetics for a CYP2C9 model reaction under microfluidic flow and determining the half-maximal inhibitory concentrations (IC50) of three human CYP2C9 inhibitors (sulfaphenazole, tienilic acid, miconazole), including evaluation of their inhibition mechanisms and nonspecific microsomal binding on chip. Overall, the enzyme kinetics of CYP2C9 metabolism on the HLM chip (KM = 127 ± 55 µM) was shown to be similar to that of static HLM incubations (KM = 114 ± 14 µM) and the IC50 values toward CYP2C9 derived from the microfluidic assays (sulfaphenazole 0.38 ± 0.09 µM, tienilic acid 3.4 ± 0.6 µM, miconazole 0.54 ± 0.09 µM) correlated well with those determined using current standard IC50 shift assays. Most importantly, the HLM chip could distinguish between reversible (sulfaphenazole) and irreversible (tienilic acid) enzyme inhibitors in a single, automated experiment, indicating the great potential of the HLM chip to simplify current workflows used in mechanistic CYP inhibition studies. Furthermore, the results suggest that the HLM chip can also identify irreversible enzyme inhibitors, which are not necessarily resulting in a time-dependent inhibition (like suicide inhibitors), but whose inhibition mechanism is based on other kind of covalent or irreversible interaction with the CYP system. With our HLM chip approach, we could identify miconazole as such a compound that nonselectively inhibits the human CYP system with a prolonged, possibly irreversible impact in vitro, even if it is not a time-dependent inhibitor according to the IC50 shift assay.

Inactivation kinetics of polyphenol oxidase and peroxidase enzymes in buffer solutions by ultraviolet light sources at multiple UV-C wavelengths

This study aimed to evaluate the effect of multiple wavelengths of UV-C light sources on the inactivation of polyphenol oxidase (PPO) and peroxidase (POD) in buffer solutions. Enzyme solutions were treated with a UV-C excimer lamp (EL) emitting at 222 nm, low-pressure mercury (LPM) lamp at 253.7 nm, four UV-LEDs sources emitting at 255, 265, 275 and 285 nm, and a novel pulsed UV-C lamp (PL) emitting at 232, 242, 261 and 272 nm. Three kinetic models were used to compare UV-C wavelengths enzymatic inactivation efficacy considering the volume average fluence for 90% reduction (HD). Validation of kinetic parameters was performed under continuous-flow treatment in a UV-C LED and EL system. EL treatment resulted in the highest inactivation efficacy of PPO and POD enzymes with HD of 52.8 and 74.8 mJ/cm2, respectively. PL inactivation efficacy for both enzymes was higher than with LPM lamp or UV-LEDs. The two-component first-order model best described the inactivation of PPO and POD in a buffer solution. The adjusted kinetic parameters for this model were validated under UV-C light continuous flow processing. This study contributes to the development of liquid food treatment by ultraviolet technology.

Role of gamma radiation as an agent modulator of Mucor subtilissimus UCP1262 Fibrinolytic Enzyme (MsFE)

Thrombosis is a dangerous condition that can result in major damage. Fibrinolytic enzymes are significant therapeutic agents for thrombosis. This study focused on MsFE, a fibrinolytic protease from M. subtilissimus UCP1262, exploring its physicochemical traits and the impact of gamma radiation on its activity. Investigations included: fibrinogenolytic activity, effects of solvents and ions, gamma rays, enzyme shelf life, FTIR, CD, clotted times determination, and enzymatic kinetics. Notably, the major results obtained show the protease retained 90 % activity in organic solvents, FTIR indicated carbonyl functional groups and Circular dichroism spectroscopic studies indicated that the secondary structure (β-sheet) of enzyme remains mostly unaffected over pH 2.0–9.0, whereas considerable changes were observed in the tertiary structure. Kinetic analysis revealed altered parameters post-irradiation. Gamma radiation initially enhanced enzyme activity (10 kGy) but progressively decreased beyond 20 kGy. Metallic ions, especially magnesium, augmented irradiated enzyme activity. This study highlights gamma radiation's potential as a modulator for enhanced fibrinolytic activity, suggesting future biomedical applications.

In vitro cleavage of tumor necrosis factor α (TNFα) by Signal-Peptide-Peptidase-like 2b (SPPL2b) resembles mechanistic principles observed in the cellular context

Members of the Signal Peptide-Peptidase (SPP) and Signal Peptide-Peptidase-like (SPPL) family are intramembrane aspartyl-proteases like their well-studied homologs, the presenilins, which comprise the catalytically active subunit within the γ-secretase complex. The lack of in vitro cleavage assays for SPPL proteases limited their biochemical characterization as well as substrate identification and validation. So far, SPPL proteases have been analyzed exclusively in intact cells or membranes, restricting mechanistic analysis to co-expression of enzyme and substrate variants colocalizing in the same subcellular compartments. We describe the details of developing an in vitro cleavage assay for SPPL2b and its model substrate TNFα and analyzed the influence of phospholipids, detergent supplements, and cholesterol on the SPPL2b in vitro activity. SPPL2b in vitro activity resembles mechanistic principles that have been observed in a cellular context, such as cleavage sites and consecutive turnover of the TNFα transmembrane domain. The novel in vitro cleavage assay is functional with separately isolated protease and substrate and amenable to a high throughput plate-based readout overcoming previous limitations and providing the basis for studying enzyme kinetics, catalytic activity, substrate recognition, and the characteristics of small molecule inhibitors. As a proof of concept, we present the first biochemical in vitro characterization of the SPPL2a and SPPL2b specific small molecule inhibitor SPL-707.

The Inhibition Activity of Natural Methoxyflavonoid from Inula britannica on Soluble Epoxide Hydrolase and NO Production in RAW264.7 Cells.

Soluble epoxide hydrolase (sEH) is an enzyme targeted for the treatment of inflammation and cardiovascular diseases. Activated inflammatory cells produce nitric oxide (NO), which induces oxidative stress and exacerbates inflammation. We identify an inhibitor able to suppress sEH and thus NO production. Five flavonoids 1-5 isolated from Inula britannica flowers were evaluated for their abilities to inhibit sEH with IC50 values of 12.1 ± 0.1 to 62.8 ± 1.8 µM and for their effects on enzyme kinetics. A simulation study using computational chemistry was conducted as well. Furthermore, five inhibitors (1-5) were confirmed to suppress NO levels at 10 µM. The results showed that flavonoids 1-5 exhibited inhibitory activity in all tests, with compound 3 exhibiting the most significant efficacy. Thus, in the development of anti-inflammatory inhibitors, compound 3 is a promising natural candidate.

Characterization of structural, biochemical, pharmacokinetic, and pharmacodynamic properties of the LSD1 inhibitor bomedemstat in preclinical models.

Lysine-specific demethylase 1 (LSD1) is emerging as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Neuroendocrine prostate cancer (NEPC) is increasingly recognized as an adaptive mechanism of resistance in mCRPC patients failing androgen receptor axis-targeted therapies. Safe and effective LSD1 inhibitors are necessary to determine antitumor response in prostate cancer models. For this reason, we characterize the LSD1 inhibitor bomedemstat to assess its clinical potential in NEPC as well as other mCRPC pathological subtypes. Bomedemstat was characterized via crystallization, flavine adenine dinucleotide spectrophotometry, and enzyme kinetics. On-target effects were assessed in relevant prostate cancer cell models by measuring proliferation and H3K4 methylation using western blot analysis. In vivo, pharmacokinetic (PK) and pharmacodynamic (PD) profiles of bomedemstat are also described. Structural, biochemical, and PK/PD properties of bomedemstat, an irreversible, orally-bioavailable inhibitor of LSD1 are reported. Our data demonstrate bomedemstat has >2500-fold greater specificity for LSD1 over monoamine oxidase (MAO)-A and -B. Bomedemstat also demonstrates activity against several models of advanced CRPC, including NEPC patient-derived xenografts. Significant intra-tumoral accumulation of orally-administered bomedemstat is measured with micromolar levels achieved in vivo (1.2 ± 0.45 µM at the 7.5 mg/kg dose and 3.76 ± 0.43 µM at the 15 mg/kg dose). Daily oral dosing of bomedemstat at 40 mg/kg/day is well-tolerated, with on-target thrombocytopenia observed that is rapidly reversible following treatment cessation. Bomedemstat provides enhanced specificity against LSD1, as revealed by structural and biochemical data. PK/PD data display an overall safety profile with manageable side effects resulting from LSD1 inhibition using bomedemstat in preclinical models. Altogether, our results support clinical testing of bomedemstat in the setting of mCRPC.