Enzymes and Enzyme Inhibitors in Drug Research

Streptococcus dysgalactiae infection can cause bovine mastitis and lead to huge economic losses for the dairy industry. The abuse of antibiotics has resulted in growing drug resistance of S. dysgalactiae, which causes hard-to-treat infections. Bacteriophage lysin, as a novel antibacterial agent, has great potential for application against drug-resistant gram-positive bacteria. However, few studies have been conducted on the prophage lysin of S. dysgalactiae. In this study, we mined a novel prophage lysin, named Lys1644, from a clinical S. dysgalactiae isolate by genome sequencing and bioinformatic analysis. Lys1644 was expressed and purified, and the lytic activity, antibacterial spectrum, optimal pH and temperature, lytic activity in milk in vitro, and synergistic bacteriostasis with antibiotics were assessed. The Lys1644 prophage lysin showed high bacteriolysis activity specifically on S. dysgalactiae, which resulted in CFU 100-fold reduction in milk. Moreover, Lys1644 maintained high activity over a wide pH range (pH 5–10) and a wide temperature range (4–42 °C). Synergistic bacteriostatic experiments showed that the combination of low-dose Lys1644 (50 μg/mL) with a subinhibitory concentration of aminoglycoside antibiotics (kanamycin or spectinomycin) can completely inhibit bacterial growth, suggesting that the combination of Lys1644 and antibiotics could be an effective therapeutic strategy against S. dysgalactiae infection. Full article

Dicerothamnus rhinocerotis (L.f.) Koekemoer, also known as rhinoceros bush and previously called Elytropappus rhinocerotis (L.f.) Less., is from the Asteraceae plant family. The plant is traditionally used to treat indigestion, stomach ulcers, influenza, and diarrhea. This study was aimed at investigating the phytochemistry, anti-glucosidase, anti-amylase, and anti-tyrosinase effects of D. rhinocerotis as research in this area is limited. The air-dried plant materials were macerated in 80% methanol (MeOH) and fractionated between hexane, dichloromethane (DCM), ethyl acetate (EtOAc), and butanol (BuOH). Column chromatography on silica gel was employed for the isolation of the compounds. A total of six compounds (1–6) were isolated from the fractions viz. acacetin (1), 15-hydroxy-cis-clerodan-3-ene-18-oic-acid (2), acacetin-7-glucoside (3), pinitol (4), apigenin (5), and β-sitosterol-3-O-glycoside (6). Compounds 2–4 and 6 are reported for the first time from this plant. Among the different fractions, the BuOH and EtOAc fractions had strong tyrosinase inhibitory activities with IC₅₀ values of 13.7 ± 1.71 and 11.6 ± 2.68 µg/mL, respectively, while among the isolated compounds, apigenin (5) had the strongest inhibitory activity, with an IC₅₀ of 14.58 µM, which competes favorably with Kojic acid (17.26 µM). The anti-glucosidase assay showed good activity in three of the fractions and compound 5, while the anti-amylase assays did not show significant inhibition activity. Full article

The antioxidant defense mechanisms play a critical role in mitigating the deleterious effects of reactive oxygen species (ROS). Catalase stands out as a paramount enzymatic antioxidant. It efficiently catalyzes the decomposition of hydrogen peroxide (H₂O₂) into water and oxygen, a potentially harmful byproduct of cellular metabolism. This reaction detoxifies H₂O₂ and prevents oxidative damage. Catalase has been extensively studied as a therapeutic antioxidant. Its applications range from direct supplementation in conditions characterized by oxidative stress to gene therapy approaches to enhance endogenous catalase activity. The enzyme’s stability, bioavailability, and the specificity of its delivery to target tissues are significant hurdles. Furthermore, studies employing conventional catalase formulations often face issues related to enzyme purity, activity, and longevity in the biological milieu. Addressing these challenges necessitates rigorous scientific inquiry and well-designed clinical trials. Such trials must be underpinned by sound experimental designs, incorporating advanced catalase formulations or novel delivery systems that can overcome existing limitations. Enhancing catalase’s stability, specificity, and longevity in vivo could unlock its full therapeutic potential. It is necessary to understand the role of catalase in disease-specific contexts, paving the way for precision antioxidant therapy that could significantly impact the treatment of diseases associated with oxidative stress. Full article

Alzheimer’s disease (AD) and diabetes are non-communicable diseases with global impacts. Inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are suitable therapies for AD, while α-amylase and α-glucosidase inhibitors are employed as antidiabetic agents. Compounds were isolated from the medicinal plant Terminalia macroptera and evaluated for their AChE, BChE, α-amylase, and α-glucosidase inhibitions. From ¹H and ¹³C NMR data, the compounds were identified as 3,3′-di-O-methyl ellagic acid (1), 3,3′,4′-tri-O-methyl ellagic acid-4-O-β-D-xylopyranoside (2), 3,3′,4′-tri-O-methyl ellagic acid-4-O-β-D-glucopyranoside (3), 3,3′-di-O-methyl ellagic acid-4-O-β-D-glucopyranoside (4), myricetin-3-O-rhamnoside (5), shikimic acid (6), arjungenin (7), terminolic acid (8), 24-deoxysericoside (9), arjunglucoside I (10), and chebuloside II (11). The derivatives of ellagic acid (1–4) showed moderate to good inhibition of cholinesterases, with the most potent being 3,3′-di-O-methyl ellagic acid, with IC₅₀ values of 46.77 ± 0.90 µg/mL and 50.48 ± 1.10 µg/mL against AChE and BChE, respectively. The compounds exhibited potential inhibition of α-amylase and α-glucosidase, especially the phenolic compounds (1–5). Myricetin-3-O-rhamnoside had the highest α-amylase inhibition with an IC₅₀ value of 65.17 ± 0.43 µg/mL compared to acarbose with an IC₅₀ value of 32.25 ± 0.36 µg/mL. Two compounds, 3,3′-di-O-methyl ellagic acid (IC₅₀ = 74.18 ± 0.29 µg/mL) and myricetin-3-O-rhamnoside (IC₅₀ = 69.02 ± 0.65 µg/mL), were more active than the standard acarbose (IC₅₀ = 87.70 ± 0.68 µg/mL) in the α-glucosidase assay. For α-glucosidase and α-amylase, the molecular docking results for 1–11 reveal that these compounds may fit well into the binding sites of the target enzymes, establishing stable complexes with negative binding energies in the range of −4.03 to −10.20 kcalmol⁻¹. Though not all the compounds showed binding affinities with cholinesterases, some had negative binding energies, indicating that the inhibition was thermodynamically favorable. Full article

The β-adrenergic drug Mirabegron, a drug initially used for the treatment of an overactive bladder, has new potential indications and is hydrolyzed by butyrylcholinesterase (BChE). This compound is one of the only arylacylamide substrates to be catabolized by BChE. A steady-state kinetic analysis at 25 °C and pH 7.0 showed that the enzyme behavior is Michaelian with this substrate and displays a long pre-steady-state phase characterized by a burst. The induction time, τ, increased with substrate concentration (τ ≈ 18 min at maximum velocity). The kinetic behavior was interpreted in terms of hysteretic behavior, resulting from a slow equilibrium between two enzyme active forms, E and E′. The pre-steady-state phase with the highest activity corresponds to action of the E form, and the steady state corresponds to action of the E′ form. The catalytic parameters were determined as k_cat = 7.3 min⁻¹ and K_m = 23.5 μM for the initial (burst) form E, and k_cat = 1.6 min⁻¹ and K_m = 3.9 μM for the final form E′. Thus, the higher affinity of E′ for Mirabegron triggers the slow enzyme state equilibrium toward a slow steady state. Despite the complexity of the reaction mechanism of Mirabegron with BChE, slow BChE-catalyzed degradation of Mirabegron in blood should have no impact on the pharmacological activities of this drug. Full article