Enzymes and Enzyme Inhibitors in Drug Research

Tyrosyl-DNA phosphodiesterases 1 and 2 (TDP1 and TDP2) are important DNA repair enzymes that remove various adducts from the 3′- and 5′-ends of DNA, respectively. The suppression of the activity of these enzymes is considered as a promising adjuvant therapy for oncological diseases in combination with topoisomerase inhibitors. The simultaneous inhibition of TDP1 and TDP2 may result in greater antitumor effects, as these enzymes can mimic each other’s functions. We have previously shown that usnic acid-based sulfides can act as dual inhibitors, with TDP1 activity in the low micromolar range and their TDP2 at 1 mM. The oxidation of their sulfide moieties to sulfoxides led to an order of magnitude decrease in their cytotoxicity potential, while their TDP1 and TDP2 activity was preserved. In this work, we synthesized new series of usnic acid-based sulfides and their oxidized analogues, i.e., sulfoxides and sulfones, to systematically study these irregularities. The new compounds inhibit TDP1 with IC₅₀ values (the concentration of inhibitor required to reduce enzyme activity by half) in the 0.33–25 μM range. Most sulfides and some sulfoxides and sulfones inhibit TDP2 with an IC₅₀ = 138−421 μM. In addition, the most active compounds synergized (×4) with topotecan on the HeLa cell line as well as causing dose-dependent DNA damage, as confirmed by Comet assay. Sulfides with the 6-methylbenzoimidazol-2-yl substituent (8f, IC₅₀ = 0.33/138 μM, TDP1/2) and sulfones containing a pyridine-2-yl fragment (12k, IC₅₀ = 2/228 μM, TDP1/2) are the most potent derivatives and, therefore, are promising for further development. Full article

The current study has yielded promising results in the evaluation of a new ciprofloxacin-chalcone hybrid (CP derivative) for its anticancer activity as potential Topoisomerases (Topo) I and II inhibitors. The in vitro results showed that the CP derivative significantly suppressed the growth of HCT-116 and LOX IMVI cells, with IC₅₀ values of 5.0 μM and 1.3 μM, respectively, outperforming Staurosporine, which had IC₅₀ values of 8.4 μM and 1.6 μM, respectively. Flow cytometry analysis revealed that the new CP derivative triggered apoptosis and cell cycle arrest at the G2/M phase, associated with the up-regulation of pro-apoptotic genes (Bax and Caspase 9) and downregulation of the anti-apoptotic gene (Bcl-2). Further investigations showed that the CP derivative inhibited Topo I and II enzymes, as expected molecular targets; docking studies further supported its dual inhibitory action on Topo I and II. These findings suggest that the ciprofloxacin-chalcone hybrid could be a promising lead compound for developing new anticancer therapy. Full article

Background/Objectives: Nanoparticles derived from medicinal plants are gaining attention for their diverse biological activities and potential therapeutic applications. Methods: This study explored the antioxidant, anti-inflammatory, anti-tumoral, and antimicrobial properties of green synthesized silver nanoparticles (AgNPs) using the aqueous leaf and root extracts of Saussurea costus (S. costus). The physicochemical characterizations of both biosynthesized AgNPs using the aqueous leaf extract (L-AgNPs) and root extract (R-AgNPs) were examined using UV spectroscopy, fluorescence spectroscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction, dynamic light scattering, and Fourier-transform infrared spectroscopy. The antioxidant activity measured using ABTS, DPPH, and FRAP assays showed that AgNPs, particularly from roots, had higher activity than aqueous extracts, attributed to phenolic compounds acting as capping and antioxidant agents. Results: Enzyme inhibition studies indicated that AgNPs exhibited remarkable anti-inflammatory effects, inhibiting COX-1, 5-LOX, and secreted PLA₂ enzymes by over 99% at 120 µg/mL, comparable to standard drugs. The anti-tumoral effects were evaluated on the human cancer cell lines HCT-116, LoVo, and MDA-MB-231, with AgNPs inhibiting cell proliferation dose-dependently and IC₅₀ values between 42 and 60 µg/mL, demonstrating greater potency than extracts. The AgNPs also showed enhanced antimicrobial activities against various microbial strains, with IC₅₀ values as low as 14 µg/mL, which could be linked to nanoparticle interactions with microbial cell membranes, causing structural damage and cell death. Conclusions: These findings suggest that S. costus-derived AgNPs are promising natural, biodegradable agents for various biological applications and potential new therapeutic agents, necessitating further research to explore their mechanisms and applications. Full article

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