Search Results (33)

The inhibition of steroid 5-alpha reductase (S5AR), a key mechanism for managing dihydrotestosterone-dependent conditions, has been demonstrated in teak (Tectona grandis L.f.) leaf extracts. Our recent clinical study confirmed the effectiveness of a hair growth formulation containing teak leaf extract in males with androgenic alopecia. However, significant variability in S5AR inhibitory activity among teak leaf samples from different regions underscores the need for quality control of raw materials. This study applied a metabolomics approach to investigate the influence of leaf age, harvesting period, and geographic origin on chemical composition and S5AR inhibitory activity, as well as to identify active S5AR inhibitors. Geographic origin emerged as the primary determinant of variations in chemical profiles and S5AR inhibitory activity. Using orthogonal partial least squares analysis, six diterpenoid S5AR inhibitors were identified, including four compounds reported for the first time as S5AR inhibitors: rhinocerotinoic acid, 7-oxo-8-labden-15-oic acid, 8-hydroxy-labd-13-en-15-oic acid, and a novel diterpene, 7-hydroxy-labd-8,13-dien-15-oic acid. These findings highlight the potential of metabolomics as a powerful tool for discovering bioactive compounds and optimizing raw material selection. By prioritizing proven geographic sources, consistent bioactivity can be achieved, supporting the therapeutic potential of teak leaves in managing S5AR-related conditions. Full article

The Atacama Desert is emerging as an unexpected source of microbial life and, thus, a source of bioactive compounds and novel enzymes. Baeyer–Villiger monooxygenases (BVMOs), a subclass of flavin-dependent monooxygenases (FPMOs), have gained attention as promising biocatalysts for the biosynthesis of industrially relevant molecules for a wide range of applications, such as pharmaceuticals and polymers, among others. BVMOs catalyze the oxidation of ketones and cyclic ketones to esters and lactones, respectively, by using molecular oxygen and NAD(P)H. BVMOs may also catalyze heteroatoms oxidation including sulfoxidations and N-oxidations. This work aims to search for novel BVMOs in the genomes of new bacterial strains isolated from the Atacama Desert. Bioinformatic analysis led to the identification of 10 putative BVMOs, where the monooxygenase named MO-G35A was selected. Genome context showed, downstream of the MO-G35A, a gene encoding for an enzyme from the short-chain dehydrogenase/reductase family, suggesting a closer redox loop between both enzymes. MO-G35A was successfully expressed in three Escherichia coli expression systems, where higher yields were achieved using the E. coli Shuffle T7 as host, suggesting that correct disulfide bond formation is necessary for correct folding. Enzyme characterization showed that it operates optimally at 35–38 °C, exhibiting a Km of 0.06 mM and a kcat of 0.15 s⁻¹ for bicyclo [3.2.0] hept-2-en-6-one (BHC). Furthermore, the study revealed high stability in the presence of organic solvents, making it suitable for applications in various industrial processes, especially when the substrates have poor solubility in aqueous solutions. These results highlight the robustness and adaptability of enzymes in extreme environments, making them valuable candidates for biotechnological applications. Full article

Industrial biotechnology offers a potential ecological solution for PET recycling under relatively mild reaction conditions via enzymatic degradation, particularly using the leaf branch compost cutinase (LCC) quadruple mutant ICCG. To improve the efficient downstream processing of this biocatalyst after heterologous gene expression with a suitable production host, protein crystallization can serve as an effective purification/capture step. Enhancing protein crystallization was achieved in recent studies by introducing electrostatic (and aromatic) interactions in two homologous alcohol dehydrogenases (Lb/LkADH) and an ene reductase (NspER1-L1,5) produced with Escherichia coli. In this study, ICCG, which is difficult to crystallize, was utilized for the application of crystal contact engineering strategies, resulting in ICCG mutant L50Y (ICCGY). Previously focused on the Lys-Glu interaction for the introduction of electrostatic interactions at crystal contacts, the applicability of the engineering strategy was extended here to an Arg-Glu interaction to increase crystallizability, as shown for ICCGY T110E. Furthermore, the rationale of the engineering approach is demonstrated by introducing Lys and Glu at non-crystal contacts or sites without potential interaction partners as negative controls. These resulting mutants crystallized comparably but not superior to the wild-type protein. As demonstrated by this study, crystal contact engineering emerges as a promising approach for rationally enhancing protein crystallization. This advancement could significantly streamline biotechnological downstream processing, offering a more efficient pathway for research and industry. Full article

Protein crystallization is an alternative to well-established but cost-intensive and time-consuming chromatography in biotechnological processes, with protein crystallization defined as an essential unit operation for isolating proteins, e.g., active pharmaceutical ingredients. Crystalline therapeutic proteins attract interest in formulation and delivery processes of biopharmaceuticals due to the high purity, concentration, and stability of the crystalline state. Although improving protein crystallization is mainly achieved by high-throughput screening of crystallization conditions, recent studies have established a rational protein engineering approach to enhance crystallization for two homologous alcohol dehydrogenases from Lactobacillus brevis (LbADH) and Lactobacillus kefiri (LkADH). As generalizing crystallization processes across a wide range of target proteins remains challenging, this study takes a further step by applying the successful crystal contact engineering strategies for LbADH/LkADH to a non-homologous protein, an NADH-binding derivative of the Nostoc sp. PCC 1720 ene reductase (NspER1-L1,5). Here, the focus lies on introducing electrostatic interactions at crystal contacts, specifically between lysine and glutamic acid. Out of the nine tested NspER1-L1,5 mutants produced in E. coli, six crystallized, while four mutants revealed an increased propensity to crystallize in static µL-batch crystallization compared to the wild type: Q204K, Q350K, D352K, and T354K. The best-performing mutant Q204K was selected for upscaling, crystallizing faster than the wild type in a stirred batch crystallizer. Even when spiked with E. coli cell lysate, the mutant maintained increased crystallizability compared to the wild type. The results of this study highlight the potential of crystal contact engineering as a reliable tool for improving protein crystallization as an alternative to chromatography, paving the way for more efficient biotechnological downstream processing. Full article

Background/Objectives: Pteridine reductase 1 (PTR1) has been one of the prime targets for discovering novel antileishmanial therapeutics in the fight against Leishmaniasis. This enzyme catalyzes the NADPH-dependent reduction of pterins to their tetrahydro forms. While chemotherapy remains the primary treatment, its effectiveness is constrained by drug resistance, unfavorable side effects, and substantial associated costs. Methods: This study addresses the urgent need for novel, cost-effective drugs by employing in silico techniques to identify potential lead compounds targeting the PTR1 enzyme. A library of 1463 natural compounds from AfroDb and NANPDB, prefiltered based on Lipinski’s rules, was used to screen against the LmPTR1 target. The X-ray structure of LmPTR1 complexed with NADP and dihydrobiopterin (Protein Data Bank ID: 1E92) was identified to contain the critical residues Arg17, Leu18, Ser111, Phe113, Pro224, Gly225, Ser227, Leu229, and Val230 including the triad of residues Asp181-Tyr194-Lys198, which are critical for the catalytic process involving the reduction of dihydrofolate to tetrahydrofolate. Results: The docking yielded 155 compounds meeting the stringent criteria of −8.9 kcal/mol instead of the widely used −7.0 kcal/mol. These compounds demonstrated binding affinities comparable to the known inhibitors; methotrexate (−9.5 kcal/mol), jatrorrhizine (−9.0 kcal/mol), pyrimethamine (−7.3 kcal/mol), hardwickiic acid (−8.1 kcal/mol), and columbamine (−8.6 kcal/mol). Protein–ligand interactions and molecular dynamics (MD) simulation revealed favorable hydrophobic and hydrogen bonding with critical residues, such as Lys198, Arg17, Ser111, Tyr194, Asp181, and Gly225. Crucial to the drug development, the compounds were physiochemically and pharmacologically profiled, narrowing the selection to eight compounds, excluding those with potential toxicities. The five selected compounds ZINC000095486253, ZINC000095486221, ZINC000095486249, 8alpha-hydroxy-13-epi-pimar-16-en-6,18-olide, and pachycladin D were predicted to be antiprotozoal (Leishmania) with Pa values of 0.642, 0.297, 0.543, 0.431, and 0.350, respectively. Conclusions: This study identified five lead compounds that showed substantial binding affinity against LmPTR1 as well as critical residue interactions. A 100 ns MD combined with molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations confirmed the robust binding interactions and provided insights into the dynamics and stability of the protein–ligand complexes. Full article

Ene-reductases (ERs) are enzymes known for catalyzing the asymmetric hydrogenation of activated alkenes. Among these, old yellow enzyme (OYE) ERs have been the most extensively studied for biocatalytic applications due to their dependence on NADH or NADPH as electron donors. These flavin-containing enzymes are highly enantio- and stereoselective, making them attractive biocatalysts for industrial use. To discover novel thermostable OYE-type ERs, we explored genomes of thermophilic fungi. Five genes encoding ERs were selected and expressed in Escherichia coli, namely AtOYE (from Aspergillus thermomutatus), CtOYE (from Chaetomium thermophilum), LtOYE (from Lachancea thermotolerans), OpOYE (from Ogatae polymorpha), and TtOYE (from Thermothielavioides terrestris). Each enzyme was purified as a soluble FMN-containing protein, allowing detailed characterization. All ERs exhibited a preference for NADPH, with AtOYE showing the broadest substrate range. Moreover, all the enzymes showed activity toward maleimide and p-benzoquinone, with TtOYE presenting the highest catalytic efficiency. The optimal pH for enzyme activity was between 6 and 7 and the enzymes displayed notable solvent tolerance and thermostability, with CtOYE and OpOYE showing the highest stability (T_m > 60 °C). Additionally, all enzymes converted R-carvone into (R,R)-dihydrocarvone. In summary, this study contributes to expanding the toolbox of robust ERs. Full article

According to recent data, one in eight people in the world struggle with obesity. Obesity management is increasingly dependent on bariatric surgical interventions, as the combination of lifestyle modifications and pharmacotherapy could have a modest long-term effect. Surgery is recommended only for individuals whose body mass index (BMI) ≥ 40 kg/m² and ≥ 35 kg/m² in the presence of weight-related comorbidities. The most commonly performed procedures are sleeve gastrectomy and roux-en-Y gastric bypass. Pharmacokinetic and pharmacodynamic alterations occur as a result of the anatomical and physiological changes caused by surgery, which further differ depending on physicochemical drug factors and factors related to the dosage form. The following modifications are distinguished based on the type of bariatric surgery performed. Most bariatric patients have accompanying comorbidities, including dyslipidemia treated with hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors or statins. Significant improvements in the lipid profile are observed early in the postoperative period. The data reported in this review on statin pharmacokinetic alterations have demonstrated substantial inter- and intravariability, making it difficult to adopt clear guidelines. Based on the current literature review, reducing the statin dose to the lowest effective with continuous monitoring is considered an optimal approach in clinical practice. Full article

Synthetic nicotinamide biomimetics (NCBs) have emerged as alternatives to the use of natural cofactors. The relatively low cost and ease of manufacture of NCBs may enable the scaling of biocatalytic reactions to produce bulk chemicals (e.g., biofuels and plastics). NCBs are also recognized by only a subset of NAD(P)/NAD(P)H-dependent enzymes, which potentially allows access to orthogonal redox cascades that can be run simultaneously within a single reactor. In the work presented here, a series of NCBs was prepared and tested for activity with alcohol dehydrogenases and ene-reductases. While the NCBs did not support enzymatic activity with the alcohol dehydrogenases, the observed rate of the ene-reductases with NCBs was greater than when incubated with the natural cofactor (consistent with previous observations). We obtained the structures of an ene-reductase and an alcohol dehydrogenase with an NCB bound in their active sites. While the NCB bound to the ene-reductases in a productive position and orientation for hydride transfer to the isoalloxazine ring of the flavin cofactor, the NCB failed to adopt a catalytically competent binding mode in the alcohol dehydrogenase. Full article

Tef (Eragrostis tef) is an orphan crop that is widely grown in East Africa, primarily in Ethiopia as a staple crop. It is becoming popular in the Western world owing to its nutritious and gluten-free grains and the forage quality of its biomass. Tef is also considered to have a high antioxidant capacity based on cell-free studies. However, the antioxidant activity of tef has never been validated using a physiologically relevant cell model. The purpose of this study was to investigate the antioxidant capacity of tef grain extracts using a mammalian cell model. We hypothesized that the tef grain extracts are capable of modulating the cellular antioxidant response via the modulation of glutathione (GSH) biosynthetic pathways. Therefore, we evaluated the antioxidant activity of purified tef grain extracts in the human acute monocytic leukemia (THP-1) cell line. Our findings revealed that the organic fraction of grain extracts increased the cellular GSH level, which was more evident for brown-colored tef than the ivory variety. Moreover, a brown-tef fraction increased the expressions of GSH-pathway genes, including γ-glutamate cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits and glutathione reductase (GR), an enzyme that plays a key role in GSH biosynthesis, suggesting that tef extracts may modulate GSH metabolism. Several compounds were uniquely identified via mass spectrometry (MS) in GSH-modulating brown-tef samples, including 4-oxo-β-apo-13-carotenone, γ-linolenic acid (methyl ester), 4,4′-(2,3-dimethyl-1,4-butanediyl)bis-phenol (also referred to as 8,8′-lignan-4,4′-diol), and (3β)-3-[[2-[4-(Acetylamino)phenoxy]acetyl]oxy]olean-12-en-28-oic acid. Tef possesses antioxidant activity due to the presence of phytochemicals that can act as direct antioxidants, as well as modulators of antioxidant-response genes, indicating its potential role in alleviating diseases triggered by oxidative stresses. To the best of our knowledge, this is the first report revealing the antioxidant ability of tef extracts in a physiologically relevant human cell model. Full article

Ginsenosides, the main active compounds in Panax species, are glycosides of protopanaxadiol (PPD) or protopanaxatriol (PPT). PPT-type ginsenosides have unique pharmacological activities on the central nervous system and cardiovascular system. As an unnatural ginsenoside, 3,12-Di-O-β-D-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (3β,12β-Di-O-Glc-PPT) can be synthesized through enzymatic reactions but is limited by the expensive substrates and low catalytic efficiency. In the present study, we successfully produced 3β,12β-Di-O-Glc-PPT in Saccharomyces cerevisiae with a titer of 7.0 mg/L by expressing protopanaxatriol synthase (PPTS) from Panax ginseng and UGT109A1 from Bacillus subtilis in PPD-producing yeast. Then, we modified this engineered strain by replacing UGT109A1 with its mutant UGT109A1-K73A, overexpressing the cytochrome P450 reductase ATR2 from Arabidopsis thaliana and the key enzymes of UDP-glucose biosynthesis to increase the production of 3β,12β-Di-O-Glc-PPT, although these strategies did not show any positive effect on the yield of 3β,12β-Di-O-Glc-PPT. However, the unnatural ginsenoside 3β,12β-Di-O-Glc-PPT was produced in this study by constructing its biosynthetic pathway in yeast. To the best of our knowledge, this is the first report of producing 3β,12β-Di-O-Glc-PPT through yeast cell factories. Our work provides a viable route for the production of 3β,12β-Di-O-Glc-PPT, which lays a foundation for drug research and development. Full article

The acetophenone-based 3,4-dihydropyrimidine-2(1H)-thione was synthesized by the reaction of 4-methylpent-3-en-2-one (1), 4-acetyl aniline (2) and potassium thiocyanate. The spectroscopic analysis including: FTIR, ¹H-NMR, and single crystal analysis proved the structure of synthesized compound (4), with the six-membered nonplanar ring in envelope conformation. In crystal structure, the intermolecular N–H ⋯ S and C–H ⋯ O hydrogen bonds link the molecule in a two-dimensional manner which is parallel to (010) the plane enclosing R₂² (8) and R₂² (10) ring motifs. After that, the Hirshfeld surfaces and their related two-dimensional fingerprint plots were used for thorough investigation of intermolecular interactions. According to Hirshfeld surface analysis, the most substantial contributions to the crystal packing are from H ⋯ H (59.5%), H ⋯ S/S ⋯ H (16.1%), and H ⋯ C/C ⋯ H (13.1%) interactions. The electronic properties and stability of the compound were investigated through density functional theory (DFT) studies using B3LYP functional and 6-31G* as a basis set. The compound 4 displayed the high chemical reactivity with chemical softness of 2.48. In comparison to the already reported known tyrosinase inhibitor, the newly synthesized derivatives exhibited almost seven-fold better inhibition of tyrosinase (IC₅₀ = 1.97 μM), which was further supported by molecular docking studies. The compound 4 inside the active pocket of ribonucleotide reductase (RNR) exhibited a binding energy of −19.68 kJ/mol, and with mammalian deoxy ribonucleic acid (DNA) it acts as an effective DNA groove binder with a binding energy of −21.32 kJ/mol. The results suggested further exploration of this compound at molecular level to synthesize more potential leads for the treatment of cancer. Full article

Chiral dimethyl 2-methylsuccinate (1) is a very important building block for the manufacturing of many active pharmaceutical ingredients and fine chemicals. The asymmetric reduction of C=C double bond of dimethyl citraconate (2), dimethyl mesaconate (3) or dimethyl itaconate (4) by ene-reductases (ERs) represents an attractive straightforward approach, but lack of high-performance ERs, especially (S)-selective ones, has limited implementing this method to prepare the optically pure dimethyl 2-methylsuccinate. Herein, three ERs (Bac-OYE1 from Bacillus sp., SeER from Saccharomyces eubayanus and AfER from Aspergillus flavus) with high substrate tolerance and stereoselectivity towards 2, 3 and 4 have been identified. Up to 500 mM of 3 was converted to (S)-dimethyl 2-methylsuccinate ((S)-1) by SeER in high yields (80%) and enantioselectivity (98% ee), and 700 mM of 2 and 400 mM of 4 were converted to (R)-1 by Bac-OYE1 and AfER, respectively, in high yields (86% and 77%) with excellent enantioselectivity (99% ee). The reductions of diethyl citraconate (5), diethyl mesaconate (6) and diethyl itaconate (7) were also tested with the three ERs. Although up to 500 mM of 5 was completely converted to (R)-diethyl 2-methylsuccinate ((R)-8) by Bac-OYE1 with excellent enantioselectivity (99% ee), the alcohol moiety of the esters had a great effect on the activity and enantioselectivity of ERs. This work provides an efficient methodology for the enantiocomplementary production of optically pure dimethyl 2-methylsuccinate from dimethyl itaconate and its isomers at high titer. Full article

A series of novel 21E-arylidene-4-azapregn-5-ene steroids has been successfully designed, synthesized and structurally characterized, and their antiproliferative activity was evaluated in four different cell lines. Within this group, the 21E-(pyridin-3-yl)methylidene derivative exhibited significant cytotoxic activity in hormone-dependent cells LNCaP (IC₅₀ = 10.20 µM) and T47-D cells (IC₅₀ = 1.33 µM). In PC-3 androgen-independent cells, the steroid 21E-p-nitrophenylidene-4-azapregn-5-ene was the most potent of this series (IC₅₀ = 3.29 µM). Considering these results, the 21E-(pyridin-3-yl)methylidene derivative was chosen for further biological studies on T47-D and LNCaP cells, and it was shown that this azasteroid seems to lead T47-D cells to apoptotic death. Finally, molecular docking studies were performed to explore the affinity of these 4-azapregnene derivatives to several steroid targets, namely 5α-reductase type 2, estrogen receptor α, androgen receptor and CYP17A1. In general, compounds presented higher affinity to 5α-reductase type 2 and estrogen receptor α. Full article

The aim of the study was to conduct phytochemical and pharmacological investigations of Wrightia coccinea (Roxb. ex Hornem.) Sims via several in vitro, in vivo, and in silico models. A total of four compounds were identified and isolated from the methanol extract of the bark and the methanol extract of the seed pulp of W. coccinea through successive chromatographic techniques and were characterized as 3β-acetyloxy-olean-12-en-28-ol (1), wrightiadione (2), 22β-hydroxylupeol (3), and β-sitosterol (4) by spectroscopic analysis. The aqueous fraction of the bark and chloroform fraction of the fruits provided the most potent antioxidant capacity (IC₅₀ = 7.22 and 4.5 µg/mL, respectively) in DPPH free radical scavenging assay compared with the standard ascorbic acid (IC₅₀ = 17.45 µg/mL). The methanol bark extract and the methanol fruit coat extract exerted anti-diarrheal activity by inhibiting 74.55 ± 0.67% and 77.78 ± 1.5% (mean ± SEM) of the diarrheal episode in mice, respectively, after four hours of loading the samples. In the hypoglycemic test, the methanol bark extract and the methanol fruit coat extract (400 mg/kg) produced a significant (p < 0.05) reduction in the blood glucose level in mice. Both doses of the plant extracts (200 mg/kg and 400 mg/kg) used in the study induced a significant (p < 0.05) increase in pain reaction time. The in vitro and in vivo findings were supported by the computational studies. The isolated compounds exhibited higher binding affinity compared with the standard drugs towards the active binding sites of glutathione reductase, epidermal growth factor receptor (EGFR), kappa opioid receptor, glucose transporter 3 (GLUT 3), Mu opioid receptor, and cyclooxygenase 2 (COX-2) proteins due to their potent antioxidant, cytotoxic, anti-diarrheal, hypoglycemic, and central and peripheral analgesic properties, respectively. The current findings concluded that W. coccinea might be a potential natural source for managing oxidative stress, diarrhea, hyperglycemia, and pain. Further studies are warranted for extensively phytochemical screening and establishing exact mechanisms of action. Full article

In this work, we describe two different biotechnological processes that provide the natural flavour dihydrocoumarin in preparative scale. Both the presented approaches are based on the enzyme-mediated reduction of natural coumarin. The first one is a whole-cell process exploiting the reductive activity of the yeast Kluyveromyces marxianus, a Generally Recognized As Safe (GRAS) microorganism that possesses high resistance to the substrate toxicity. Differently, the second is based on the reduction of natural coumarin by nicotinamide adenine dinucleotide phosphate (NADPH) and using the Old Yellow Enzyme reductase OYE2 as catalyst. NADPH is used in catalytic amount since the co-factor regeneration is warranted employing an enzymatic system based on glucose oxidation, in turn catalysed by a further enzyme, namely glucose dehydrogenase (GDH). Both processes compare favourably over the previously reported industrial method as they work with higher coumarin concentration (up to 3 g/L for the enzymatic process) yet allowing the complete conversion of the substrate. Furthermore, the two approaches have significant differences. The microbial reduction is experimentally simple but the isolated dihydrocoumarin yield does not exceed 60%. On the contrary, the enzymatic approach requires the use of two specially prepared recombinant enzymes, however, it is more efficient, affording the product in 90% of isolated yield. Full article