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Dihydroorotase (DHOase; EC 3.5.2.3) is a zinc-dependent metalloenzyme that plays a key role in the de novo pyrimidine biosynthesis pathway, catalyzing the reversible cyclization of N-carbamoyl aspartate to dihydroorotate. This reaction is essential for the production of uridine monophosphate, the precursor of all pyrimidine nucleotides required for DNA and RNA synthesis. Despite its conserved enzymatic function, DHOase exhibits significant structural diversity across species, particularly in its oligomeric states, gene fusion patterns, and active site architecture. A crucial structural feature of DHOase is its flexible active site loop, which undergoes dynamic conformational changes during catalysis. Previously, the loop-in conformation was associated with substrate binding, whereas the loop-out conformation was linked to product release and non-substrate ligand binding. However, recent crystallographic studies challenge this paradigm, revealing that certain non-substrate ligands and inhibitors, including malate, 5-fluoroorotate, plumbagin, 5-aminouracil, and 5-fluorouracil, interact with DHOase via a loop-in binding mechanism rather than the previously assumed loop-out mode. These findings necessitate a reassessment of the catalytic mechanism of DHOase and underscore the active site loop as a potential target for drug development. This review revisits the structural and biochemical mechanisms of DHOase, with a focus on recent crystallographic insights that redefine the loop-in binding mode for ligand interaction. By leveraging the unique conformational dynamics of the active site loop, novel inhibitors may be developed to selectively target pyrimidine biosynthesis in cancer cells and microbial pathogens. These insights emphasize the crucial role of structural biology in therapeutic design and highlight DHOase as a promising drug target. [ABSTRACT FROM AUTHOR]

The tamarillo, or Solanum betaceum, recognized for its comprehensive nutritional profile, has long been valued for its diverse ethnobotanical uses. This study delves into the potential therapeutic applications of S. betaceum by analyzing its polyphenolic content (TPC), total flavonoid content (TFC), anti-skin aging activities against key enzymes like elastase, tyrosinase, and hyaluronidase, and its cytotoxic effects on oral carcinoma cells. Extracts from the seeds, pulp, and peel of red and yellow fruits were prepared using methanol, ethanol, and acetone. The highest TPC was found in the methanol extract from red fruit seeds (9.89 mg GAE/g), and the highest TFC was found in the methanol extract of yellow fruit peel (3.02 mg QUE/g). Some of these extracts significantly inhibited skin aging-associated enzymes with the red fruit seed extract (100 μg/mL) showing up to 50.4% inhibition of tyrosinase. Additionally, the red fruit seed extract obtained using methanol demonstrated potential anticancer effects against Ca9-22 oral carcinoma cells by inhibiting cell survival, migration, and proliferation as well as inducing apoptosis. These results underscore the potential of S. betaceum fruit extracts, especially from red fruit seeds, as promising agents for anti-skin aging and anticancer applications, meriting further exploration for therapeutic uses. [ABSTRACT FROM AUTHOR]

The passion fruit, Passiflora edulis, recognized for its rich nutritional properties, has long been used for its varied ethnobotanical applications. This study investigates the therapeutic potential of P. edulis var. Tainung No. 1 rind extracts by examining their polyphenolic content (TPC), total flavonoid content (TFC), anti-skin aging activities against key enzymes such as elastase, tyrosinase, and hyaluronidase, and their ability to inhibit bacterial growth, single-stranded DNA-binding protein (SSB), and their cytotoxic effects on oral carcinoma cells. The acetone extract from the rind exhibited the highest levels of TPC, TFC, anti-SSB, and antibacterial activities. The antibacterial effectiveness of the acetone-extracted rind was ranked as follows: Escherichia coli > Pseudomonas aeruginosa > Staphylococcus aureus. A titration curve for SSB inhibition showed an IC50 value of 313.2 μg/mL, indicating the potency of the acetone extract in inhibiting SSB. It also significantly reduced the activity of enzymes associated with skin aging, particularly tyrosinase, with a 54.5% inhibition at a concentration of 100 μg/mL. Gas chromatography–mass spectrometry (GC–MS) analysis tentatively identified several major bioactive compounds in the acetone extract, including stigmast-5-en-3-ol, vitamin E, palmitic acid, stigmasterol, linoleic acid, campesterol, and octadecanoic acid. Molecular docking studies suggested some of these compounds as potential inhibitors of tyrosinase and SSB. Furthermore, the extract demonstrated anticancer potential against Ca9-22 oral carcinoma cells by inhibiting cell survival, migration, and proliferation and inducing apoptosis. These results underscore the potential of P. edulis (Tainung No. 1) rind as a promising candidate for anti-skin aging, antibacterial, and anticancer applications, meriting further therapeutic investigation. [ABSTRACT FROM AUTHOR]

Carnivorous pitcher plants from the genus Nepenthes are renowned for their ethnobotanical uses. This research explores the therapeutic potential of Nepenthes miranda leaf extract against nonstructural protein 9 (Nsp9) of SARS-CoV-2 and in treating human non-small cell lung carcinoma (NSCLC) cell lines. Nsp9, essential for SARS-CoV-2 RNA replication, was expressed and purified, and its interaction with ssDNA was assessed. Initial tests with myricetin and oridonin, known for targeting ssDNA-binding proteins and Nsp9, respectively, did not inhibit the ssDNA-binding activity of Nsp9. Subsequent screenings of various N. miranda extracts identified those using acetone, methanol, and ethanol as particularly effective in disrupting Nsp9's ssDNA-binding activity, as evidenced by electrophoretic mobility shift assays. Molecular docking studies highlighted stigmast-5-en-3-ol and lupenone, major components in the leaf extract of N. miranda, as potential inhibitors. The cytotoxic properties of N. miranda leaf extract were examined across NSCLC lines H1975, A549, and H838, focusing on cell survival, apoptosis, and migration. Results showed a dose-dependent cytotoxic effect in the following order: H1975 > A549 > H838 cells, indicating specificity. Enhanced anticancer effects were observed when the extract was combined with afatinib, suggesting synergistic interactions. Flow cytometry indicated that N. miranda leaf extract could induce G2 cell cycle arrest in H1975 cells, potentially inhibiting cancer cell proliferation. Gas chromatography–mass spectrometry (GC–MS) enabled the tentative identification of the 19 most abundant compounds in the leaf extract of N. miranda. These outcomes underscore the dual utility of N. miranda leaf extract in potentially managing SARS-CoV-2 infection through Nsp9 inhibition and offering anticancer benefits against lung carcinoma. These results significantly broaden the potential medical applications of N. miranda leaf extract, suggesting its use not only in traditional remedies but also as a prospective treatment for pulmonary diseases. Overall, our findings position the leaf extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and antiviral therapies, warranting further investigation into its molecular mechanisms and potential clinical applications. [ABSTRACT FROM AUTHOR]

5-Fluorouracil (5-FU) stands as one of the most widely prescribed chemotherapeutics. Despite over 60 years of study, a systematic synopsis of how 5-FU binds to proteins has been lacking. Investigating the specific binding patterns of 5-FU to proteins is essential for identifying additional interacting proteins and comprehending their medical implications. In this review, an analysis of the 5-FU binding environment was conducted based on available complex structures. From the earliest complex structure in 2001 to the present, two groups of residues emerged upon 5-FU binding, classified as P- and R-type residues. These high-frequency interactive residues with 5-FU include positively charged residues Arg and Lys (P type) and ring residues Phe, Tyr, Trp, and His (R type). Due to their high occurrence, 5-FU binding modes were simplistically classified into three types, based on interactive residues (within <4 Å) with 5-FU: Type 1 (P-R type), Type 2 (P type), and Type 3 (R type). In summary, among 14 selected complex structures, 8 conform to Type 1, 2 conform to Type 2, and 4 conform to Type 3. Residues with high interaction frequencies involving the N1, N3, O4, and F5 atoms of 5-FU were also examined. Collectively, these interaction analyses offer a structural perspective on the specific binding patterns of 5-FU within protein pockets and contribute to the construction of a structural interactome delineating the associations of the anticancer drug 5-FU. [ABSTRACT FROM AUTHOR]

The carnivorous pitcher plants of the genus Nepenthes have long been known for their ethnobotanical applications. In this study, we prepared various extracts from the pitcher, stem, and leaf of Nepenthes miranda using 100% ethanol and assessed their inhibitory effects on key enzymes related to skin aging, including elastase, tyrosinase, and hyaluronidase. The cytotoxicity of the stem extract of N. miranda on H838 human lung carcinoma cells were also characterized by effects on cell survival, migration, proliferation, apoptosis induction, and DNA damage. The cytotoxic efficacy of the extract was enhanced when combined with the chemotherapeutic agent 5-fluorouracil (5-FU), indicating a synergistic effect. Flow cytometry analysis suggested that the stem extract might suppress H838 cell proliferation by inducing G2 cell cycle arrest, thereby inhibiting carcinoma cell proliferation. Gas chromatography–mass spectrometry (GC–MS) enabled the tentative identification of the 15 most abundant compounds in the stem extract of N. miranda. Notably, the extract showed a potent inhibition of the human RPA32 protein (huRPA32), critical for DNA replication, suggesting a novel mechanism for its anticancer action. Molecular docking studies further substantiated the interaction between the extract and huRPA32, highlighting bioactive compounds, especially the two most abundant constituents, stigmast-5-en-3-ol and plumbagin, as potential inhibitors of huRPA32's DNA-binding activity, offering promising avenues for cancer therapy. Overall, our findings position the stem extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and anti-skin-aging treatments, warranting further investigation into its molecular mechanisms and potential clinical applications. [ABSTRACT FROM AUTHOR]

Plinia cauliflora has been associated with numerous ethnobotanical applications. In this study, we uncovered that the fruit extract of P. cauliflora, obtained using 50% ethanol, possesses inhibition activity against the Klebsiella pneumoniae single-stranded DNA-binding protein (KpSSB). SSB plays a critical role in cell survival, making it an attractive target for the development of anti-infective drugs. The inhibition activity against KpSSB by the P. cauliflora extract demonstrated an IC50 value of 73 ± 8 μg/mL. By using gas chromatography–mass spectrometry, the chemical content of this extract was tentatively determined. The top 15 compounds (>0.7%) were as follows: 5-hydroxymethylfurfural, 2,3-dihydro-3,5-dihydroxy-6-methyl-4h-pyran-4-one, 2,5-diformylfuran, furfural, ace-tic acid, citraconic anhydride, formic acid, ethyl 4-hydroxy-3-methylbut-2-enoate, furfuryl alcohol, furyl hydroxymethyl ketone, 3-acetyl-3-hydroxyoxolane-2-one, 2,3-dihydro-5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one, 2(3H)-furanone, dihy-dro-4-hydroxy-, hydroxyacetone, and 1-hydroxybut-3-en-2-one. To analyze the possible binding modes, the three most abundant compounds were then subjected to docking analysis. We also investigated whether the P. cauliflora extract exhibited any cytotoxic and antiproliferative effects on the survival of B16F10 melanoma cells. Additionally, we found that the extract of P. cauliflora could inhibit the migration and induce apoptosis of B16F10 cells. The results of this study collectively suggest that P. cauliflora holds potential pharmacological benefits, warranting further exploration for therapeutic applications. [ABSTRACT FROM AUTHOR]

The carnivorous pitcher plants of the genus Nepenthes exhibit many ethnobotanical uses, including treatments of stomachache and fever. In this study, we prepared different extracts from the pitcher, stem, and leaf extracts of Nepenthes miranda obtained using 100% methanol and analyzed their inhibitory effects on recombinant single-stranded DNA-binding protein (SSB) from Klebsiella pneumoniae (KpSSB). SSB is essential for DNA replication and cell survival and thus an attractive target for potential antipathogen chemotherapy. Different extracts prepared from Sinningia bullata, a tuberous member of the flowering plant family Gesneriaceae, were also used to investigate anti-KpSSB properties. Among these extracts, the stem extract of N. miranda exhibited the highest anti-KpSSB activity with an IC50 value of 15.0 ± 1.8 μg/mL. The cytotoxic effects of the stem extract of N. miranda on the survival and apoptosis of the cancer cell lines Ca9-22 gingival carcinoma, CAL27 oral adenosquamous carcinoma, PC-9 pulmonary adenocarcinoma, B16F10 melanoma, and 4T1 mammary carcinoma cells were also demonstrated and compared. Based on collective data, the cytotoxic activities of the stem extract at a concentration of 20 μg/mL followed the order Ca9-22 > CAL27 > PC9 > 4T1 > B16F10 cells. The stem extract of N. miranda at a concentration of 40 μg/mL completely inhibited Ca9-22 cell migration and proliferation. In addition, incubation with this extract at a concentration of 20 μg/mL boosted the distribution of the G2 phase from 7.9% to 29.2% in the Ca9-22 cells; in other words, the stem extract might suppress Ca9-22 cell proliferation by inducing G2 cell cycle arrest. Through gas chromatography–mass spectrometry, the 16 most abundant compounds in the stem extract of N. miranda were tentatively identified. The 10 most abundant compounds in the stem extract of N. miranda were used for docking analysis, and their docking scores were compared. The binding capacity of these compounds was in the order sitosterol > hexadecanoic acid > oleic acid > plumbagin > 2-ethyl-3-methylnaphtho[2,3-b]thiophene-4,9-dione > methyl α-d-galactopyranoside > 3-methoxycatechol > catechol > pyrogallol > hydroxyhydroquinone; thus, sitosterol might exhibit the greatest inhibitory capacity against KpSSB among the selected compounds. Overall, these results may indicate the pharmacological potential of N. miranda for further therapeutic applications. [ABSTRACT FROM AUTHOR]

Nepenthes are carnivorous pitcher plants that have several ethnobotanical uses, such as curing stomachache and fever. Here, we prepared different extracts from the stem, leaf, and pitcher of Nepenthes miranda to further investigate their pharmacological potential. The leaf extract of N. miranda obtained by 100% acetone (N. miranda-leaf-acetone) was used in this study to analyze the cytotoxic activities, antioxidation capacity, antibacterial activity, and allantoinase (ALLase) inhibitory effect of this plant. The cytotoxic effects of N. miranda-leaf-acetone on the survival, apoptosis, and migration of the cancer cell lines PC-9 pulmonary adenocarcinoma, B16F10 melanoma, and 4T1 mammary carcinoma cells were demonstrated. Based on collective data, the cytotoxic activities of N. miranda-leaf-acetone followed the order: B16F10 > 4T1 > PC-9 cells. In addition, the cytotoxic activities of N. miranda-leaf-acetone were synergistically enhanced when co-acting with the clinical anticancer drug 5-fluorouracil. N. miranda-leaf-acetone could also inhibit the activity of ALLase, a key enzyme in the catabolism pathway for purine degradation. Through gas chromatography–mass spectrometry, the 16 most abundant ingredients in N. miranda-leaf-acetone were identified. The top six compounds in N. miranda-leaf-acetone, namely, plumbagin, lupenone, palmitic acid, stigmast-5-en-3-ol, neophytadiene, and citraconic anhydride, were docked to ALLase, and their docking scores were compared. The docking results suggested plumbagin and stigmast-5-en-3-ol as potential inhibitors of ALLase. Overall, these results may indicate the pharmacological potential of N. miranda for further medical applications. [ABSTRACT FROM AUTHOR]

The carnivorous pitcher plant Sarracenia purpurea exhibits many ethnobotanical uses, including the treatments of type 2 diabetes and tuberculosis-like symptoms. In this study, we prepared different extracts from the leaves (pitchers), stems, and roots of S. purpurea and investigated their antioxidant and anticancer properties. To evaluate the extraction efficiency, we individually used different solvents, namely methanol, ethanol, acetone, and distilled water, for S. purpurea extract preparations. The root extract of S. purpurea, obtained by 100% acetone (S. purpurea-root-acetone), had the highest anticancer activities, antioxidation capacity (the DPPH activity with IC50 of 89.3 ± 2.2 μg/mL), antibacterial activities, total phenolic content (33.4 ± 0.7 mg GAE/g), and total flavonoid content (107.9 ± 2.2 mg QUE/g). The most abundant compounds in S. purpurea-root-acetone were identified using gas chromatography–mass spectrometry; 7,8-Dihydro-α-ionone was the major compound present in S. purpurea-root-acetone. In addition, the co-cytotoxicity of S. purpurea-root-acetone (combined with the clinical anticancer drug 5-fluorouracil (5-FU) on the survival, apoptosis, proliferation, and migration of the 4T1 mammary carcinoma) was examined. The combination of 5-FU with S. purpurea-root-acetone could be highly efficient for anti-4T1 cells. We also found that S. purpurea-root-acetone could inhibit the enzymatic activity of human dihydroorotase (huDHOase), an attractive target for potential anticancer chemotherapy. The sic most abundant compounds in S. purpurea-root-acetone were tested using an in silico analysis via MOE-Dock software for their binding affinities. The top-ranked docking conformations were observed for 7,8-dihydro-α-ionone and stigmast-5-en-3-ol, suggesting the inhibition potential against huDHOase. Overall, the collective data in this study may indicate the pharmacological potentials of S. purpurea-root-acetone for possible medical applications. [ABSTRACT FROM AUTHOR]

Single-stranded DNA-binding proteins (SSBs) are essential to cells because they participate in DNA metabolic processes, such as DNA replication, repair, and recombination. Some bacteria possess more than one paralogous SSB. Three similar SSBs, namely, SsbA, SsbB, and SsbC, are found in Staphylococcus aureus. Whether the FDA-approved clinical drug 5-fluorouracil (5-FU) that is used to target the enzyme thymidylate synthase for anticancer therapy can also bind to SSBs remains unknown. In this study, we found that 5-FU could form a stable complex with S. aureus SsbB (SaSsbB). We cocrystallized 5-FU with SaSsbB and solved complex structures to assess binding modes. Two complex forms of the structures were determined, namely, the individual asymmetric unit (two SaSsbB monomers) containing one (PDB entry 7D8J) or two 5-FU molecules (PDB entry 7DEP). The locations of 5-FU in these two SaSsbB complexes were similar regardless of the binding ratio. The structures revealed that residues T12, K13, T30, F48, and N50 of SaSsbB were involved in 5-FU binding. The mutations of T12, K13, and F48 caused the low 5-FU binding activity of SaSsbB, a result consistent with the structural analysis results. Taken together, the complexed structure and the binding mode analysis of SaSsbB extended the anticancer drug 5-FU interactome to include the oligonucleotide/oligosaccharide-binding fold protein. • The FDA-approved clinical drug 5-FU could form a stable complex with SaSsbB. • Two complex forms of the crystal structures were determined. • Residues T12, K13, T30, F48, and N50 of SaSsbB were involved in 5-FU binding. • The 5-FU interactome extended to include the OB fold protein. [ABSTRACT FROM AUTHOR]

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC–MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies. [ABSTRACT FROM AUTHOR]

Dihydropyrimidinase (DHPase) catalyzes the reversible cyclization of dihydrouracil to N -carbamoyl-β-alanine in the second step of the pyrimidine degradation pathway. Whether 5-fluorouracil (5-FU), the best-known fluoropyrimidine that is used to target the enzyme thymidylate synthase for anticancer therapy, can bind to DHPase remains unknown. In this study, we found that 5-FU can form a stable complex with Pseudomonas aeruginosa DHPase (PaDHPase). The crystal structure of PaDHPase complexed with 5-FU was determined at 1.76 Å resolution (PDB entry 6KLK). Various interactions between 5-FU and PaDHPase were examined. Six residues, namely, His61, Tyr155, Asp316, Cys318, Ser289 and Asn337, of PaDHPase were involved in 5-FU binding. Except for Cys318, these residues are also known as the substrate-binding sites of DHPase. 5-FU interacts with the main chains of residues Ser289 (3.0 Å) and Asn337 (3.2 Å) and the side chains of residues Tyr155 (2.8 Å) and Cys318 (2.9 Å). Mutation at either Tyr155 or Cys318 of PaDHPase caused a low 5-FU binding activity of PaDHPase. This structure and the binding mode provided molecular insights into how the dimetal center in DHPase undergoes a conformational change during 5-FU binding. Further research can directly focus on revisiting the role of DHPase in anticancer therapy. • The crystal structure of PaDHPase complexed with 5-FU was determined at 1.76 Å resolution. • His61, Tyr155, Asp316, Cys318, Ser289 and Asn337 of PaDHPase were involved in 5-FU binding. • Mutation at either Tyr155 or Cys318 of PaDHPase caused a low 5-FU binding activity of PaDHPase. • The dimetal center in PaDHPase undergoes a conformational change during 5-FU binding. [ABSTRACT FROM AUTHOR]

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides and considered an attractive target for potential antimalarial, anticancer, and antipathogen chemotherapy. Whether the FDA-approved clinical drug 5-fluorouracil (5-FU) that is used to target the enzyme thymidylate synthase for anticancer therapy can also bind to DHOase remains unknown. Here, we report the crystal structures of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with malate, 5-FU, and 5-aminouracil (5-AU). ScDHOase shares structural similarity with Escherichia coli DHOase. We also characterized the binding of 5-FU and 5-AU to ScDHOase by using the fluorescence quenching method. These complexed structures revealed that residues Arg18, Asn43, Thr106, and Ala275 of ScDHOase were involved in the 5-FU (PDB entry 6L0B) and 5-AU binding (PDB entry 6L0F). Overall, these results provide structural insights that may facilitate the development of new inhibitors targeting DHOase and constitute the 5-FU and 5-AU interactomes for further clinical chemotherapies. • The FDA-approved clinical drug 5-FU could form a stable complex with DHOase. • The crystal structures of ScDHOase complexed with malate, 5-FU, and 5-AU were determined. • Residues Arg18, Asn43, Thr106, Kcx98, and Ala275 of ScDHOase were involved in 5-FU binding. • The 5-FU interactome extended to include DHOase. [ABSTRACT FROM AUTHOR]