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1.
Mol Cell ; 84(11): 2036-2052.e7, 2024 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-38688279

RESUMEN

Alterations of bases in DNA constitute a major source of genomic instability. It is believed that base alterations trigger base excision repair (BER), generating DNA repair intermediates interfering with DNA replication. Here, we show that genomic uracil, a common type of base alteration, induces DNA replication stress (RS) without being processed by BER. In the absence of uracil DNA glycosylase (UNG), genomic uracil accumulates to high levels, DNA replication forks slow down, and PrimPol-mediated repriming is enhanced, generating single-stranded gaps in nascent DNA. ATR inhibition in UNG-deficient cells blocks the repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells upregulates UNG2 to suppress genomic uracil and limit RS, and these cancer cells are hypersensitive to co-treatment with ATR inhibitors and drugs increasing genomic uracil. These results reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability of cancer cells.


Asunto(s)
Reparación del ADN , Replicación del ADN , Inestabilidad Genómica , Uracil-ADN Glicosidasa , Uracilo , Humanos , Uracilo/metabolismo , Uracil-ADN Glicosidasa/metabolismo , Uracil-ADN Glicosidasa/genética , Reparación del ADN/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Daño del ADN , Línea Celular Tumoral , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo
2.
Mol Cell ; 83(23): 4398-4412.e4, 2023 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-37995690

RESUMEN

The DNA replication of mpox virus is performed by the viral polymerase F8 and also requires other viral factors, including processivity factor A22, uracil DNA glycosylase E4, and phosphoprotein H5. However, the molecular roles of these viral factors remain unclear. Here, we characterize the structures of F8-A22-E4 and F8-A22-E4-H5 complexes in the presence of different primer-template DNA substrates. E4 is located upstream of F8 on the template single-stranded DNA (ssDNA) and is catalytically active, highlighting a functional coupling between DNA base-excision repair and DNA synthesis. Moreover, H5, in the form of tetramer, binds to the double-stranded DNA (dsDNA) region downstream of F8 in a similar position as PCNA (proliferating cell nuclear antigen) does in eukaryotic polymerase complexes. Omission of H5 or disruption of its DNA interaction showed a reduced synthesis of full-length DNA products. These structures provide snapshots for the working cycle of the polymerase and generate insights into the mechanisms of these essential factors in viral DNA replication.


Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Monkeypox virus/genética , Monkeypox virus/metabolismo , Replicación Viral , ADN Viral/genética , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo
3.
Plant J ; 113(3): 610-625, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36565011

RESUMEN

Base editing enables precise gene editing without requiring donor DNA or double-stranded breaks. To facilitate base editing tools, a uracil DNA glycosylase inhibitor (UGI) was fused to cytidine deaminase-Cas nickase to inhibit uracil DNA glycosylase (UDG). Herein, we revealed that the bacteriophage PBS2-derived UGI of the cytosine base editor (CBE) could not inhibit archaic Type IV UDG in oligoploid cyanobacteria. To overcome the limitation of the CBE, dCas12a-assisted gene repression of the udg allowed base editing at the desired targets with up to 100% mutation frequencies, and yielded correct phenotypes of desired mutants in cyanobacteria. Compared with the original CBE (BE3), base editing was analyzed within a broader C4-C16 window with a strong TC-motif preference. Using multiplexed CyanoCBE, while udg was repressed, simultaneous base editing at two different sites was achieved with lower mutation frequencies than single CBE. Our discovery of a Type IV UDG that is not inhibited by the UGI of the CBE in cyanobacteria and the development of dCas12a-mediated base editing should facilitate the application of base editing not only in cyanobacteria, but also in archaea and green algae that possess Type IV UDGs. We revealed the bacteriophage-derived UGI of the base editor did not repress Type IV UDG in cyanobacteria. To overcome the limitation, orthogonal dCas12a interference was successfully applied to repress the UDG gene expression in cyanobacteria during base editing occurred, yielding a premature translational termination at desired targets. This study will open a new opportunity to perform base editing with Type IV UDGs in archaea and green algae.


Asunto(s)
Cianobacterias , Uracil-ADN Glicosidasa , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/metabolismo , Edición Génica , ADN , Reparación del ADN , Cianobacterias/genética , Cianobacterias/metabolismo , Citosina
4.
Mol Microbiol ; 120(2): 298-306, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37452011

RESUMEN

DNA glycosylases protect genetic fidelity during DNA replication by removing potentially mutagenic chemically damaged DNA bases. Bacterial Lhr proteins are well-characterized DNA repair helicases that are fused to additional 600-700 amino acids of unknown function, but with structural homology to SecB chaperones and AlkZ DNA glycosylases. Here, we identify that Escherichia coli Lhr is a uracil-DNA glycosylase (UDG) that depends on an active site aspartic acid residue. We show that the Lhr DNA helicase activity is functionally independent of the UDG activity, but that the helicase domains are required for fully active UDG activity. Consistent with UDG activity, deletion of lhr from the E. coli chromosome sensitized cells to oxidative stress that triggers cytosine deamination to uracil. The ability of Lhr to translocate single-stranded DNA and remove uracil bases suggests a surveillance role to seek and remove potentially mutagenic base changes during replication stress.


Asunto(s)
Escherichia coli , Uracil-ADN Glicosidasa , Uracil-ADN Glicosidasa/genética , Uracil-ADN Glicosidasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Secuencia de Aminoácidos , ADN/metabolismo , Uracilo/química , Reparación del ADN , ADN Helicasas/metabolismo , Proteínas Bacterianas/metabolismo
5.
BMC Biotechnol ; 24(1): 17, 2024 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-38566117

RESUMEN

Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10-5 bp-1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity - an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.


Asunto(s)
ADN Polimerasa Dirigida por ADN , Uracilo , Reacción en Cadena de la Polimerasa , ADN Polimerasa Dirigida por ADN/genética , Uracilo/metabolismo , Plásmidos , ADN
6.
Anal Biochem ; 696: 115697, 2024 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-39442604

RESUMEN

PCR-based DNA walking is of efficacy for capturing unknown flanking genomic sequences. Here, an uracil base PCR (UB-PCR) with satisfying specificity has been devised for DNA walking. Primary UB-PCR replaces thymine base with uracil base, resulting in a primary PCR product composed of U-DNAs. A single-primer (primary nested sequence-specific primer) single-cycle amplification, using the four normal bases (adenine, thymine, cytosine, and guanine) as substrate, is then performed on the primary PCR product. Clearly, only those U-DNAs, ended by the primary nested sequence-specific primer at least at one side, will produce the corresponding normal single strands. Next, the single-cycle product undergoes uracil-DNA glycosylase treatment to destroy the U-DNAs, while the normal single strands are unaffected. Afterward, secondary even tertiary PCR is performed to exclusively enrich the target product. The feasibility of UB-PCR has been checked by obtaining unknown sequences bordering the three selected genetic sites.

7.
Anal Biochem ; 692: 115569, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38750682

RESUMEN

Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.


Asunto(s)
Técnicas de Amplificación de Ácido Nucleico , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/genética , Endonucleasas/metabolismo , Endonucleasas/genética
8.
Mol Pharm ; 2024 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-39392428

RESUMEN

An integrated strategy by combining cocrystallization with nanotechnology is developed to optimize in vitro/vivo performances of marine antitumor drug cytarabine (ARA) and further obtain innovative insights into the exploitation of cocrystal alloy nanoformulation. Therein, the optimization of properties and synergistic effects of ARA mainly depends on assembling with uracil (U) and antitumor drug 5-fluorouracil (FU) into the same crystal by cocrystallization technology, while the long-term efficacy is primarily maintained by playing the superiority of nanotechnology. Along this line, the first cocrystal alloy of ARA, viz., ARA-FU-U (0.6:0.4), is successfully obtained and then transformed into a nanocrystal. Single-crystal X-ray diffraction analysis demonstrates that this cocrystal alloy consists of two isomorphic cocrystals of ARA, namely, ARA-FU and ARA-U, in 0.6:0.4 ratio. An R22(8) hydrogen-bonding cyclic system formed by a cytosine fragment of ARA with U or FU can protect and stabilize the amine group on ARA, laying the foundation for regulating its properties. The in vitro/in vivo properties of the cocrystal alloy and its nanocrystals are investigated by theoretical and experimental means. It reveals that both the alloy and nanocrystal can improve physicochemical properties and promote drug absorption, thus bringing to optimized pharmacokinetic behaviors. The nanocrystal produces superior effects than the alloy that helps to extend therapeutic time and action. Particularly, relative to the corresponding binary cocrystal, the synergistic antitumor activity of ARA and FU in the cocrystal alloy is heightened obviously. It may be that U contributes to reducing the degradation of FU, specifically increasing its concentration in tumors to enhance the synergistic effects of FU and ARA. These findings provide new thoughts for the application of cocrystal alloys in the marine drug field and break fresh ground for cocrystal alloy formulations to optimize drug properties.

9.
Purinergic Signal ; 2024 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-38526670

RESUMEN

The P2Y6 receptor, activated by uridine diphosphate (UDP), is a target for antagonists in inflammatory, neurodegenerative, and metabolic disorders, yet few potent and selective antagonists are known to date. This prompted us to use machine learning as a novel approach to aid ligand discovery, with pharmacological evaluation at three P2YR subtypes: initially P2Y6 and subsequently P2Y1 and P2Y14. Relying on extensive published data for P2Y6R agonists, we generated and validated an array of classification machine learning model using the algorithms deep learning (DL), adaboost classifier (ada), Bernoulli NB (bnb), k-nearest neighbors (kNN) classifier, logistic regression (lreg), random forest classifier (rf), support vector classification (SVC), and XGBoost (XGB) classifier models, and the common consensus was applied to molecular selection of 21 diverse structures. Compounds were screened using human P2Y6R-induced functional calcium transients in transfected 1321N1 astrocytoma cells and fluorescent binding inhibition at closely related hP2Y14R expressed in CHO cells. The hit compound ABBV-744, an experimental anticancer drug with a 6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine scaffold, had multifaceted interactions with the P2YR family: hP2Y6R inhibition in a non-surmountable fashion, suggesting that noncompetitive antagonism, and hP2Y1R enhancement, but not hP2Y14R binding inhibition. Other machine learning-selected compounds were either weak (experimental anti-asthmatic drug AZD5423 with a phenyl-1H-indazole scaffold) or inactive in inhibiting the hP2Y6R. Experimental drugs TAK-593 and GSK1070916 (100 µM) inhibited P2Y14R fluorescent binding by 50% and 38%, respectively, and all other compounds by < 20%. Thus, machine learning has led the way toward revealing previously unknown modulators of several P2YR subtypes that have varied effects.

10.
Clin Chem Lab Med ; 62(12): 2415-2424, 2024 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-38896022

RESUMEN

OBJECTIVES: Dihydropyrimidine dehydrogenase (DPD) deficiency is the main cause of severe fluoropyrimidine-related toxicities. The best strategy for identifying DPD-deficient patients is still not defined. The EMA recommends targeted DPYD genotyping or uracilemia (U) testing. We analyzed the concordance between both approaches. METHODS: This study included 19,376 consecutive French patients with pre-treatment plasma U, UH2 and targeted DPYD genotyping (*2A, *13, D949V, *7) analyzed at Eurofins Biomnis (2015-2022). RESULTS: Mean U was 9.9 ± 10.1 ng/mL (median 8.7, range 1.6-856). According to French recommendations, 7.3 % of patients were partially deficient (U 16-150 ng/mL) and 0.02 % completely deficient (U≥150 ng/mL). DPYD variant frequencies were *2A: 0.83 %, *13: 0.17 %, D949V: 1.16 %, *7: 0.05 % (2 homozygous patients with U at 22 and 856 ng/mL). Variant carriers exhibited higher U (median 13.8 vs. 8.6 ng/mL), and lower UH2/U (median 7.2 vs. 11.8) and UH2/U2 (median 0.54 vs. 1.37) relative to wild-type patients (p<0.00001). Sixty-six% of variant carriers exhibited uracilemia <16 ng/mL, challenging correct identification of DPD deficiency based on U. The sensitivity (% patients with a deficient phenotype among variant carriers) of U threshold at 16 ng/mL was 34 %. The best discriminant marker for identifying variant carriers was UH2/U2. UH2/U2<0.942 (29.7 % of patients) showed enhanced sensitivity (81 %) in identifying deleterious genotypes across different variants compared to 16 ng/mL U. CONCLUSIONS: These results reaffirm the poor concordance between DPD phenotyping and genotyping, suggesting that both approaches may be complementary and that targeted DPYD genotyping is not sufficiently reliable to identify all patients with complete deficiency.


Asunto(s)
Deficiencia de Dihidropirimidina Deshidrogenasa , Dihidrouracilo Deshidrogenasa (NADP) , Genotipo , Uracilo , Humanos , Dihidrouracilo Deshidrogenasa (NADP)/genética , Uracilo/análogos & derivados , Uracilo/sangre , Estudios Retrospectivos , Masculino , Femenino , Anciano , Persona de Mediana Edad , Deficiencia de Dihidropirimidina Deshidrogenasa/genética , Deficiencia de Dihidropirimidina Deshidrogenasa/diagnóstico , Adulto , Anciano de 80 o más Años , Adulto Joven , Adolescente
11.
Jpn J Clin Oncol ; 2024 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-39255996

RESUMEN

BACKGROUND: The use of adjuvant osimertinib for epidermal growth factor receptor (EGFR) mutants is expected to expand to earlier stage I in the future, potentially competing with the current standard of care, oral tegafur/uracil (UFT), in Japan. However, the effect of EGFR mutation status on the therapeutic effect of UFT remains unclear. This study was conducted as an exploratory analysis of a retrospective observational study that investigated the real-world data of postoperative adjuvant chemotherapy in Japan (CSPOR-LC03). METHODS: Between 2008 and 2013, 1812 patients with completely resected adenocarcinoma diagnosed as pathologic stage I (T1 > 2 cm, TNM classification, sixth edition) who have maintained organ function, and no history of other cancers were included. The primary endpoint was the 5-year disease-free survival (DFS) rate, and we compared this rate between four groups classified based on the administration of adjuvant UFT and EGFR mutation status. RESULTS: Of the 933 (51%) patients with EGFR mutations, 394 underwent adjuvant UFT therapy. Of the 879 (49%) patients without EGFR mutations, 393 underwent adjuvant UFT therapy. The 5-year DFS of UFT+/EGFR+ and UFT-/EGFR+ patients were 82.0 and 87.1%, respectively, and those of UFT+/EGFR- and UFT-/EGFR- patients were 80.0 and 86.9%, respectively. DFS was significantly worse in the UFT+ group than in the UFT- group (P = 0.015). Adjuvant UFT therapy was not an independent prognostic factor for DFS, regardless of the EGFR mutation status. CONCLUSION: In pathologic stage I (>2 cm) lung adenocarcinomas with EGFR mutation, the survival benefit of adjuvant UFT was not observed.

12.
Bioorg Chem ; 144: 107176, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38330721

RESUMEN

Repurposing drugs can significantly reduce the time and costs associated with drug discovery and development. However, many drug compounds possess intrinsic fluorescence, resulting in aberrations such as auto-fluorescence, scattering and quenching, in fluorescent high-throughput screening assays. To overcome these drawbacks, time-resolved technologies have received increasing attention. In this study, we have developed a rapid and efficient screening platform based on time-resolved emission spectroscopy in order to screen for inhibitors of the DNA repair enzyme, uracil-DNA glycosylase (UDG). From a database of 1456 FDA/EMA-approved drugs, sodium stibogluconate was discovered as a potent UDG inhibitor. This compound showed synergistic cytotoxicity against 5-fluorouracil-resistant cancer cells. This work provides a promising future for time-resolved technologies for high-throughput screening (HTS), allowing for the swift identification of bioactive compounds from previously overlooked scaffolds due to their inherent fluorescence properties.


Asunto(s)
Neoplasias de la Próstata , Uracil-ADN Glicosidasa , Humanos , Masculino , Uracil-ADN Glicosidasa/química , Oligonucleótidos , Gluconato de Sodio Antimonio , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Detección Precoz del Cáncer
13.
Biol Pharm Bull ; 47(7): 1275-1281, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38987176

RESUMEN

The generation of DNA damage causes mutations and consequently cancer. Reactive oxygen species are important sources of DNA damage and some mutation signatures found in human cancers. 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the most abundant oxidized bases and induces a G→T transversion mutation at the modified site. The damaged G base also causes untargeted base substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations) in human cells, and the cytosine deaminase apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is involved in the mutation process. The deaminated cytosine, i.e., uracil, bases are expected to be removed by uracil DNA glycosylase. Most of the substitution mutations at the G bases of 5'-GpA-3' might be caused by abasic sites formed by the glycosylase. In this study, we expressed the uracil DNA glycosylase inhibitor from Bacillus subtilis bacteriophage PBS2 in human U2OS cells and examined the effects on the GO-induced action-at-a-distance mutations. The inhibition of uracil DNA glycosylase increased the mutation frequency, and in particular, the frequency of G→A transitions. These results indicated that uracil DNA glycosylase, in addition to APOBEC3, is involved in the untargeted mutation process induced by GO.


Asunto(s)
Guanina , Mutación , Uracil-ADN Glicosidasa , Humanos , Guanina/análogos & derivados , Guanina/metabolismo , Uracil-ADN Glicosidasa/metabolismo , Uracil-ADN Glicosidasa/genética , Línea Celular Tumoral , Daño del ADN , Bacillus subtilis/genética , Bacteriófagos/genética
14.
Int J Clin Oncol ; 29(9): 1284-1292, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38833114

RESUMEN

BACKGROUND: The efficacy of adjuvant chemotherapy for high-risk stage II colon cancer (CC) has not been well established. Using propensity score matching, we previously reported that the 3-year disease-free survival (DFS) rate was significantly higher in patients treated with uracil and tegafur plus leucovorin (UFT/LV) against surgery alone. We report the final results, including updated 5-year overall survival (OS) rates and risk factor analysis outcomes. METHODS: In total, 1902 high-risk stage II CC patients with T4, perforation/penetration, poorly differentiated adenocarcinoma/mucinous carcinoma, and/or < 12 dissected lymph nodes were enrolled in this prospective, non-randomized controlled study based on their self-selected treatment. Oral UFT/LV therapy was administered for six months after surgery. RESULTS: Of the 1880 eligible patients, 402 in Group A (surgery alone) and 804 in Group B (UFT/LV) were propensity score-matched. The 5-year DFS rate was significantly higher in Group B than in Group A (P = 0.0008). The 5-year OS rates were not significantly different between groups. The inverse probability of treatment weighting revealed significantly higher 5-year DFS (P = 0.0006) and 5-year OS (P = 0.0122) rates in group B than in group A. Multivariate analyses revealed that male sex, age ≥ 70 years, T4, < 12 dissected lymph nodes, and no adjuvant chemotherapy were significant risk factors for DFS and/or OS. CONCLUSION: The follow-up data from our prospective non-randomized controlled study revealed a considerable survival advantage in DFS offered by adjuvant chemotherapy with UFT/LV administered for six months over surgery alone in individuals with high-risk stage II CC. TRIAL REGISTRATION: Japan Registry of Clinical Trials: jRCTs031180155 (date of registration: 25/02/2019), UMIN Clinical Trials Registry: UMIN000007783 (date of registration: 18/04/2012).


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias del Colon , Leucovorina , Recurrencia Local de Neoplasia , Estadificación de Neoplasias , Puntaje de Propensión , Tegafur , Uracilo , Humanos , Tegafur/administración & dosificación , Tegafur/uso terapéutico , Masculino , Femenino , Anciano , Uracilo/administración & dosificación , Uracilo/uso terapéutico , Persona de Mediana Edad , Leucovorina/uso terapéutico , Leucovorina/administración & dosificación , Quimioterapia Adyuvante , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Estudios Prospectivos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Factores de Riesgo , Recurrencia Local de Neoplasia/tratamiento farmacológico , Adulto , Supervivencia sin Enfermedad , Anciano de 80 o más Años
15.
Arch Pharm (Weinheim) ; 357(1): e2300374, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37902389

RESUMEN

A series of salicylidene uracil (1-18) derived from 5-aminouracil and substituted salicylaldehydes were analyzed for cytotoxic activity and enzyme inhibitory potency. Nine out of eighteen derivatives (6-8, 10, 12-15, 18) are novel molecules synthesized for the first time in this work, and other derivatives were previously synthesized by our group. The compounds were characterized by Proton nuclear magnetic resonance, carbon nuclear magnetic resonance, fourier transform infrared spectroscopy, and elemental analysis. All compounds were tested for their in vitro cytotoxicity against PC-3 (human prostate adenocarcinoma), A549 (human alveolar adenocarcinoma), and SHSY-5Y (human neuroblastoma) cancer cell lines and the nontumorigenic HEK293 (human embryonic kidney cells) cell line. The 3,5-di-tert-butylsalicylaldehyde derived compound (8) was toxic to PC-3 human prostate adenocarcinoma cells, showing a promising IC50 value at 7.05 ± 0.76 µM. The present study also aimed to evaluate the inhibitory effects of the compounds against several key enzymes, namely carbonic anhydrase I and II (CA I and CA II), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and glutathione reductase (GR), which are implicated in various global disorders, such as Alzheimer's disease, epilepsy, cancer, malaria, diabetes, and glaucoma. The inhibitory profiles of the tested compounds were assessed by determining their Ki values, which ranged from 2.96 to 9.24 nM for AChE, 3.78 to 12.57 nM for BChE, 8.42 to 25.74 nM for CA I, 7.24 to 19.74 nM for CA II, and 0.541 to 1.124 µM for GR. Molecular docking studies were also performed for all compounds. Most derivatives exhibited much more effective inhibitory action compared with clinically used standards. Thus, our findings indicate that the salicylidene derivatives presented in this study are promising drug candidates that need further evaluation.


Asunto(s)
Adenocarcinoma , Antineoplásicos , Humanos , Butirilcolinesterasa/metabolismo , Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/química , Inhibidores de Anhidrasa Carbónica , Simulación del Acoplamiento Molecular , Células HEK293 , Relación Estructura-Actividad , Antineoplásicos/farmacología , Estructura Molecular
16.
J Asian Nat Prod Res ; 26(2): 259-268, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38347748

RESUMEN

A series of novel substituted uracil-1'(N)-acetic acid esters (5-9) and 4-pyridone-1'(N)-acetic acid esters (10-11) of 20(S)-camptothecins (CPTs) have been synthesized by the acylation method. All of these new esters were assayed for in vitro cytotoxicity against five human cancer cell lines A549, Bel7402, BGC-823, HCT-8 and A2780. The in vitro bioassay results showed that all the synthesized compounds 5-11 had cytotoxities that were higher than TPT and comparable to CPT on these five tumor cell lines, some of them even showed comparable or superior cytotoxic activity to CPT. The in vitro data exhibited the cytotoxicity of the ester depended on that of its parent compound. The ester 5, 6, 8, 10, 11 even possessed the cytotoxity activity comparable to or even a little better than CPT on A549, HCT-8 and A2780. The compound 11 had the same level of cytoxity on Bel7402 as that of CPT. Here the synthesis and the in vitro antitumor evaluation of a series of novel 20-O-linked substituted uracil-1'(N)-acetic acid and 4-pyridone-1'(N)-acetic acid esters derivatives of CPTs are reported.


Asunto(s)
Antineoplásicos , Neoplasias Ováricas , Piridonas , Humanos , Femenino , Ácido Acético , Línea Celular Tumoral , Uracilo/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Camptotecina/farmacología , Antineoplásicos/farmacología , Ésteres/farmacología , Relación Estructura-Actividad
17.
Int J Mol Sci ; 25(17)2024 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-39273338

RESUMEN

The pyrimidine heterocycle plays an important role in anticancer research. In particular, the pyrimidine derivative families of uracil show promise as structural scaffolds relevant to cervical cancer. This group of chemicals lacks data-driven machine learning quantitative structure-activity relationships (QSARs) that allow for generalization and predictive capabilities in the search for new active compounds. To achieve this, a dataset of pyrimidine and uracil compounds from ChEMBL were collected and curated. A workflow was developed for data-driven machine learning QSAR using an intuitive dataset design and forwards selection of molecular descriptors. The model was thoroughly externally validated against available data. Blind validation was also performed by synthesis and antiproliferative evaluation of new synthesized uracil-based and pyrimidine derivatives. The most active compound among new synthesized derivatives, 2,4,5-trisubstituted pyrimidine was predicted with the QSAR model with differences of 0.02 compared to experimentally tested activity.


Asunto(s)
Antineoplásicos , Proliferación Celular , Pirimidinas , Relación Estructura-Actividad Cuantitativa , Uracilo , Uracilo/química , Uracilo/análogos & derivados , Uracilo/farmacología , Uracilo/síntesis química , Pirimidinas/química , Pirimidinas/farmacología , Pirimidinas/síntesis química , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Proliferación Celular/efectos de los fármacos , Aprendizaje Automático , Línea Celular Tumoral
18.
Mol Biol (Mosk) ; 58(2): 325-332, 2024.
Artículo en Ruso | MEDLINE | ID: mdl-39355889

RESUMEN

We have previously shown that 5-arylaminouracil derivatives can inhibit HIV-1, herpesviruses, mycobacteria, and other pathogens through various mechanisms. The purpose of this study was to evaluate the potential of 5-arylaminouracils and their derivatives against leukemia, neuroblastoma, and glial brain tumors. 5-Aminouracils with various substituents and their 5'-norcabocyclic and ribo derivatives were screened for cytotoxicity against two neuroblastoma cell lines (SH-SY5Y and IMR-32), K-562 lymphoblastic cells, HL-60 promyeoloblastic cells, and low-passage variants of well-differentiated glioblastoma multiforme (GBM5522 and GBM6138). Cytotoxicity assessment by the standard MTT test showed that most of the compounds lack significant toxicity towards the above cells. However, 5-(4-isopropylphenylamine)uracil and 5-(4-tert-butylphenylamine)uracil exhibited a dose-dependent toxic effect towards the GBM6138 cell line with half-maximal inhibitory concentrations (IC50) of 9 and 2.3 µÐœ, respectively. Antitumor activity was for the first time demonstrated for compounds of this type and can serve as a starting point for further research.


Asunto(s)
Uracilo , Humanos , Uracilo/análogos & derivados , Uracilo/farmacología , Uracilo/toxicidad , Antineoplásicos/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología
19.
Biochem Biophys Res Commun ; 639: 126-133, 2023 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-36481356

RESUMEN

Aquaporin (AQP) water channels facilitate water transport across cellular membranes and are essential in regulation of body water balance. Moreover, several AQPs are overexpressed or ectopically expressed in breast cancer. Interestingly, several in vitro studies have suggested that AQPs can affect the response to conventional anticancer chemotherapies. Therefore, we took a systematic approach to test how AQP1, AQP3 and AQP5, which are often over-/ectopically expressed in breast cancer, affect total viability of 3-dimensional (3D) breast cancer cell spheroids when treated with the conventional anticancer chemotherapies Cisplatin, 5-Fluorouracil (5-FU) and Doxorubicin, a Combination of the three drugs as well as the Combination plus the Ras inhibitor Salirasib. Total viability of spheroids overexpressing AQP1 were decreased by all treatments except for 5-FU, which increased total viability by 20% compared to DMSO treated controls. All treatments reduced viability of spheroids overexpressing AQP3. In contrast, only Doxorubicin, Combination and Combination + Salirasib reduced total viability of spheroids overexpressing AQP5. Thus, this study supports a significant role of AQPs in the response to conventional chemotherapies. Evaluating the role of individual proteins that contribute to resistance to chemotherapies is essential in advancing personalized medicine in breast carcinomas.


Asunto(s)
Acuaporinas , Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Acuaporinas/metabolismo , Fluorouracilo/farmacología , Doxorrubicina/farmacología , Acuaporina 1/genética , Acuaporina 1/metabolismo , Acuaporina 5/metabolismo , Acuaporina 3/genética , Acuaporina 3/metabolismo , Acuaporina 4 , Acuaporina 2
20.
Chembiochem ; 24(10): e202200765, 2023 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-36883884

RESUMEN

DNA repair proteins participate in extensive protein-protein interactions that promote the formation of DNA repair complexes. To understand how complex formation affects protein function during base excision repair, we used SpyCatcher/SpyTag ligation to produce a covalent complex between human uracil DNA glycosylase (UNG2) and replication protein A (RPA). Our covalent "RPA-Spy-UNG2" complex could identify and excise uracil bases in duplex areas next to ssDNA-dsDNA junctions slightly faster than the wild-type proteins, but this was highly dependent on DNA structure, as the turnover of the RPA-Spy-UNG2 complex slowed at DNA junctions where RPA tightly engaged long ssDNA sections. Conversely, the enzymes preferred uracil sites in ssDNA where RPA strongly enhanced uracil excision by UNG2 regardless of ssDNA length. Finally, RPA was found to promote UNG2 excision of two uracil sites positioned across a ssDNA-dsDNA junction, and dissociation of UNG2 from RPA enhanced this process. Our approach of ligating together RPA and UNG2 to reveal how complex formation affects enzyme function could be applied to examine other assemblies of DNA repair proteins.


Asunto(s)
Reparación del ADN , Proteína de Replicación A , Uracil-ADN Glicosidasa , Humanos , ADN/metabolismo , Replicación del ADN , ADN de Cadena Simple , Cinética , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Uracilo/metabolismo , Uracil-ADN Glicosidasa/genética
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