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1.
Viruses ; 13(10)2021 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-34696413

RESUMO

The causative agent of COVID-19, SARS-CoV-2, gains access to cells through interactions of the receptor-binding domain (RBD) on the viral S protein with angiotensin-converting enzyme 2 (ACE2) on the surface of human host cells. Systematic evolution of ligands by exponential enrichment (SELEX) was used to generate aptamers (nucleic acids selected for high binding affinity to a target) to the RBD made from 2'-fluoro-arabinonucleic acid (FANA). The best selected ~79 nucleotide aptamers bound the RBD (Arg319-Phe541) and the larger S1 domain (Val16-Arg685) of the 1272 amino acid S protein with equilibrium dissociation constants (KD,app) of ~10-20 nM, and binding half-life for the RBD, S1 domain, and full trimeric S protein of 53 ± 18, 76 ± 5, and 127 ± 7 min, respectively. Aptamers inhibited the binding of the RBD to ACE2 in an ELISA assay. Inhibition, on a per weight basis, was similar to neutralizing antibodies that were specific for RBD. Aptamers demonstrated high specificity, binding with about 10-fold lower affinity to the related S1 domain from the original SARS virus, which also binds to ACE2. Overall, FANA aptamers show affinities comparable to previous DNA aptamers to RBD and S1 protein and directly block receptor interactions while using an alternative Xeno-nucleic acid (XNA) platform.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Arabinonucleotídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Ligação Viral/efeitos dos fármacos , COVID-19/tratamento farmacológico , Humanos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Domínios Proteicos/genética , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
2.
Acc Chem Res ; 54(9): 2287-2297, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33861067

RESUMO

This Account highlights the structural features that render 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA) an ideal tool for mimicking DNA secondary structures and probing biomolecular interactions relevant to chemical biology.The high binding affinity of FANA to DNA and RNA has had implications in therapeutics. FANA can hybridize to complementary RNA, resulting in a predominant A-form helix stabilized by a network of 2'F-H8(purine) pseudohydrogen bonding interactions. We have shown that FANA/RNA hybrids are substrates of RNase H and Ago2, both implicated in the mechanism of action of antisense oligonucleotides (ASOs) and siRNA, respectvely. This knowledge has helped us study the conformational preferences of ASOs and siRNA as well as crRNA in CRISPR-associated Cas9, thereby revealing structural features crucial to biochemical activity.Additionally, FANA is of particular use in stabilizing noncanonical DNA structures. For instance, we have taken advantage of the anti N-glycosidic bond conformation of FANA monomers to induce a parallel topology in telomeric G-quadruplexes. Subsequent single-molecule FRET studies elucidated the mechanism by which these parallel G-quadruplexes are recognized and extended by telomerase. Similarly, we have utilized FANA to stabilize elusive telomeric i-motifs in the presence of concomitant parallel G-quadruplexes and under physiological conditions, thereby reinforcing their potential relevance to telomere biology. In another study, we adapted microarray technology and used FANA substitutions to enhance the binding affinity of the G-quadruplex thrombin-binding aptamer to its thrombin target.Finally, we discovered that DNA polymerases can synthesize FANA strands from DNA templates. On the basis of this property, other groups demonstrated that FANA, like DNA, can store hereditary information. They did so by engineering polymerases to efficiently transfer genetic information from DNA to FANA and retrieve it back into DNA. Subsequent studies showed that FANA could be evolved to acquire ribozyme-like endonuclease or ligase activity and to form high-affinity aptamers.Overall, the implications of these studies are remarkable because they promise a deeper understanding of human biochemistry for innovative therapeutic avenues. This Account summarizes past achievements and provides an outlook for inspiring the increased use of FANA in biological applications and fostering interdisciplinary collaborations.


Assuntos
Arabinonucleotídeos/química , DNA/química , RNA/química , Arabinonucleotídeos/biossíntese , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Conformação de Ácido Nucleico , RNA/metabolismo
3.
Sci Rep ; 11(1): 2760, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531619

RESUMO

Candidatus Liberibacter asiaticus (CLas), a bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, is the causal agent of citrus greening disease, or Huanglongbng (HLB). Currently, vector population suppression with insecticides and tree removal are the most effective strategies for managing the HLB pathosystem. In this study, we assessed the bactericidal capabilities of 2'-deoxy-2'-fluoro-D-arabinonucleic acid antisense oligonucleotides (FANA ASO) both in vitro and in vivo by (1) confirming their capacity to penetrate insect cells, (2) silencing bacterial essential genes, and (3) quantifying reductions in bacterial titer and D. citri transmission. We confirmed that FANA ASO are able to penetrate insect cells without the use of a delivery agent. Expression of an essential gene in the D. citri endosymbiont, Wolbachia (wDi), significantly decreased by 30% following incubation with a wDi-specific FANA ASO. Viability of isolated wDi cells also decreased in response to the FANA ASO treatment. Delivery of a CLas-specific FANA ASO to infected adult D. citri in feeding assays resulted in significant silencing of a CLas essential gene. CLas relative density and transmission were significantly lower among D. citri fed FANA ASO in diet compared to untreated insects. Root infusions of a CLas-specific FANA ASO into infected Citrus trees significantly reduced CLas titer during a 30-day trial. Our results suggest that FANA ASO targeting insect-transmitted plant bacteria or insect endosymbionts may be useful tool for integrated management of agricultural pathogens.


Assuntos
Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Hemípteros/microbiologia , Oligonucleotídeos Antissenso/administração & dosagem , Doenças das Plantas/prevenção & controle , Rhizobiaceae/efeitos dos fármacos , Animais , Arabinonucleotídeos/administração & dosagem , Arabinonucleotídeos/genética , Linhagem Celular , Citrus/microbiologia , Drosophila , Inativação Gênica , Hemípteros/efeitos dos fármacos , Insetos Vetores/efeitos dos fármacos , Insetos Vetores/microbiologia , Oligonucleotídeos Antissenso/genética , Doenças das Plantas/microbiologia , Rhizobiaceae/genética , Rhizobiaceae/patogenicidade , Simbiose/efeitos dos fármacos , Simbiose/genética
4.
J Am Chem Soc ; 142(5): 2317-2326, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31913615

RESUMO

The abiotic synthesis of ribonucleotides is thought to have been an essential step toward the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2'-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2'-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2'-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabinonucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2'-deoxy- or arabinonucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.


Assuntos
Arabinonucleotídeos/química , Desoxirribonucleotídeos/química , Modelos Químicos , Prebióticos , RNA/química , Ribonucleotídeos/química , Polimerização
5.
Chembiochem ; 21(7): 1001-1006, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31680396

RESUMO

The discovery of synthetic genetic polymers (XNAs) with catalytic activity demonstrates that natural genetic polymers are not unique in their ability to function as enzymes. However, all known examples of in vitro selected XNA enzymes function with lower activity than their natural counterparts, suggesting that XNAs might be limited in their ability to fold into structures with high catalytic activity. To explore this problem, we evaluated the catalytic potential of FANAzyme 12-7, an RNA-cleaving catalyst composed entirely of 2'-fluoroarabino nucleic acid (FANA) that was evolved to cleave RNA at a specific phosphodiester bond located between an unpaired guanine and a paired uracil in the substrate recognition arm. Here, we show that this activity extends to chimeric DNA substrates that contain a central riboguanosine (riboG) residue at the cleavage site. Surprisingly, FANAzyme 12-7 rivals known DNAzymes that were previously evolved to cleave chimeric DNA substrates under physiological conditions. These data provide convincing evidence that FANAzyme 12-7 maintains the catalytic potential of equivalent DNAzymes, which has important implications for the evolution of XNA catalysts and their contributions to future applications in synthetic biology.


Assuntos
Arabinonucleotídeos/química , DNA Catalítico/metabolismo , RNA/metabolismo , Catálise , DNA Catalítico/química , Cinética , Conformação de Ácido Nucleico , Polímeros/química , Especificidade por Substrato , Temperatura de Transição
6.
Phys Chem Chem Phys ; 21(41): 22869-22878, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31599901

RESUMO

The non-biological 2'-deoxy-2'-fluoro-arabinonucleic acid (2'F-ANA) may be used as a valid alternative to DNA in biomedical and electronic applications because of its higher resistance to hydrolysis and nuclease degradation. However, the advantage of using 2'F-ANA in such applications also depends on its charge-transfer properties compared to DNA. In this study, we compare the charge conduction properties of model 2'F-ANA and DNA double-strands, using structural snapshots from MD simulations to calculate the electronic couplings and reorganization energies associated with the hole transfer steps between adjacent nucleobase pairs. Inserting these charge-transfer parameters into a kinetic model for charge conduction, we find similar conductive properties for DNA and 2'F-ANA. Moreover, we find that 2'F-ANA's enhanced chemical stability does not correspond to a reduction in the nucleobase π-stack structural flexibility relevant to both electronic couplings and reorganization free energies. Our results promote the use of 2'F-ANA in applications that can be based on charge transport, such as biosensing and chip technology, where its chemical stability and conductivity can advantageously combine.


Assuntos
Arabinonucleotídeos/química , Biotecnologia/métodos , DNA/química , Eletrônica , Simulação de Dinâmica Molecular
7.
ACS Chem Biol ; 14(10): 2166-2175, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31560515

RESUMO

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is the iterative process by which nucleic acids that can bind with high affinity and specificity (termed aptamers) to specific protein targets are selected. Using a SELEX protocol adapted for Xeno-Nucleic Acid (XNA) as a suitable substrate for aptamer generation, 2'-fluoroarabinonucleic acid (FANA) was used to select several related aptamers to HIV-1 integrase (IN). IN bound FANA aptamers with equilibrium dissociation constants (KD,app) of ∼50-100 pM in a buffer with 200 mM NaCl and 6 mM MgCl2. Comparisons to published HIV-1 IN RNA and DNA aptamers as well as IN genomic binding partners indicated that FANA aptamers bound more than 2 orders of magnitude more tightly to IN. Using a combination of RNA folding algorithms and covariation analysis, all strong binding aptamers demonstrated a common four-way junction structure, despite significant sequence variation. IN aptamers were selected from the same starting library as FA1, a FANA aptamer that binds with pM affinity to HIV-1 Reverse Transcriptase (RT). It contains a 20-nucleotide 5' DNA sequence followed by 59 FANA nucleotides. IN-1.1 (one of the selected aptamers) potently inhibited IN activity and intasome formation in vitro. Replacing the FANA nucleotides of IN-1.1 with 2'-fluororibonucleic acid (F-RNA), which has the same chemical formula but with a ribose rather than arabinose sugar conformation, dramatically reduced binding, suggesting that FANA adopts unique structural conformations that promote binding to HIV-1 IN.


Assuntos
Aptâmeros de Nucleotídeos/química , Arabinonucleotídeos/química , Integrase de HIV/química , HIV-1/enzimologia , RNA/química , Sequência de Bases , Técnica de Seleção de Aptâmeros , Alinhamento de Sequência
8.
Nucleic Acids Res ; 47(13): 6973-6983, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31170294

RESUMO

Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2'-deoxy-2'-fluoro-ß-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.


Assuntos
Proteínas de Bactérias/química , DNA Polimerase I/química , Geobacillus stearothermophilus/enzimologia , DNA Polimerase Dirigida por RNA/química , Arabinonucleotídeos/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , DNA Polimerase I/isolamento & purificação , DNA Polimerase I/metabolismo , DNA Bacteriano/metabolismo , Modelos Moleculares , Hibridização de Ácido Nucleico , Nucleosídeos/metabolismo , Ligação Proteica , Conformação Proteica , DNA Polimerase Dirigida por RNA/isolamento & purificação , DNA Polimerase Dirigida por RNA/metabolismo , Relação Estrutura-Atividade , Moldes Genéticos
9.
Phys Chem Chem Phys ; 20(41): 26063-26067, 2018 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-30191207

RESUMO

The non-biological nucleic acid 2'-deoxy-2'-fluoro-arabinonucleic acid (2'F-ANA) may be of use because of its higher chemical stability than DNA in terms of resistance to hydrolysis and nuclease degradation. In order to investigate the charge transfer characteristics of 2'F-ANA, of relevance to applications in nucleic acid-based biosensors and chip technologies, we compare the electronic couplings for hole transfer between stacked nucleobase pairs in DNA and 2'F-ANA by carrying out density functional theory (DFT) calculations on geometries taken from molecular dynamics simulations. We find similar averages and distribution widths of the base-pair couplings in the two systems. On the basis of this result, 2'F-ANA is expected to have charge transfer properties similar to those of DNA, while offering the advantage of enhanced chemical stability. As such, 2'F-ANA may serve as a possible alternative to DNA for use in a broad range of nanobiotechnological applications. Furthermore, we show that the (experimentally observed) enhanced chemical stability resulting from the backbone modifications does not cause reduced fluctuations of the base-pair electronic couplings around the values found for "ideal" B-DNA (with standard step parameter values). Our study also supports the use of a DFT implementation, with the M11 functional, of the wave function overlap method to compute effective electronic couplings in nucleic acid systems.


Assuntos
Arabinonucleotídeos/química , Arabinonucleotídeos/metabolismo , Pareamento de Bases , DNA/química , DNA/metabolismo , Transporte de Elétrons , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismo , RNA/química , RNA/metabolismo
10.
Biol Blood Marrow Transplant ; 23(10): 1701-1713, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28684371

RESUMO

A prospective multicenter study was conducted to characterize the pharmacokinetics (PK) and pharmacodynamics (PD) of fludarabine plasma (f-ara-a) and intracellular triphosphate (f-ara-ATP) in children undergoing hematopoietic cell transplantation (HCT) and receiving fludarabine with conditioning. Plasma and peripheral blood mononuclear cells (PBMCs) were collected over the course of therapy for quantitation of f-ara-a and f-ara-ATP. Nonlinear mixed-effects modeling was used to develop the PK model, including identification of covariates impacting drug disposition. Data from a total of 133 children (median age, 5 years; range, .2 to 17.9) undergoing HCT for a variety of malignant and nonmalignant disorders were available for PK-PD modeling. The implementation of allometric scaling of PK parameters alone was insufficient to describe drug clearance, particularly in very young children. Renal impairment was predicted to increase drug exposure across all ages. The rate of f-ara-a entry into PBMCs (expressed in pmoles per million cells) decreased over the course of therapy, resulting in 78% lower f-ara-ATP after the fourth dose (1.7 pmoles/million cells [range, .2 to 7.2]) compared with first dose (7.9 pmoles/million cells [range, .7 to 18.2]). The overall incidence of treatment-related mortality (TRM) was low at 3% and 8% at days 60 and 360, respectively, and no association with f-ara-a exposure and TRM was found. In the setting of malignancy, disease-free survival was highest at 1 year after HCT in subjects achieving a systemic f-ara-a cumulative area under the curve (cAUC) greater than 15 mg*hour/L compared to patients with a cAUC less than 15 mg*hour/L (82.6% versus 52.8% P = .04). These results suggest that individualized model-based dosing of fludarabine in infants and young children may reduce morbidity and mortality through improved rates of disease-free survival and limiting drug-related toxicity. ClinicalTrials.gov Identifier: NCT01316549.


Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Modelagem Computacional Específica para o Paciente , Vidarabina/análogos & derivados , Adolescente , Arabinonucleotídeos , Biomarcadores Farmacológicos , Criança , Pré-Escolar , Intervalo Livre de Doença , Humanos , Lactente , Taxa de Depuração Metabólica , Medicina de Precisão , Estudos Prospectivos , Transplantados , Condicionamento Pré-Transplante/métodos , Vidarabina/administração & dosagem , Vidarabina/farmacocinética , Vidarabina/uso terapêutico , Vidarabina/toxicidade
11.
Nucleic Acids Res ; 45(4): 1619-1632, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28100695

RESUMO

In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2΄-Fluoroarabinonucleic acid (2΄F-ANA) is a prime candidate for such use in microarrays. Indeed, 2΄F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered. We thus synthesized the first microarrays containing 2΄F-ANA and 2΄F-ANA/DNA chimeric sequences to fully map the binding affinity landscape of the TBA1 thrombin-binding G-quadruplex aptamer containing all 32 768 possible DNA-to-2΄F-ANA mutations. The resulting microarray was screened against thrombin to identify a series of promising 2΄F-ANA-modified aptamer candidates with Kds significantly lower than that of the unmodified control and which were found to adopt highly stable, antiparallel-folded G-quadruplex structures. The solution structure of the TBA1 aptamer modified with 2΄F-ANA at position T3 shows that fluorine substitution preorganizes the dinucleotide loop into the proper conformation for interaction with thrombin. Overall, our work strengthens the potential of 2΄F-ANA in aptamer research and further expands non-genomic applications of nucleic acids microarrays.


Assuntos
Aptâmeros de Nucleotídeos/química , Arabinonucleotídeos/química , DNA/química , Quadruplex G , Sequência de Bases , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Análise de Sequência com Séries de Oligonucleotídeos , Compostos Organofosforados/química
12.
Bioorg Med Chem Lett ; 26(10): 2418-2421, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27080186

RESUMO

Chemically modified oligonucleotides are routinely used as diagnostic and therapeutic agents due to their enhanced biological stability relative to natural DNA and RNA. Here, we examine the biological stability of α-l-threofuranosyl nucleic acid (TNA), an artificial genetic polymer composed of repeating units of α-l-threofuranosyl sugars linked by 2',3'-phosphodiester bonds. We show that TNA remains undigested after 7days of incubation in the presence of either 50% human serum or human liver microsomes and is stable against snake venom phosphordiesterase (a highly active 3' exonuclease). We further show that TNA will protect internal DNA residues from nuclease digestion and shield complementary RNA strands from RNA degrading enzymes. Together, these results demonstrate that TNA is an RNA analogue with high biological stability.


Assuntos
Oligonucleotídeos/química , Tetroses/química , Arabinonucleotídeos/farmacocinética , Estabilidade de Medicamentos , Meia-Vida , Humanos , Espectroscopia de Ressonância Magnética , Microssomos Hepáticos/efeitos dos fármacos , Oligonucleotídeos/farmacocinética , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Ribose/química
13.
Artigo em Inglês | MEDLINE | ID: mdl-25965330

RESUMO

The substrate specificity of recombinant full-length diguanylate cyclase (DGC) of Thermotoga maritima with mutant allosteric site was investigated. It has been originally shown that the enzyme could use GTP closest analogues - 2'-deoxyguanosine-5'-triphosphate (dGTP) and 9-ß-D-arabinofuranosyl-guanine-5'-triphosphate (araGTP) as the substrates. The first demonstrations of an enzymatic synthesis of bis-(3'-5')-cyclic dimeric deoxyguanosine monophosphate (c-di-dGMP) and the previously unknown bis-(3'-5')-cyclic dimeric araguanosine monophosphate (c-di-araGMP) using DGC of T. maritima in the form of inclusion bodies have been provided.


Assuntos
Arabinonucleotídeos/química , Proteínas de Bactérias/química , GMP Cíclico/análogos & derivados , Nucleotídeos de Desoxiguanina/química , Proteínas de Escherichia coli/química , Guanosina Trifosfato/análogos & derivados , Fósforo-Oxigênio Liases/química , Thermotoga maritima/enzimologia , GMP Cíclico/síntese química , GMP Cíclico/química , Guanosina Trifosfato/química
14.
J Hematol Oncol ; 8: 5, 2015 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-25652695

RESUMO

The prognosis of relapsed acute myeloid leukemia (AML) in elderly patients is dismal, even if the AML exhibits a good prognostic karyotype, such as inv(16)(p13.1q22). We present a 72-year-old female with AML with inv(16)(p13.1q22) who suffered five episodes of relapse with temporary complete remission. Maintenance chemotherapy with oral cytarabine ocfosfate hydrate eventually produced persistent molecular complete remission of her AML that had not been induced by conventional regimens including intensive chemotherapy and low dose cytarabine therapy. The high level of tolerability to oral cytarabine ocfosfate hydrate may offer elderly patients with this type of AML a good chance for a cure.


Assuntos
Antineoplásicos/uso terapêutico , Arabinonucleotídeos/uso terapêutico , Cromossomos Humanos Par 16/genética , Citidina Monofosfato/análogos & derivados , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Idoso , Inversão Cromossômica , Citidina Monofosfato/uso terapêutico , Feminino , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Indução de Remissão
15.
ChemMedChem ; 9(9): 2138-49, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25125220

RESUMO

A group of acidic nucleosides were synthesized to develop a new class of ribonuclease A (RNase A) inhibitors. Our recent study on carboxymethylsulfonyl-modified nucleosides revealed some interesting results in RNase A inhibition. This positive outcome triggered an investigation of the role played by secondary sugar hydroxy groups in inhibiting RNase A activity. Uridines and cytidines modified with SO2 CH2 COOH groups at the 2'- and 3'-positions show good inhibitory properties with low inhibition constant (Ki ) values in the range of 109-17 µM. The present work resulted in a set of inhibitors that undergo more effective interactions with the RNase A active site, as visualized by docking studies.


Assuntos
Arabinonucleotídeos/síntese química , Arabinonucleotídeos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Ribonuclease Pancreático/antagonistas & inibidores , Domínio Catalítico/efeitos dos fármacos , Biologia Computacional , Citidina/química , Cinética , Modelos Moleculares , Uridina/química
16.
Nucleic Acid Ther ; 24(5): 336-43, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25162466

RESUMO

We have investigated, for the first time, short interfering duplexes containing arabinonucleotides (ANA; the 2'-stereoisomer of RNA), as well as combinations of ANA with RNA, and their 2'-fluorinated derivatives 2F-ANA and/or 2'F-RNA. The results show that ANA is especially well accommodated in the sense strand of small interfering RNA (siRNA) duplexes, which can be extensively modified with little effect on potency. Furthermore, combining ANA with RNA and 2'F-ANA in siRNA passenger strands, particularly in patterns that bias duplex thermal stability, produces duplexes with similar (and sometimes enhanced) potency compared with native siRNA. Effective patterns of modification were identified against firefly luciferase screens in HeLa cells and then applied to knockdown of down-regulated in renal cell carcinoma (DRR), a novel and clinically tractable target for the treatment of glioblastoma.


Assuntos
Arabinonucleotídeos/genética , Luciferases/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , RNA Interferente Pequeno/genética , Arabinonucleotídeos/síntese química , Arabinonucleotídeos/metabolismo , Linhagem Celular Tumoral , Expressão Gênica , Genes Reporter , Genes Supressores de Tumor , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/metabolismo , Estereoisomerismo
17.
Chemotherapy ; 59(2): 152-8, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24080768

RESUMO

BACKGROUND: Elderly acute myeloid leukemia (AML) patients and patients with higher-risk myelodysplastic syndromes (MDS) have a much poorer prognosis than younger patients despite intensive chemotherapy. METHODS: Ten patients with higher-risk MDS and 12 patients with AML over 65 years of age were enrolled into this study and received oral induction therapy with cytarabine ocfosfate and etoposide. RESULTS: The therapy response rates were 60% in the MDS group and 41.7% in the AML group. The difference in overall survival among MDS and AML patients was not statistically significant. The difference in the median survival times of the responsive and nonresponsive groups, which included MDS and AML patients, was statistically significant (790 and 174 days, respectively). CONCLUSIONS: Based on a comparison of the data of this therapy in elderly higher-risk MDS patients versus elderly AML patients, we conclude that this therapy is well tolerated and can be cost-effective and useful for higher-risk MDS in elderly patients.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Arabinonucleotídeos/administração & dosagem , Citidina Monofosfato/análogos & derivados , Etoposídeo/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Administração Oral , Idoso , Idoso de 80 Anos ou mais , Citidina Monofosfato/administração & dosagem , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/mortalidade , Masculino , Síndromes Mielodisplásicas/mortalidade , Resultado do Tratamento
18.
Mol Vis ; 18: 1907-17, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22876115

RESUMO

PURPOSE: The objectives of this study were to synthesize and characterize two types of cytarabine (Ara-C) lipid produgs and evaluate the prodrugs for sustained intraocular delivery after administration by intravitreal injection. METHODS: Hexadecyloxypropyl cytarabine 5'-monophosphate (HDP-P-Ara-C) and hexadecyloxypropyl cytarabine 3',5'-cyclic monophosphate (HDP-cP-Ara-C) were synthesized starting from cytarabine (1-ß-D-arabinofuranosylcytosine). Their vitreal clearance profile was simulated using a custom dissolution chamber, in vitro cytotoxicity was evaluated using cell proliferation assays, and in vivo ocular properties in rat and rabbit eyes were assessed using biomicroscopy, indirect ophthalmoscopy, tonometry, electroretinography, and histology. RESULTS: HDP-P-Ara-C was cleared from the dissolution chamber (flow rate 2 µL/min) within 7 days. In contrast, HDP-cP-Ara-C, a much more insoluble prodrug, was still detectable 36 days after the dissolution process was started. HDP-P-Ara-C had a 50% cytotoxicity concentration of 52±2.6 µM in human retinal pigment epithelium (ARPE-19) and 32±2.2 µM in a rat Müller cell line, rMC-1. The 50% cytotoxicity concentration values for HDP-cP-Ara-C in ARPE-19 and rMC-1 cells were 50 µM and 25 µM, respectively. HDP-P-Ara-C was not detectable 2 weeks after the highest intravitreal dose (228 µg/rat eye) was injected, and no ocular toxicity was found. With HDP-cP-Ara-C, the drug depot was visible for 26 weeks following a single intravitreal injection (800 µg/rabbit eye). For both compounds, the electroretinogram, intraocular pressure, and other toxicity studies were negative except for the highest dose of HDP-cP-Ara-C (800 µg/eye), which had focal toxicity from the direct touch of the retina and decreased dark adapted a-waves and decreased flicker electroretinogram amplitudes (generalized estimating equations, p=0.039 and 0.01). CONCLUSIONS: The cyclic monophosphate prodrug, HDP-cP-Ara-C, was found to have physiochemical properties better suited for sustained delivery of cytarabine to posterior segments of the eye. These properties included limited aqueous solubility, in vitro antiproliferative activity, and good tolerability after injection into rabbit eyes.


Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Arabinonucleotídeos/administração & dosagem , Citidina Monofosfato/análogos & derivados , Preparações de Ação Retardada/administração & dosagem , Pró-Fármacos/administração & dosagem , Retina/efeitos dos fármacos , Vitreorretinopatia Proliferativa/tratamento farmacológico , Corpo Vítreo/efeitos dos fármacos , Animais , Antimetabólitos Antineoplásicos/síntese química , Arabinonucleotídeos/síntese química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citidina Monofosfato/administração & dosagem , Citidina Monofosfato/síntese química , Preparações de Ação Retardada/síntese química , Cultura em Câmaras de Difusão , Eletrorretinografia , Humanos , Injeções Intravítreas , Cinética , Oftalmoscopia , Pró-Fármacos/síntese química , Coelhos , Ratos , Retina/metabolismo , Retina/patologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Solubilidade , Vitreorretinopatia Proliferativa/metabolismo , Vitreorretinopatia Proliferativa/patologia , Corpo Vítreo/metabolismo , Corpo Vítreo/patologia
19.
Nucleic Acids Res ; 40(18): 9329-39, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22798499

RESUMO

We report here the first structure of double helical arabino nucleic acid (ANA), the C2'-stereoisomer of RNA, and the 2'-fluoro-ANA analogue (2'F-ANA). A chimeric dodecamer based on the Dickerson sequence, containing a contiguous central segment of arabino nucleotides, flanked by two 2'-deoxy-2'F-ANA wings was studied. Our data show that this chimeric oligonucleotide can adopt two different structures of comparable thermal stabilities. One structure is a monomeric hairpin in which the stem is formed by base paired 2'F-ANA nucleotides and the loop by unpaired ANA nucleotides. The second structure is a bimolecular duplex, with all the nucleotides (2'F-ANA and ANA) forming Watson-Crick base pairs. The duplex structure is canonical B-form, with all arabinoses adopting a pure C2'-endo conformation. In the ANA:ANA segment, steric interactions involving the 2'-OH substituent provoke slight changes in the glycosidic angles and, therefore, in the ANA:ANA base pair geometry. These distortions are not present in the 2'F-ANA:2'F-ANA regions of the duplex, where the -OH substituent is replaced by a smaller fluorine atom. 2'F-ANA nucleotides adopt the C2'-endo sugar pucker and fit very well into the geometry of B-form duplex, allowing for favourable 2'F···H8 interactions. This interaction shares many features of pseudo-hydrogen bonds previously observed in 2'F-ANA:RNA hybrids and in single 2'F-ANA nucleotides.


Assuntos
Arabinonucleotídeos/química , Arabinose/química , Pareamento de Bases , Configuração de Carboidratos , DNA/química , Flúor/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oligonucleotídeos/química
20.
Artigo em Inglês | MEDLINE | ID: mdl-22257207

RESUMO

A murine P388 leukemia line fully resistant to thiarabine was obtained after five courses of intraperitoneal treatment (daily for nine consecutive days). The subline was sensitive as was the parental P388/0 line to 5-fluorouracil, gemcitabine, cyclophosphamide, cisplatin, melphalan, BCNU, mitomycin C, doxorubicin, mitoxantrone, etoposide, irinotecan, vincristine, and paclitaxel, but was cross resistant (at least marginally) to three antimetabolites: palmO-ara-C, fludarabine phosphate, and methotrexate. The deoxycytidine kinase activity in the subline was comparable to that for P388/0, whereas the dCMP deaminase activity was 43% of that for P388/0. No deoxycytidine deaminase activity was detected in either of the leukemias. There appeared to be little, if any, difference in the metabolism of deoxycytidine, cytidine, or thiarabine in the two leukemias.


Assuntos
Antimetabólitos/administração & dosagem , Antineoplásicos/administração & dosagem , Arabinonucleotídeos/administração & dosagem , Linhagem Celular Tumoral/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Animais , Antimetabólitos/síntese química , Antineoplásicos/química , Arabinonucleotídeos/síntese química , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/enzimologia , DCMP Desaminase/metabolismo , Desoxicitidina Quinase/metabolismo , Feminino , Leucemia P388 , Camundongos , Transplante de Neoplasias , Transplante Heterólogo
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