Biomedical subjects

Balkrishen Bhat

Publications and source records attributed to Balkrishen Bhat.

17 recordsLinked to original sources

PubMed2006

Competition for RISC binding predicts in vitro potency of siRNA.

Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2'-O-methyl and 2'-deoxy-2'-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.

Binding, Competitive↗

PubMed2006

RNA interference by 2',5'-linked nucleic acid duplexes in mammalian cells.

Synthetic small interfering RNA (siRNA) mediated silencing of a specific gene is emerging as a powerful tool for gene regulation. However, their utility is limited for therapeutic applications primarily due to poor stability. The 2',5'-linked oligonucleotides are known to be more stable to nucleolytic degradation than 3',5'-linked oligonucleotides. The 2',5'-linkage is tolerated in the sense strand of the siRNA duplex. However, the 2',5'-linkage is not tolerated in the antisense strand of the siRNA duplex.

Animals↗

PubMed2006

Improving RNA interference in mammalian cells by 4'-thio-modified small interfering RNA (siRNA): effect on siRNA activity and nuclease stability when used in combination with 2'-O-alkyl modifications.

A systematic structure-activity relationship study of 4'-thioribose containing small interfering RNAs (siRNAs) has led to the identification of highly potent and stable antisense constructs. To enable this optimization effort for both in vitro and in vivo applications, we have significantly improved the yields of 4'-thioribonucleosides by using a chirally pure (R)-sulfoxide precursor. siRNA duplexes containing strategically placed regions of 4'-thio-RNA were synthesized and evaluated for RNA interference activity and plasma stability. Stretches of 4'-thio-RNA were well tolerated in both the antisense and sense strands. However, optimization of both the number and placement of 4'-thioribonucleosides was necessary for maximal potency. These optimized siRNAs were generally equipotent or superior to native siRNAs and exhibited increased thermal and plasma stability. Furthermore, significant improvements in siRNA activity and plasma stability were achieved by judicious combination of 4'-thioribose with 2'-O-methyl and 2'-O-methoxyethyl modifications. These optimized 4'-thio-siRNAs may be valuable for developing stable siRNAs for therapeutic applications.

Animals↗

PubMed2005

Positional effect of chemical modifications on short interference RNA activity in mammalian cells.

A systematic study on the effect of 2'-sugar modifications (2'-F (2'-F-2'-deoxy-nucleoside residues), 2'-O-Me (2'-O-methyl-nucleoside residues), and 2'-O-MOE [2'-O-(2-methoxyethyl)]-nucleoside residues) in the antisense and sense strands of short interference RNA (siRNA) was performed in HeLa cells. The study of the antisense strand of siRNAs demonstrated that activity depends on the position of the modifications in the sequence. The siRNAs with modified ribonucleotides at the 5'-end of the antisense strand were less active relative to the 3'-modified ones. The 2'-F sugar was generally well-tolerated on the antisense strand, whereas the 2'-O-Me showed significant shift in activity depending on the position of modification. The 2'-O-MOE modification in the antisense strand resulted in less active siRNA constructs regardless of placement position in the construct. The incorporation of the modified residues, e.g., 2'-O-Me and 2'-O-MOE, in the sense strand of siRNA did not show a strong positional preference. These results may provide guidelines to design effective and stable siRNAs for RNA interference mediated therapeutic applications.

Animals↗

PubMed2005

Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA.

We have identified a small interfering RNA (siRNA) motif, consisting entirely of 2'-O-methyl and 2'-fluoro nucleotides, that displays enhanced plasma stability and increased in vitro potency. At one site, this motif showed remarkable >500-fold improvement in potency over the unmodified siRNA. This marks the first report of such a potent fully modified motif, which may represent a useful design for therapeutic oligonucleotides.

Animals↗

PubMed2005

Synthesis and evaluation of S-acyl-2-thioethyl esters of modified nucleoside 5'-monophosphates as inhibitors of hepatitis C virus RNA replication.

Several triphosphates of modified nucleosides (1-6) were identified as inhibitors (IC(50) = 0.08-3.8 microM) of hepatitis C virus RNA-dependent RNA polymerase (RdRp). Although the initial SAR developed by determining the ability of the triphosphates to inhibit the in vitro activity of the HCV RdRp identified several potent inhibitors, none of the corresponding nucleosides exhibited significant inhibitory potency in a cell-based replicon assay. To improve upon the activity, bis(tBu-S-acyl-2-thioethyl) nucleoside 5'-monophosphate esters (7-12) were synthesized, and these derivatives exhibited improved potency compared to the corresponding nucleosides in the cell-based assay. Analysis of the intracellular metabolism demonstrated that the S-acyl-2-thioethyl (SATE) prodrug is metabolized to the 5'-triphosphate 40- to 155-fold more efficiently compared to the corresponding nucleoside. The prodrug approach involving bis(tBuSATE)cytidine 5'-monophosphate ester significantly reduced the deamination of cytidine derivatives by cellular deaminases. Additionally, chromosomal aberration studies with the SATE prodrug in cells showed no statistically relevant increase in aberrations compared to the concurrent controls.

Animals↗

PubMed2005

Inhibitory effect of 2'-substituted nucleosides on hepatitis C virus replication correlates with metabolic properties in replicon cells.

Nucleosides have been widely used in the treatment of viral diseases, but relatively few have been identified as inhibitors of hepatitis C virus (HCV). The modified ribonucleosides, 2'-C-methyl-adenosine and 2'-O-methyl-cytidine, are potent inhibitors of HCV replication which specifically target the NS5B polymerase. Herein, a more extensive characterization of the effect of these compounds upon HCV replication in subgenomic replicons is reported. A highly selective antireplicative effect induced by the nucleosides in replicon-containing cell lines was maintained during an exponential growth period with potencies which paralleled the reduction of both positive- and negative-strand RNA replication. Moreover, the inhibitory effect closely correlated with the intrinsic metabolic properties of differing replicon clonal lines. Interestingly, while 2'-C-methyl-adenosine elicited similar inhibitory potencies in different cell lines, 2'-O-methyl-cytidine was found to be inactive in one replicon cell line tested, although the corresponding triphosphates comparably inhibited the in vitro activity of replication complexes isolated from these cells and the activity of NS5B polymerase using synthetic templates. The lack of antireplicative effect, attributed to poor intracellular conversion of the 2'-O-methyl-cytidine nucleoside to the active 5'-triphosphate, was reversed using a monophosphate prodrug. Thus, although replicon cells are useful for evaluating the effect of inhibitors upon HCV replication, these findings have important implications for their use in the identification and characterization of nucleosides and other chemotherapeutic agents requiring cellular metabolism.

Antiviral Agents↗

PubMed2004

Synthesis and biological evaluation of 5R- and 5S-methyl substituted D- and L-configuration 1,3-dioxolane nucleoside analogs.

1,3-Dioxolane and 1,3-oxathiolane nucleoside analogs play an important role in anti-viral and anti-neoplastic chemotherapy. We report here the synthesis of 2-hydroxymethyl-5-methyl-1,3-dioxolanylpurine nucleosides from 4-acetoxy-2-(benzyloxymethyl)-5-methyldioxolane. Dioxolanes of alpha-D-, beta-D-, alpha-L-, and beta-L-configuration were prepared, that included 5-methyl derivatives of both 5R and 5S configuration. Molecular mechanics calculations indicate that the 5S and 5R diastereoisomeric 1,3-dioxolanes possess distinct conformational bias, suggesting that methyl substitution may alter the conformational preference of 1,3-dioxolanes. The ability of the 1,3-dioxolanes to inhibit HCV RNA replication was evaluated in a cell-based, subgenomic replicon assay. In addition, activity against vaccinia and HIV was evaluated in cell-based assays. The 2-hydroxymethyl-5-methyl-1,3-dioxolanes were found to be inactive.

Antiviral Agents↗

PubMed2004

Structure-activity relationship of heterobase-modified 2'-C-methyl ribonucleosides as inhibitors of hepatitis C virus RNA replication.

Hepatitis C virus infection constitutes a significant health problem in need of more effective therapies. We have recently identified 2'-C-methyladenosine and 2'-C-methylguanosine as potent nucleoside inhibitors of HCV RNA replication in vitro. However, both of these compounds suffered from significant limitations. 2'-C-Methyladenosine was found to be susceptible to enzymatic conversions by adenosine deaminase and purine nucleoside phosphorylase, and it displayed limited oral bioavailability in the rat. 2'-C-Methylguanosine, on the other hand, was neither efficiently taken up in cells nor phosphorylated well. As part of an attempt to address these limitations, we now report upon the synthesis and evaluation of a series of heterobase-modified 2'-C-methyl ribonucleosides. The structure-activity relationship within this series of nucleosides reveals 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine as potent and noncytotoxic inhibitors of HCV RNA replication. Both 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine display improved enzymatic stability profiles as compared to that of 2'-C-methyladenosine. Consistent with these observations, the most potent compound, 4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine ribonucleoside, is orally bioavailable in the rat. Together, the potency of the 2'-C-methyl-4-amino-pyrrolo[2,3-d]pyrimidine ribonucleosides and their improved pharmacokinetic properties relative to that of 2'-C-methyladenosine suggests that this class of compounds may have clinical utility.

Adenosine Deaminase↗

PubMed2004

Structural requirements at the catalytic site of the heteroduplex substrate for human RNase H1 catalysis.

Human RNase H1 cleaves RNA exclusively in an RNA/DNA duplex; neither double-strand DNA nor double-strand RNA is a viable substrate. Previous studies suggest that the helical geometry and sugar conformation of the DNA and RNA may play a role in the selective recognition of the heteroduplex substrate by the enzyme. We systematically evaluated the influence of sugar conformation, minor groove bulk, and conformational flexibility of the heteroduplex on enzyme efficiency. Modified nucleotides were introduced into the oligodeoxyribonucleotide at the catalytic site of the heteroduplex and consisted of southern, northern, and eastern biased sugars with and without 2'-substituents, non-hydrogen bonding base modifications, abasic deoxyribonucleotides, intranucleotide hydrocarbon linkers, and a ganciclovir-modified deoxyribonucleotide. Heteroduplexes containing modifications exhibiting strong northern or southern conformational biases with and without a bulky 2'-substituent were cleaved at a significantly slower rate than the unmodified substrate. Modifications imparting the greatest degree of conformational flexibility were the poorest substrates, resulting in dramatically slower cleavage rates for the ribonucleotide opposing the modification and the surrounding ribonucleotides. Finally, modified heteroduplexes containing modifications predicted to mimic the sugar pucker and conformational flexibility of the deoxyribonucleotide exhibited cleavage rates comparable with those of the unmodified substrate. These data suggest that sugar conformation, minor groove width, and the relative positions of the intra- and internucleotide phosphates are the crucial determinants in the selective recognition of the heteroduplex substrate by human RNase H1 and offer immediate steps to improve the performance of DNA-like antisense oligonucleotides.

Binding Sites↗

PubMed2004

Structure-activity relationship of purine ribonucleosides for inhibition of hepatitis C virus RNA-dependent RNA polymerase.

As part of a continued effort to identify inhibitors of hepatitis C viral (HCV) replication, we report here the synthesis and evaluation of a series of nucleoside analogues and their corresponding triphosphates. Nucleosides were evaluated for their ability to inhibit HCV RNA replication in a cell-based, subgenomic replicon system, while nucleoside triphosphates were evaluated for their ability to inhibit in vitro RNA synthesis mediated by the HCV RNA-dependent RNA polymerase, NS5B. 2'-C-Methyladenosine and 2'-C-methylguanosine were identified as potent inhibitors of HCV RNA replication, and the corresponding triphosphates were found to be potent inhibitors of HCV NS5B-mediated RNA synthesis. The data generated in the cell-based assay demonstrated a fairly stringent structure-activity relationship around the active nucleosides. Increase in steric bulk beyond methyl on C2, change in the stereo- or regiochemistry of the methyl substituent, or change of identity of the heterobase beyond that of the endogenous adenine or guanine was found to lead to loss of inhibitory activity. The results highlight the importance of the ribo configuration 2'- and 3'-hydroxy pharmacophores for inhibition of HCV RNA replication in the cell-based assay and demonstrate that inclusion of the 2'-C-methylribonucleoside pharmacophore leads to increased resistance to adenosine deaminase and purine nucleoside phosphorylase mediated metabolism.

Adenosine Deaminase↗

PubMed2004

Synthesis of 2'-substituted MMI linked nucleosidic dimers: an optimization study in search of high affinity oligonucleotides for use in antisense constructs.

The synthesis of a series of methylene(methylimino) (MMI) linked oligodeoxyribonucleotide dimers modified at the 2'-position with fluoro and/or methoxy groups and their incorporation into different sequences has been accomplished. From these dimers, bis 2'-OMe MMI dimer was selected for further studies based on its synthetic accessibility and the increased thermodynamic stability conferred upon oligonucleotides incorporating this modification.

DNA, Antisense↗

PubMed2004

A 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties.

Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2'-C-methyl-adenosine and 2'-C-methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2'-C-methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5'-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2'-C-methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2'-C-methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2'-C-methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.

Animals↗

PubMed2003

Synthesis and evaluation of optically pure dioxolanes as inhibitors of hepatitis C virus RNA replication.

A series of optically pure 1,3-dioxolane nucleoside mimics was synthesized by a synthetic route that allowed incorporation of a 5R-methyl substituent from commercially available starting materials. The pyrrolo[2,3-d]pyrimidine heterocycle was chosen as a substitute for the purine derivative. Coupling of the pyrrolo[2,3-d]pyrimidine and the dioxolane was performed under solid-liquid phase transfer conditions. The ability to inhibit HCV RNA replication was assessed in a cell based subgenomic replicon assay. None of the described compounds displayed significant anti-HCV activity.

Antiviral Agents↗

PubMed2003

Characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro.

The urgent need for efficacious drugs to treat chronic hepatitis C virus (HCV) infection requires a concerted effort to develop inhibitors specific for virally encoded enzymes. We demonstrate that 2'-C-methyl ribonucleosides are efficient chain-terminating inhibitors of HCV genome replication. Characterization of drug-resistant HCV replicons defined a single S282T mutation within the active site of the viral polymerase that conferred loss of sensitivity to structurally related compounds in both replicon and isolated polymerase assays. Biochemical analyses demonstrated that resistance at the level of the enzyme results from a combination of reduced affinity of the mutant polymerase for the drug and an increased ability to extend the incorporated nucleoside analog. Importantly, the combination of these agents with interferon-alpha results in synergistic inhibition of HCV genome replication in cell culture. Furthermore, 2'-C-methyl-substituted ribonucleosides also inhibited replication of genetically related viruses such as bovine diarrhea virus, yellow fever, and West African Nile viruses. These observations, together with the finding that 2'-C-methyl-guanosine in particular has a favorable pharmacological profile, suggest that this class of compounds may have broad utility in the treatment of HCV and other flavivirus infections.

Animals↗

PubMed2003

Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs.

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.

Adenosine↗

PubMed1996

Synthesis of Novel Nucleic Acid Mimics via the Stereoselective Intermolecular Radical Coupling of 3'-Iodo Nucleosides and Formaldoximes(1).

A highly convergent free radical coupling of alkyl iodides and oximes, mediated by bis(trimethylstannyl) benzopinacolate (8), has been utilized to prepare a series of dimeric nucleosides as mimics of natural nucleic acids. The systematic optimization of the reaction conditions allowed for the single-step conversion of the appropriate iodides and oximes into the 2'-deoxy dimers 9 in moderate to excellent yields. For example, the reaction of 3'-deoxy-3'-iodo-5'-(triphenylmethyl)thymidine (6a) with 3'-O-(tert-butyldiphenylsilyl)-5'-O-(methyleneimino)thymidine (7a) in the presence of 8 in degassed benzene gave an 81% yield of 3'-de(oxyphosphinico)-3'-(methyleneimino)-5'-O-(triphenylmethyl)thymidylyl-(3'-->5')-3'-O-(tert-butyldiphenylsilyl)thymidine (9a). Similarly prepared were dimers containing both pyrimidine (thymine, 5-methylcytosine) and purine (adenine, guanine) bases. The reaction was highly stereoselective, giving only a single dimeric species having the ribo-configuration of the newly introduced C-3'-branched methylene moiety. Also prepared were dimers 16, incorporating 2'-O-methyl ribonucleosides in both halves of the dimer. This required the synthesis of 3'-deoxy-3'-iodo-2'-O-methyl nucleosides 12 as well as 2'-O-methyl-5'-O-methyleneimino nucleosides 15. For example, 5'-O-(tert-butyldiphenylsilyl)-3'-deoxy-3'-iodo-2'-O-methyl-5-methyluridine (12e) was prepared in 80% yield by displacement of the corresponding triflate with Bu(4)NI. Also prepared were the suitably protected 3'-deoxy-3'-iodo adenosine and guanosine derivatives. Compounds 15 were prepared in high yield by a regioselective Mitsunobu reaction to give the corresponding 5'-O-phthalimido nucleosides 13, which were subsequently converted to the requisite oximes 15. In the 2'-O-methyl series, the pinacolate coupling reaction proceeded with efficiency equal to that observed for the 2'-deoxy series 9, but with slightly less stereoselectivity, giving predominantly the C-3'ribo products 16, contaminated with 5-25% of the epimeric material. Mixed base dimers containing both pyrimidine and purine bases at all possible positions, including purine-purine dimers were prepared. The hydroxylamine or methyleneimino (MI) backbone of several representative dimers so prepared was converted via methylation to give the corresponding methylenemethylimino (MMI)-linked compounds, which are novel phosphate surrogates for use in antisense oligonucleotides.

Journal Article↗