Balkrishen Bhat

Ionis Pharmaceuticals, Carlsbad, California, United States

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Publications (58)191.59 Total impact

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    ABSTRACT: The ss-siRNA activity in vivo requires a metabolically stable 5'-phosphate analog. In this report we used crystal structure of the 5'-phosphate binding pocket of Ago-2 bound with guide strand to design and synthesize ss-siRNAs containing various 5'-phosphate analogs. Our results indicate that the electronic and spatial orientation of the 5'-phosphate analog was critical for ss-siRNA activity. Chemically modified ss-siRNA targeting human apoC III mRNA demonstrated good potency for inhibiting ApoC III mRNA and protein in transgenic mice. Moreover, ApoC III ss-siRNAs were able to reduce the triglyceride and LDL cholesterol in transgenic mice demonstrating pharmacological effect of ss-siRNA. Our study provides guidance to develop surrogate phosphate analog for ss-siRNA and demonstrates that ss-siRNA provides an alternative strategy for therapeutic gene silencing. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
    Full-text · Article · Mar 2015 · Nucleic Acids Research
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    ABSTRACT: We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.Molecular Therapy - Nucleic Acids (2012) 1, e47; doi:10.1038/mtna.2012.34; published online 18 September 2012.
    Full-text · Article · Oct 2012 · Molecular Therapy
  • K. Steffy · C. Allerson · B. Bhat

    No preview · Article · Oct 2011 · Biopharm International
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    ABSTRACT: The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F···H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.
    Full-text · Article · Sep 2011 · Journal of the American Chemical Society
  • Kevin Steffy · Charles Allerson · Balkrishen Bhat
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    ABSTRACT: Decades of research and development have produced a rich, deep pipeline of preclinical and clinical programs based on oligonucleotide therapeutics. In particular, anti-miR therapeutics represent an exciting opportunity in the field of microRNA drug discovery. The authors provide further insight into microRNA biology, and the simplicity of anti-miR oligonucleotide drug delivery, which can restore balance and function to dysregulated microRNA pathways of gene expression.
    No preview · Article · May 2011 · Biopharm International
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    ABSTRACT: Provided herein are novel 5'-(S)-CH3 substituted bicyclic nucleosides I, wherein B is nucleobase; one of T1 and T2 is H or a hydroxy protecting group and the other of T1 and T2 is H, a hydroxy protecting group or a reactive phosphorus group; G is a diradical moiety selected from -[C(R1R2)]n-, -C(R1)=C(R2)-, -C(R1)=C(R2)-C(R3R4)-, -C[=C(R1R2)]-C(R3R4)-, -C(R1R2)-C[=C(R3R4)]-, -C(O)-N(R7)-C(R1R2)-, -C(R1R2)-N(R7)-C(R3R4)-, -C(R1R2)-N(R7)-O-, -C(R1R2)-O-N(R7)-, -C(R1R2)-N(OR3)-, -C(R1R2)-C(R3R4)-N(R7)-, -C(R1R2)-O-OC(R3R4)-, -C(R1R2)-O-C(R3R4)-O-, -C(R1R2)-C(R3R4)-O-, -C(R1R2)-C(R3R4)-C(R5R6)-O-; R1-R6 are each independently, H, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl; OJ1, SJ1,SOJ1, SO2J1, CN, NJ1J2, N3, C(O)J1, C(O)OJ1, C(O)NJ1J2, protecting group;each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ3, N(J3)(J4), =NJ3, SJ3, N33, CN, OC(=L)J3, OC(=L)N(J3)(J4) and C(=L)N(J3)(J4); L is O, S, NJ5; each J1-J5 is independently, H or C1-C6 alkyl; and is from 1 to 3, and oligomeric compds. prepd. therefrom and methods of using the oligomeric compds. More particularly, the furanose ring of each of the novel 5'-(S)-CH3 substituted bicyclic nucleosides includes a 2' to 4' bridging group. The 5'-(S)-CH3 substituted bicyclic nucleosides are expected to be useful for enhancing one or more properties of the oligomeric compds. they are incorporated into such as for example increasing the binding affinity. In certain embodiments, the oligomeric compds. provided herein hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. Thus, nucleoside II was prepd. and incorporated into RNA duplexes. [on SciFinder(R)]
    No preview · Patent · Jan 2011
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    ABSTRACT: A high-resolution solution structure of a stable 42-nt RNA dimeric construct has been derived based on a high number of NMR observables including nuclear overhauser effects (NOEs), J-coupling constants and residual dipolar couplings (RDCs), which were all obtained with isotopically unlabeled molecules. Two 21-nt siRNA that efficiently hybridize consist of ribose units that were alternately substituted by 2′-fluoro or 2′-methoxy groups. Structure calculations utilized a set of H-F RDC values for all 21 2′-fluoro modified nucleotides under conditions of weak alignment achieved by Pf1 phages. A completely 2′-F/2′-OMe modified dimeric RNA construct adopts an antiparallel double-helical structure consisting of 19 Watson–Crick base pairs with additional 3′ UU overhangs and a 5′ phosphate group on the antisense strand. NMR data suggest that the stability of individual base pairs is not uniform throughout the construct. While most of the double helical segment exhibits well dispersed imino resonances, the last three base pairs either display uncharacteristic chemical shifts of imino protons or absence of imino resonances even at lower temperatures. Accessibility of imino protons to solvent exchange suggests a difference in stability of duplex ends, which might be of importance for incorporation of the guide siRNA strand into a RISC.
    Full-text · Article · Nov 2010 · Nucleic Acids Research
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    ABSTRACT: As part of a program aimed at exploring the structure- activity relationships of 2',4'-bridged nucleic acid (BNA) containing antisense oligonucleotides (ASOs), we report the synthesis and biophysical and biological properties of R- and S-5'-Me LNA modified oligonucleotides. We show that introduction of a methyl group in the (S) configuration at the 5'-position is compatible with the high affinity recognition of complementary nucleic acids observed with LNA. In contrast, introduction of a methyl group in the (R) configuration reversed the stabilization effect of LNA. NMR studies indicated that the R-5'-Me group changes the orientation around torsion angle γ from the +sc to the ap range at the nucleoside level, and this may in part be responsible for the poor hybridization behavior exhibited by this modification. In animal experiments, S-5'-Me-LNA modified gapmer antisense olignucleotides showed slightly reduced potency relative to the sequence matched LNA ASOs while improving the therapeutic profile.
    Full-text · Article · Nov 2010 · Journal of Medicinal Chemistry
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    ABSTRACT: 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.
    No preview · Article · Mar 2010 · The Journal of Organic Chemistry
  • B. BHAT · Y. S. SANGHVI
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    No preview · Article · Mar 2010 · ChemInform
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    No preview · Article · Mar 2010 · ChemInform
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    ABSTRACT: To identify chemistries and strategies to improve the potency of MOE second generation ASOs, we have evaluated gapmer antisense oligonucleotides containing BNAs having N-O bonds. These modifications include N-MeO-amino BNA, N-Me-aminooxy BNA, 2',4'-BNA(NC)[NMe], and 2',4'-BNA(NC) bridged nucleoside analogues. These modifications provided increased thermal stability and improved in vitro activity compared to the corresponding ASO containing the MOE modification. Additionally, ASOs containing N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] modifications showed improved in vivo activity (>5-fold) compared to MOE ASO. Importantly, toxicity parameters, such as AST, ALT, liver, kidney, and body weights, were found to be normal for N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] ASO treated animals. The data generated in these experiments suggest that N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] are useful modifications for applications in both antisense and other oligonucleotide based drug discovery efforts.
    Full-text · Article · Feb 2010 · Journal of Medicinal Chemistry
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    ABSTRACT: We have recently shown that combining the structural elements of 2'O-methoxyethyl (MOE) and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE, 2',4'-constrained MOE; cEt, 2',4'-constrained ethyl) that display improved potency over MOE and an improved therapeutic index relative to that of LNA antisense oligonucleotides. In this report we present details regarding the synthesis of the cMOE and cEt nucleoside phosphoramidites and the biophysical evaluation of oligonucleotides containing these nucleoside modifications. The synthesis of the cMOE and cEt nucleoside phosphoramidites was efficiently accomplished starting from inexpensive commercially available diacetone allofuranose. The synthesis features the use of a seldom used 2-naphthylmethyl protecting group that provides crystalline intermediates during the synthesis and can be cleanly deprotected under mild conditions. The synthesis was greatly facilitated by the crystallinity of a key mono-TBDPS-protected diol intermediate. In the case of the cEt nucleosides, the introduction of the methyl group in either configuration was accomplished in a stereoselective manner. Ring closure of the 2'-hydroxyl group onto a secondary mesylate leaving group with clean inversion of stereochemistry was achieved under surprisingly mild conditions. For the S-cEt modification, the synthesis of all four (thymine, 5-methylcytosine, adenine, and guanine) nucleobase-modified phosphoramidites was accomplished on a multigram scale. Biophysical evaluation of the cMOE- and cEt-containing oligonucleotides revealed that they possess hybridization and mismatch discrimination attributes similar to those of LNA but greatly improved resistance to exonuclease digestion.
    Full-text · Article · Feb 2010 · The Journal of Organic Chemistry
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    No preview · Article · Jan 2010 · ChemInform
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    ABSTRACT: DNA consisting of 12 to 30 linked monomers were prepd., wherein the oligomeric compd. comprises at least 4 regions, wherein each monomer within each region comprises the same type of sugar moiety and wherein the sugar moieties of the monomers of adjacent regions are different from one another; and wherein: at least one region comprises 2-20 linked monomers and each of the other regions independently comprises 1-20 linked monomers; and wherein at least one region is a tetrahydropyran region, wherein each tetrahydropyran region independently comprises one or more tetrahydropyran nucleoside analog I, wherein Bx is a heterocyclic base moiety; T3 and T4 are independently internucleoside linking group or one of T3 and T4 is H, hydroxy protecting group, a linked conjugate group or a 5' or 3'-terminal group; q1-q7 are independently, H, alkyl, alkenyl, alkynyl; R3 and R4 are independently H, hydroxy, halogen, alkyl, alkoxy. Certain such antisense compds. are useful as RNase H antisense compds., as RNAi compds., and/or as modulators of splicing. Thus, nucleoside phosphoramidite II was prepd. and used in prepn. of antisense DNA as modulators of splicing and for treating a disease by inhibiting or altering gene expression. [on SciFinder(R)]
    No preview · Patent · Jan 2010
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    ABSTRACT: In a search to identify chemical modifications to improve the properties of siRNA, we have investigated the effect of the 2 '-O-methyl-2-thiouridine modification on the biological activity of siRNA. Our results indicate that judicious placement of 2 '-O-methyl-2-thiouridine residues could lead to modified siRNA with activity in mammalian cells.
    No preview · Article · Oct 2009 · Nucleosides Nucleotides & Nucleic Acids
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    ABSTRACT: The present disclosure describes tetrahydropyran nucleoside analogs I, wherein: B is a heterocyclic base moiety; R is a hydroxy protecting group; L1 is H, halogen, alkyl; Z1 is O or OE1; Z2 is OH, OE1 or N(E1)(E2); each E1l and E2 is independently alkyl or substituted alkyl; q1-q7 are each, independently, H, alkyl, , alkenyl, alkynyl; wherein each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ1,, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, wherein each J1-J3 is independently, H, alkyl; X is O, S or NJ1, and tetrahydropyran nucleic acid analogs were prepd. Thus, nucleoside II was prepd. and incorporated into tetrahydropyran nucleic acid analogs. More particularly, tetrahydropyran nucleoside analogs are provided, having one or more chiral substituents, that are useful for enhancing properties of oligomeric compds. including nuclease resistance and binding affinity. In some embodiments, the oligomeric compds. provided herein hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. The use of title oligomeric compds. for the manuf. of a medicament for the treatment of a disease characterized by inhibiting gene expression, is reported. [on SciFinder(R)]
    No preview · Patent · Jan 2009
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    ABSTRACT: A process for the prepn. of bicyclic nucleosides is presented such that intermediates I, wherein R is H or an acceptable hydroxyl protecting group; R1 and R2 are independently an (un)substituted C1-C6 alkyl group; and Q is an optionally substituted polycyclic arom. or heteroarom. moiety are characteristic synthons. Specifically, II and III were successfully employed toward the prepn. of bicyclic nucleic acid analog. The bicyclic nucleosides are useful for prepg. chem. modified oligomeric compds. Oligomeric compds. comprising these bicyclic nucleosides have enhanced properties such as increased nuclease resistance. [on SciFinder(R)]
    No preview · Patent · Jan 2009
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    ABSTRACT: Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2′-fluoro/2′-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.
    Preview · Article · Dec 2008 · Nucleic Acids Research
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    ABSTRACT: A number of 2'- O-modified antisense oligonucleotides have been reported for their potential use in oligonucleotide-based therapeutics. To date, most of the in vivo data has been generated for 2'-O-MOE (2'-O-methoxyethyl)- and 2'-O-Me (2'-O-methyl)-modified ASOs (antisense oligonucleotides). We now report the synthesis and biological activity of another 2'-O-modification, namely 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA). This modification resulted in an increase in the affinity of antisense oligonucleotides to complementary RNA similar to 2'-O-MOE-modified ASOs as compared to first-generation antisense oligodeoxynucleotides. The ASO modified with 2'-O-NMA reduced expression of PTEN mRNA in vitro and in vivo in a dose-dependent manner similar to 2'-O-MOE modified ASO. Importantly, toxicity parameters such as AST, ALT, organ weights, and body weights were found to be normal similar to 2'-O-MOE ASO-treated animal models. The data generated in these experiments suggest that 2'-O-NMA is a useful modification for potential application in both antisense and other oligonucleotide-based drug discovery efforts.
    No preview · Article · Jun 2008 · Journal of Medicinal Chemistry

Publication Stats

2k Citations
191.59 Total Impact Points

Institutions

  • 1997-2015
    • Ionis Pharmaceuticals
      Carlsbad, California, United States
  • 2001-2011
    • Isis Pharmaceuticals, Inc.
      Carlsbad, California, United States
    • McGill University
      • Department of Chemistry
      Montréal, Quebec, Canada
  • 2010
    • University of Ljubljana
      • Faculty of Chemistry and Chemical Technology
      Lubliano, Ljubljana, Slovenia
  • 2008
    • Allegheny College
      Мийдвил, Pennsylvania, United States