Jyoti Chattopadhyaya

Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat

Huntingtons disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD.

A critical survey of the structure-function of the antisense oligo/RNA heteroduplex as substrate for RNase H

The aim of this review is to draw a correlation between the structure of the DNA/RNA hybrid and its properties as a substrate for the RNase H, as well as to point the crucial structural requirements for the modified AONs to preserve their RNase H potency. The review is divided into the following parts: (1) mechanistic considerations, (2) target RNA folding-AON folding-RNase H assistance in AON/RNA hybrid formation, (3) carbohydrate modifications, (4) backbone modifications, (5) base modifications, (6) conjugated AONs, (7) importance of the tethered chromophore in AON for the AON/RNA hybrid interactions with the RNase H. The structural changes in the AON/RNA hybrid duplexes brought by different modifications of the sugar, backbone or base in the antisense strand, and the effect of these changes on the RNase H recognition of the modified substrates have been addressed. Only those AON modifications and the corresponding AON/RNA hybrids, which have been structurally characterized by spectroscopic means and functionally analyzed by their ability to elicit RNase H potency in comparison with the native counterpart have been presented here.

Novel quinoline and naphthalene derivatives as potent antimycobacterial agents

We have designed and synthesized both the quinoline and naphthalene based molecules influenced by the unique structural make-up of mefloquine and TMC207, respectively. These compounds were evaluated for their anti-mycobacterial activity against drug sensitive Mycobacterium tuberculosis H37Rv in vitro at single-dose concentration (6.25 microg/mL). The compounds 22, 23, 26 and 27 inhibited the growth of M. tuberculosis H37Rv 99%, 90%, 98% and 91% respectively. Minimum inhibitory concentration of compounds 22, 23, 26 and 27 was found to be 6.25 microg/mL. Our molecular modeling and docking studies of designed compounds showed hydrogen bonding with Glu-61, Tyr-64 and Asn-190 amino acid residues at the putative binding site of ATP synthase, these interactions were coherent as shown by Mefloquine and TMC207, where hydrogen bonding was found with Tyr-64 and Glu-61 respectively. SAR analysis indicates importance of hydroxyl group and nature of substituents on piperazinyl-phenyl ring was critical in dictating the biological activity of newly synthesized compounds.

Design, synthesis and biological evaluation of novel triazole, urea and thiourea derivatives of quinoline against Mycobacterium tuberculosis

A new series of 20 quinoline derivatives possessing triazolo, ureido and thioureido substituents have been synthesized and their antimycobacterial properties have been evaluated. Compounds 10, 22 and 24 inhibited Mycobacterium tuberculosis H37Rv up to 96%, 98% and 94% respectively, at a fixed concentration of 6.25 microg/mL. Minimum inhibitory concentration of 3.125 microg/mL was obtained for compound 10 and 24, while for compound 22 it was 6.25 microg/mL. Molecular docking calculations suggest critical hydrogen bonding and electrostatic interactions between polar functional groups (such as quinoline-nitrogen, urea-carbonyl and hydroxyl) of anti-mycobacterial (anti-TB) compounds and amino acids (Arg186 and Glu61) of ATP-synthase of M. tuberculosis, could be the probable reason for observed anti-mycobacterial action.

Design, synthesis, biological evaluation and molecular modelling studies of novel quinoline derivatives against Mycobacterium tuberculosis ☆

We herein describe the synthesis and antimycobacterial activity of a series of 27 different derivatives of 3-benzyl-6-bromo-2-methoxy-quinolines and amides of 2-[(6-bromo-2-methoxy-quinolin-3-yl)-phenyl-methyl]-malonic acid monomethyl ester. The antimycobacterial activity of these compounds was evaluated in vitro against Mycobacterium tuberculosis H37Rv for nine consecutive days upon a fixed concentration (6.25 microg/mL) at day one in Bactec assay and compared to untreated TB cell culture as well as one with isoniazide treated counterpart, under identical experimental conditions. The compounds 3, 8, 17 and 18 have shown 92-100% growth inhibition of mycobacterial activity, with minimum inhibitory concentration (MIC) of 6.25 microg/mL. Based on our molecular modelling and docking studies on well-known diarylquinoline antitubercular drug R207910, the presence of phenyl, naphthyl and halogen moieties seem critical. Comparison of docking studies on different stereoisomers of R207910 as well as compounds from our data set, suggests importance of electrostatic interactions. Further structural analysis of docking studies on our compounds suggests attractive starting point to find new lead compounds with potential improvements.

Rapid and quantitative recovery of DNA fragments from gels by displacement electrophoresis (isotachophoresis)

The use of displacement electrophoresis (synonymous to isotachophoresis, steady-state stacking, and moving boundary electrophoresis) for recovery of DNA fragments from agarose and polyacrylamide gels is described. Complete recovery of DNA molecules ranging from oligonucleotides to 20 000-basepairs-long fragments was achieved. The DNA is recovered in a small volume (0.1-0.3 ml) and can be used directly in enzyme-mediated cleavage and ligation reactions. The recovered DNA contained no inhibitory contaminants as revealed by ligation or restriction enzyme cleavage.

Computational and NMR study of quaternary ammonium ion conformations in solution

Conformations around C–N bonds at the quaternary centre in tetraalkylammonium ions in water solution are investigated. Structures of Me4N+, Et4N+, n-Pr4N+, n-Bu4N+, and n-Pe4N+ are calculated using quantum mechanical HF and DFT methods together with the PCM solvent model. Relative solvation free energies of tetraalkylammonium ions are further estimated from microscopic molecular dynamics free energy perturbation simulations using the Gromos-87 and Amber-95 force fields. The predicted free energy difference in solution between two stable conformations of Et4N+, D2d and S4, is 0.6–1.0 kcal mol−1 (in favour of D2d), which is in quantitative agreement with the recent Raman spectroscopy results. The energies of the g+g− conformations of Et4N+ are 3.6–4.0 kcal mol−1 higher. The ions with longer hydrocarbon chains show quite similar energy gap between D2d and S4. The torsion barrier for a two-step interconversion between the D2d and S4 structures is 9.5 kcal mol−1 (HF/6-31G(d) calculations). The computational results are augmented by NMR measurements of the Et4N+–I− salt in aqueous solution, which predict a symmetric structure of Et4N+ in water. However, the D2d and S4 conformers are not discernible due to presumably high similarity of chemical shifts. The calculated conformational energetics in solution together with previously observed D2d, S4 and high-energy g+g−-type structures of Et4N+, n-Pr4N+, and n-Bu4N+ in the solid state indicate that the carbon chain conformations at the quaternary ammonium centre sensitively depend on the actual microenvironment.

Michael addition reactions of α β-ene-3′-phenylselenone of uridine. New synthesis of 2′,3′-dideoxy-ribo-aziridino-, 2′,3′-dideoxy-2′, 3′-ribo-cyclopropyl- & 2,2′-O-anhydro-3′-deoxy-3′-amino uridine derivatives

A high-yielding synthesis of 1- [5′-O-(4-monomethoxytrityl)-2′,3′-dideoxy-3′-phenylselenonyl-β-D-glyceropent-2′-enofuranosyl]uracil (6) is described starting from 5′-O-(4-monomethoxytrityl)-2′,3′-O-anhydro-β-D-lyxofuranosyl uracil 1. The α, β-ene-3′-phenylselenone 6 can be easily deprotected to give 7. The synthetic utilities of 6 and 7 as synthetic equivalent of a dication [ CH2+ - CH2+ ] have been demonstrated from the fact that they act as Michael acceptors and undergo conjugate addition reactions at C-2′with ammonia, methylamine, benzylamine and glycine methyl ester, followed by a direct intramolecular SN2 type displacement reaction at C-3′ in the adduct, to give various 2′,3′-dideoxy-ribo-aziridino uridines 8a – d or 13a – c, while dimethylamine, pyrrolidine, and morpholine give 2,2′-O-anhydro-3′-deoxy-3′-substituted-aminouridines 10a – c. Carbon-nucleophiles such as sodium methyl malonate and conjugate bases of nitromethane and acetophenone upon reaction with 6 provides a convenient access to 2′,3′-dideoxy-2′,3′- cyclopropyl(bicyclo[3.1.0] system) derivatives of uridine 14 – 16, while a reaction of 6 with methylacetoacetate gives an unusual 2′,3′-fused furano(bicyclo[3.3.0] system) derivative 19. Compounds 8a – c, 10a – c, 14, 15 and 19 were deprotected with 80% aqueous acetic acid to give various 5′-hydroxy derivatives 9a – d, 11a – c, 17, 18 and 20. 2′,3′-Dideoxy-ribo-aziridines 13a – c were, however, obtained by direct reactions of 7 with ammonia, methylamine and benzylamine. The methodology described herein constitute a new general approach to fuctionalize the 2′- and 3′-carbons of β-D-nucleosides simultaneously. All new 2′,3′-disubstituted nucleosides with free 5′-hydroxyl group are potential inhibitors of HIV-specific reverse transcriptase.

How do the energetics of the stereoelectronic gauche and anomeric effects modulate the conformation of nucleos(t)ides

The determination of the energetics of the temperature-dependent two-state N reversible arrow S pseudorotational equilibrium through (3)J(HH) analysis in 36 nucleos(t)ides and 3 abasic sugars has a ...

New stereocontrolled synthesis of isomeric C-branched-β-D-nucleosides by intramolecular free-radical cyclization- opening reactions based on temporary silicon connection

Abstract Silicon-bearing allyl group tethered to a 2′ or 3′-hydroxyl group onto the radical generated at the vicinal 2′ or 3′ center in the free-radical precursors 11, 15, 19 and 23 were used to promote intramolecular stereocontrolled free-radical-cyclization to give 12a + 12b, 16, 20 and 24 in 60–70% yields. The configuration at the 2′ or the 3′ center of the allylsiloxane group dictated the stereochemical outcome of the radical cyclization reaction to give cisfused seven-membered rings in compounds 16, 20 and 24 (from 15, 19 and 23, respectively) due to relatively long SiO bond and large CSiO bond angle leading to exclusive 7-endo cyclization. The only exception to this was found in the radical-cyclization of 11 in which both cis-fused and trans-fused seven-membered rings 12a and 12b were formed as inseparable mixture almost in equal amounts. The seven-membered siloxane ring in the radical-cyclized products 12a + 12b, 16, 20 and 24 were then opened up by a simple oxidation reaction to give different 1,5-diols 13a + 13b, 17, 21 and 25 in high yields. The 5′-O-(4-methoxytrityl) group from 13a + 13b, 17, 21 and 25 was then removed to give pure and isomeric C-branched nucleosides 14a, 14b, 18, 22 and 26, respectively. The acid catalyzed isomerization of the pentofuranose ring in 17 to a pyranose system in 18 has been concluded on the basis of comparative structural analysis of 17 and 18 by 500 MHz1H-NMR spectroscopy. The configurations of triol 18 are C-2′(S), C-4′(R), C-5(R), C-6(S), C-7(S) which are also the configurations of the corresponding chiral centers in the precursors 16 and 17. Note that the acid catalyzed isomerization of furanose in 17 to pyranose ring in 18 has been achieved with full retention of anomeric configuration. The configuration of C-3′ in compounds 14a. 14b, 22, and C-2′ in 26 has been elucidated by 1D differential nOe experiments by 1H-NMR spectroscopy at 500 MHz in D2O solution at 293K. The estimation of the 3JHH coupling constants led us to calculate dihedral angles of 14a, 14b, 17, 18, 22 and 26 using the Karplus-Altona algorithm which have allowed us to define the conformational parameters of their constituent sugar moieties. Molecular mechanics calculations have been subsequently performed on the initial NMR structures of 17 and 18 to give their energy minimized conformations. The structures of 18 has been finally confirmed by estimating proton-proton distances derived from their nOe build-up rates by 2D NOESY experiments at 293K at different mixing times.