Kinetic profiling of novel spirobenzo-oxazinepiperidinone derivatives as equilibrative nucleoside transporter 1 inhibitors

Evaluation of kinetic parameters of drug-target binding, kon, koff, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving increasing attention in the early stages of drug discovery. Target binding kinetics emerges as a meaningful concept for the evaluation of a ligand's duration of action and more generally drug efficacy and safety. We report the biological evaluation of a novel series of spirobenzo-oxazinepiperidinone derivatives as inhibitors of the human equilibrative nucleoside transporter 1 (hENT1, SLC29A1). The compounds were evaluated in radioligand binding experiments, i.e., displacement, competition association, and washout assays, to evaluate their affinity and binding kinetic parameters. We also linked these pharmacological parameters to the compounds' chemical characteristics, and learned that separate moieties of the molecules governed target affinity and binding kinetics. Among the 29 compounds tested, 28 stood out with high affinity and a long residence time of 87 min. These findings reveal the importance of supplementing affinity data with binding kinetics at transport proteins such as hENT1.Medicinal Chemistr

Molecular-dynamics studies of single-stranded hexitol, altritol, mannitol, and ribose nucleic acids (HNA, MNA, ANA, and RNA, resp,) and of the stability of HNA center dot RNA, ANA center dot RNA, and MNA center dot RNA duplexes

The influence of the orientation of a 3'-OH group on the conformation and stability of hexitol oligonucleotides in complexes with RNA and as single strands in aqueous solution was investigated by molecular-dynamics (MD)simulations with AMBER 4.1. The particle mesh Ewald (PME) method was used for the treatment of long-range electrostatic interactions. An equatorial orientation of the 3'-OH group in the single-stranded D-mannitol nucleic acid (MNA) m(GCGTAGCG) and in the complex with the RNA r(CGCAUCGC) has an unfavorable influence on the helical stability. Frequent I-I-bonds between the 3'-OH group and the O-C(6') of the phosphate backbone of the following nucleotide explain the distorted conformation of the MNA RNA complex as well as that of the single MNA strand. This is consistent with experimental results that show lowered hybridization potentials for MNA RNA complexes.status: publishe

Oligonucleotide analogues with 4-hydroxy-N-acetylprolinol as sugar substitute

Modified oligonucleotides incorporating trans-4-hydroxy-N-acetyl-L-prolinol (trans-4-HO-L-NAP) or its D-analogue as sugar substitute were synthesised with adenine and thymine as nucleobases. All-adenine oligonucleotides built from (2S,4S) or (2R,4R)-cis-4-hydroxy-N-acetylprolinol were likewise prepared. Hybridisation studies revealed that heterocomplexes formed between polyU and homochiral trans-4-hydroxy-N-acetylprolinol-based oligomers of the same as well as of opposite chirality (polyU/trans-DA(13)* and polyU/trans-LA(13)*). The former, however, were triple-stranded. Other complexes with ribonucleic acids were polyA/trans-LT13* and polyU/cis-LA(13)*. Heteroduplexes with deoxynucleic acids were formed between trans-LA(13)* and oligothymidylate. Interaction was also observed for cis-LA(13)* and oligothymidylate, but not with the D-hydroxyprolinol analogues. Microcalorimetry proved this interaction to be the formation of a triple-stranded complex. Two heteroduplexes, trans-LA(13)*/dT(13) and trans-LA(13)*/polyU, had similar or slightly increased stability when compared to the natural dA(13)/dT(13) or dA(13)/polyU systems. Microcalorimetry clearly indicated the formation of a duplex, in contrast to interactions with N-acetylprolinol oligonucleotides of different stereochemistry. Moreover, the enthalpy change was of the same magnitude but the association constant was slightly lower. Natural nucleicacids thus clearly prefer hybridisation with L-hydroxyprolinol oligomers over D-hydroxyprolinol oligomers. For the series investigated, the L-trans oligomers (Figure 1) seem best to mimic natural oligonucleotides. These modified oligonucleotides formed homocomplexes if both strands were of the same chirality, that is, homocomplexes formed between trans-LA* and trans-LT* and between trans-DA* and trans-DT*, reflecting the isochiral pu-py pairing found in natural nucleic acids. Once more, however, calorimetry proved these to be triplex interactions. Heterochiral pairing was not observed between modified oligonucleotides, but only between modified oligonucleotides and natural polyU. The thermal stabilities of these heterochiral complexes differed clearly

Molecular-dynamics studies of single-stranded hexitol, altritol, mannitol, and ribose nucleic acids (HNA, MNA, ANA, and RNA, resp.) and of the stability of HNA·RNA, ANA·RNA, and MNA·RNA duplexes

The influence of the orientation of a 3'-OH group on the conformation and stability of hexitol oligonucleotides in complexes with RNA and as single strands in aqueous solution was investigated by molecular-dynamics (MD) simulations with AMBER 4.1. The particle mesh Ewald (PME) method was used for the treatment of long-range electrostatic interactions. An equatorial orientation of the 3'-OH group in the single-stranded D-mannitol nucleic acid (MNA) m(GCGTAGCG) and in the complex with the RNA r(CGCAUCGC) has an unfavorable influence on the helical stability. Frequent H-bonds between the 3'-OH group and the O-C(6') of the phosphate backbone of the following nucleotide explain the distorted conformation of the MNA·RNA complex as well as that of the single MNA strand. This is consistent with experimental results that show lowered hybridization potentials for MNA·RNA complexes. An axial orientation of the 3'-OH group in the D-altritol nucleic acid (ANA) a(GCGTAGCG) leads to a stable complex with the complementary RNA r(CGCAUCGC), as well as to a more highly preorganized single-stranded ANA chain. The averaged conformation of the ANA·RNA complex is similar to that of A-RNA, with only minor changes in groove width, helical curvature, and H-bonding pattern. The relative stabilities of ANA·RNA vs. HNA·RNA (HNA = D-hexitol nucleic acid without 3'-OH group) can be explained by differences in restricted movements, H-bonds, and solvation effects

Oligonucleotides composed of 2 '-deoxy-1 ',5 '-anhydro-D-mannitol nucleosides with a purine base moiety

2'-Deoxy-D-mannitol nucleosides with a purine base moiety have been conveniently synthesized starting from 1,5-anhydro-4,6-O-benzylidene-D-glucito The 3-OH function of 1,5-anhydro-4,6-O-benzylidene-D-glucitol was selectively protected with tert-butyldimethylsilyl group, and the 2'-OH function was subsequently converted to the corresponding O-triflate derivative for the introduction of the nucleobase moieties. These nucleoside derivatives were transformed to 1,5-anhydro-4-O-(P-(2 -cyanoethyl)-P-(N,N-diisopropylamino)phosphinyl)-2-deoxy-6-O-monomethoxytrityl-3-O-(tert-butyldimethylsilyl)-D-mannitol with either a 2-(N-6-benzoyladenin-9-yl) or a 2-(N-2-isobutyrylguanin-9-yl) substituent as the building blocks for oligonucleotide synthesis. The corresponding fully modified oligonucleotides afford considerably less stable duplexes with RNA as compared to the 3-deoxy hexitol nucleic acid analogues described previously, The reason for the lower stability was investigated using molecular modeling, MD simulations of single strand MNA(GCGTAGCG) and MNA(GCGTAGCG) complexed with RNA(CGCAUCGC) in aqueous solution were performed by use of AMBER 4.1 with the particle mesh Ewald (PME) method for the treatment of long-range electrostatic interactions, Frequent hydrogen bonds between the 3'-hydroxyl and the 6'-O of the phosphate backbone of the following base changed the conformation of the single strand as well as the MNA:RNA complex. The MNA:RNA backbone widens up and shows partial unwinding and disruption of base pair hydrogen bonds consistent with their low hybridization potential.status: publishe

Oligonucleotides composed of 2'-deoxy-1',5'-anhydro-D-mannitol nucleosides with a purine base moiety

2'-Deoxy-D-mannitol nucleosides with a purine base moiety have been conveniently synthesized starting from 1,5-anhydro-4,6-O-benzylidene-D-glucito The 3-OH function of 1,5-anhydro-4,6-O-benzylidene-D-glucitol was selectively protected with tert-butyldimethylsilyl group, and the 2'-OH function was subsequently converted to the corresponding O-triflate derivative for the introduction of the nucleobase moieties. These nucleoside derivatives were transformed to 1,5-anhydro-4-O-(P-(2 -cyanoethyl)-P-(N,N-diisopropylamino)phosphinyl)-2-deoxy-6-O-monomethoxytrityl-3-O-(tert-butyldimethylsilyl)-D-mannitol with either a 2-(N(6)-benzoyladenin-9-yl) or a 2-(N(2)-isobutyrylguanin-9-yl) substituent as the building blocks for oligonucleotide synthesis. The corresponding fully modified oligonucleotides afford considerably less stable duplexes with RNA as compared to the 3-deoxy hexitol nucleic acid analogues described previously, The reason for the lower stability was investigated using molecular modeling, MD simulations of single strand MNA(GCGTAGCG) and MNA(GCGTAGCG) complexed with RNA(CGCAUCGC) in aqueous solution were performed by use of AMBER 4.1 with the particle mesh Ewald (PME) method for the treatment of long-range electrostatic interactions, Frequent hydrogen bonds between the 3'-hydroxyl and the 6'-O of the phosphate backbone of the following base changed the conformation of the single strand as well as the MNA:RNA complex. The MNA:RNA backbone widens up and shows partial unwinding and disruption of base pair hydrogen bonds consistent with their low hybridization potential

Oligonucleotide analogues with 4-hydroxy-N-acetylprolinol as sugar substitute

Modified oligonucleotides incorporating trans-4-hydroxy-N-acetyl-L-prolinol (trans-4-HO-L-NAP) or its D-analogue as sugar substitute were synthesised with adenine and thymine as nucleobases. All-adenine oligonucleotides built from (2S,4S) or (2R,4R)-cis-4-hydroxy-N-acetylprolinol were likewise prepared. Hybridisation studies revealed that heterocomplexes formed between polyU and homochiral trans-4-hydroxy-N-acetylprolinol-based oligomers of the same as well as of opposite chirality (polyU/trans-DA(13)* and polyU/trans-LA(13)*). The former, however, were triple-stranded. Other complexes with ribonucleic acids were polyA/trans-LT13* and polyU/cis-LA(13)*. Heteroduplexes with deoxynucleic acids were formed between trans-LA(13)* and oligothymidylate. Interaction was also observed for cis-LA(13)* and oligothymidylate, but not with the D-hydroxyprolinol analogues. Microcalorimetry proved this interaction to be the formation of a triple-stranded complex. Two heteroduplexes, trans-LA(13)*/dT(13) and trans-LA(13)*/polyU, had similar or slightly increased stability when compared to the natural dA(13)/dT(13) or dA(13)/polyU systems. Microcalorimetry clearly indicated the formation of a duplex, in contrast to interactions with N-acetylprolinol oligonucleotides of different stereochemistry. Moreover, the enthalpy change was of the same magnitude but the association constant was slightly lower. Natural nucleicacids thus clearly prefer hybridisation with L-hydroxyprolinol oligomers over D-hydroxyprolinol oligomers. For the series investigated, the L-trans oligomers (Figure 1) seem best to mimic natural oligonucleotides. These modified oligonucleotides formed homocomplexes if both strands were of the same chirality, that is, homocomplexes formed between trans-LA* and trans-LT* and between trans-DA* and trans-DT*, reflecting the isochiral pu-py pairing found in natural nucleic acids. Once more, however, calorimetry proved these to be triplex interactions. Heterochiral pairing was not observed between modified oligonucleotides, but only between modified oligonucleotides and natural polyU. The thermal stabilities of these heterochiral complexes differed clearly.status: publishe