Four hydrogen-bonding motifs in oligonucleotides

The design and synthesis of imidazo[5’,4’:4,5]pyrido[2,3-d]pyrimidine nucleosides and 1,8-naphthyridine C-nucleosides with the aim of developing new base pairing motifs consisting of four hydrogen bonds is described.Part (vii): Special Issue Plenary and Invited Lectures ICHC-2

ホスフィン酸型ジヌクレオチド誘導体を含むsiRNA分子の創製

生体内で安定なsiRNA分子の創製を目的として,3\u27-突出末端をホスフィン酸型ジヌクレオチド誘導体で置換した新規修飾型siRNA分子を設計し,それらの合成を検討した.二つ合成法についてそれぞれ検討したが,いずれの場合においても目的とするホスフィン酸型ジヌクレオチドユニット(ジヌクレオチド誘導体)を効率良く合成することができなかった.For the production of stable siRNA molecules in vivo, We have designed novel modified siRNA molecules that contain phosphinate type dinucleotide derivatives at 3\u27-end. Though we examined various synthetic routes of phosphinate type dinucleotide derivatives, we could not find effective synthetic route

1.45\AA resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state

Low molecular mass purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) are homotrimeric enzymes that are tightly inhibited by immucillins. Due to the positive charge on the ribose like part (iminoribitol moiety) and protonation of the N7 atom of the purine ring, immucillins are believed to act as transition state analogues. Over a wide range of concentrations, immucillins bind with strong negative cooperativity to PNPs, so that only every third binding site of the enzyme is occupied (third-of-the-sites binding). 9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) shares with immucillins the protonation of the N7, but not the positive charge on the ribose like part of the molecule. We have previously shown that DFPP-DG interacts with PNPs with subnanomolar inhibition constant. Here, we report additional biochemical experiments to demonstrate that the inhibitor can be bound with the same K(d) ( approximately 190pM) to all three substrate binding sites of the trimeric PNP, and a crystal structure of PNP in complex with DFPP-DG at 1.45A resolution, the highest resolution published for PNPs so far. The crystals contain the full PNP homotrimer in the asymmetric unit. DFPP-DG molecules are bound in superimposable manner and with full occupancies to all three PNP subunits. Thus the postulated third-of-the-sites binding of immucillins should be rather attribute to the second feature of the transition state, ribooxocarbenium ion character of the ligand or to the coexistence of both features characteristic for the transition state. The DFPP-DG/PNP complex structure confirms the earlier observations, that the loop from Pro57 to Gly66 covering the phosphate-binding site cannot be stabilized by phosphonate analogues. The loop from Glu250 to Gln266 covering the base-binding site is organized by the interactions of Asn243 with the Hoogsteen edge of the purine base of analogues bearing one feature of the postulated transition state (protonated N7 position)

Acid sphingomyelinase inhibition suppresses lipopolysaccharide-mediated release of inflammatory cytokines from macrophages and protects against disease pathology in dextran sulphate sodium-induced colitis in mice

Lipopolysaccharide (LPS) and inflammatory cytokines cause activation of sphingomyelinases (SMases) and subsequent hydrolysis of sphingomyelin (SM) to produce a lipid messenger ceramide. The design of SMase inhibitors may offer new therapies for the treatment of LPS- and cytokine-related inflammatory bowel disease. We synthesized a series of difluoromethylene analogues of SM (SMAs). We report here the effects of the most potent SMase inhibitor, SMA-7, on the LPS-mediated release of tumour necrosis factor-α, interleukin-1β and interleukin-6 from THP-1 macrophages and the pathology of dextran sulphate sodium (DSS)-induced colitis in mice. SMA-7 suppressed the LPS-induced cytokine release and nuclear factor-κB activation. LPS stimulation caused a four-fold increase in acid SMase activation, but little increase in neutral SMase activity. The presence of 10 μm SMA-7 caused acid SMase to remain at the control levels and reduced the formation of ceramide. HT-29 cells had significantly decreased cell viability when incubated with media from LPS-stimulated THP-1 macrophages. However, incubating the colon cells in media from both SMA-7 and LPS-treated macrophages caused little decrease in viability, suggesting that ceramide has a role in the LPS-stimulated signalling that releases cytotoxic factors against colon cells. Oral administration of SMA-7 to mice with 2% DSS in the drinking water, for 10 or 21 consecutive days, reduced significantly the cytokine levels in the colon and the severity of colonic injury. These findings suggest a central role for acid SMase/ceramide signalling in the pathology of DSS-induced colitis in mice, indicating a possible preventive or therapeutic role for SMase inhibitor in inflammatory bowel disease