Naphthyridine tetramer with a pre-organized structure for 1:1 binding to a CGG/CGG sequence.
ABSTRACT: A naphthyridine carbamate dimer (NCD) is a synthetic ligand for DNA containing a CGG/CGG sequence. Although NCD can bind selectively and tightly to a CGG/CGG sequence, the highly cooperative 2:1 binding mode has hampered precise analysis of the binding. We describe herein the synthesis of a series of naphthyridine tetramers consisting of two NCD molecules connected with various linkers to seek a ligand that binds to a CGG/CGG sequence exclusively with a 1:1 stoichiometry. Among the tested ligands, NCTB and Z-NCTS, which have linker moieties with restricted conformational flexibility [biphenyl and (Z)-stilbene linker, respectively], gave the exclusive formation of a 1:1 ligand-CGG/CGG complex. The (Z)-stilbene linker in Z-NCTS was designed to have pre-organized conformation appropriate for the binding and, in fact, resulted in the highest binding affinity. Thermodynamic parameters obtained by isothermal titration calorimetry indicated that the stronger binding of Z-NCTS was attributed to its lower entropic cost. The present study provides not only a novel 1:1 binding ligand, but also valuable feedback for subsequent molecular design of DNA and RNA binding ligands.
Project description:Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder caused by a limited expansion of CGG repeats in the FMR1 gene. Degeneration of neurons in FXTAS cell models can be triggered by accumulation of polyglycine protein (FMRpolyG), a by-product of translation initiated upstream to the repeats. Specific aims of our work included testing if naphthyridine-based molecules could (i) block FMRpolyG synthesis by binding to CGG repeats in RNA, (ii) reverse pathological alterations in affected cells and (iii) preserve the content of FMRP, translated from the same FMR1 mRNA. We demonstrate that cyclic mismatch binding ligand CMBL4c binds to RNA structure formed by CGG repeats and attenuates translation of FMRpolyG and formation of nuclear inclusions in cells transfected with vectors expressing RNA with expanded CGG repeats. Moreover, our results indicate that CMBL4c delivery can reduce FMRpolyG-mediated cytotoxicity and apoptosis. Importantly, its therapeutic potential is also observed once the inclusions are already formed. We also show that CMBL4c-driven FMRpolyG loss is accompanied by partial FMRP reduction. As complete loss of FMRP induces FXS in children, future experiments should aim at evaluation of CMBL4c therapeutic intervention in differentiated tissues, in which FMRpolyG translation inhibition might outweigh adverse effects related to FMRP depletion.
Project description:Synthetic small molecules modulating RNA structure and function have therapeutic potential for RNA diseases. Here we report our discovery that naphthyridine carbamate dimer (NCD) targets disease-causing r(UGGAA)<sub>n</sub> repeat RNAs in spinocerebellar ataxia type 31 (SCA31). Structural analysis of the NCD-UGGAA/UGGAA complex by nuclear magnetic resonance (NMR) spectroscopy clarifies the mode of binding that recognizes four guanines in the UGGAA/UGGAA pentad by hydrogen bonding with four naphthyridine moieties of two NCD molecules. Biological studies show that NCD disrupts naturally occurring RNA foci built on r(UGGAA)<sub>n</sub> repeat RNA known as nuclear stress bodies (nSBs) by interfering with RNA-protein interactions resulting in the suppression of nSB-mediated splicing events. Feeding NCD to larvae of the Drosophila model of SCA31 alleviates the disease phenotype induced by toxic r(UGGAA)<sub>n</sub> repeat RNA. These studies demonstrate that small molecules targeting toxic repeat RNAs are a promising chemical tool for studies on repeat expansion diseases.
Project description:The CGG repeats are present in the 5'-untranslated region (5'-UTR) of the fragile X mental retardation gene FMR1 and are associated with two diseases: fragile X-associated tremor ataxia syndrome (FXTAS) and fragile X syndrome (FXS). FXTAS occurs when the number of repeats is 55-200 and FXS develops when the number exceeds 200. FXTAS is an RNA-mediated disease in which the expanded CGG tracts form stable structures and sequester important RNA binding proteins. We obtained and analysed three crystal structures of double-helical CGG repeats involving unmodified and 8-Br modified guanosine residues. Despite the presence of the non-canonical base pairs, the helices retain an A-form. In the G-G pairs one guanosine is always in the syn conformation, the other is anti. There are two hydrogen bonds between the Watson-Crick edge of G(anti) and the Hoogsteen edge of G(syn): O6·N1H and N7·N2H. The G(syn)-G(anti) pair shows affinity for binding ions in the major groove. G(syn) causes local unwinding of the helix, compensated elsewhere along the duplex. CGG helical structures appear relatively stable compared with CAG and CUG tracts. This could be an important factor in the RNA's ligand binding affinity and specificity.
Project description:Copper boryl species have been widely invoked as reactive intermediates in Cu-catalysed C-H borylation reactions, but their isolation and study have been challenging. Use of the robust dinucleating ligand DPFN (2,7-bis(fluoro-di(2-pyridyl)methyl)-1,8-naphthyridine) allowed for the isolation of two very thermally stable dicopper(i) boryl complexes, [(DPFN)Cu<sub>2</sub>(μ-Bpin)][NTf<sub>2</sub>] (2) and [(DPFN)Cu<sub>2</sub>(μ-Bcat)][NTf<sub>2</sub>] (4) (pin = 2,3-dimethylbutane-2,3-diol; cat = benzene-1,2-diol). These complexes were prepared by cleavage of the corresponding diborane <i>via</i> reaction with the alkoxide [(DPFN)Cu<sub>2</sub>(μ-O <sup><i>t</i></sup> Bu)][NTf<sub>2</sub>] (3). Reactivity studies illustrated the exceptional stability of these boryl complexes (thermal stability in solution up to 100 °C) and their role in the activation of C(sp)-H bonds. X-ray diffraction and computational studies provide a detailed description of the bonding and electronic structures in these complexes, and suggest that the dinucleating character of the naphthyridine-based ligand is largely responsible for their remarkable stability.
Project description:Nucleotide repeat expansions can elicit neurodegeneration as RNA by sequestering specific RNA-binding proteins, preventing them from performing their normal functions. Conversely, mutations in RNA-binding proteins can trigger neurodegeneration at least partly by altering RNA metabolism. In Fragile X-associated tremor/ataxia syndrome (FXTAS), a CGG repeat expansion in the 5'UTR of the fragile X gene (FMR1) leads to progressive neurodegeneration in patients and CGG repeats in isolation elicit toxicity in Drosophila and other animal models. Here, we identify the amyotrophic lateral sclerosis (ALS)-associated RNA-binding protein TAR DNA-binding protein (TDP-43) as a suppressor of CGG repeat-induced toxicity in a Drosophila model of FXTAS. The rescue appears specific to TDP-43, as co-expression of another ALS-associated RNA-binding protein, FUS, exacerbates the toxic effects of CGG repeats. Suppression of CGG RNA toxicity was abrogated by disease-associated mutations in TDP-43. TDP-43 does not co-localize with CGG RNA foci and its ability to bind RNA is not required for rescue. TDP-43-dependent rescue does, however, require fly hnRNP A2/B1 homologues Hrb87F and Hrb98DE. Deletions in the C-terminal domain of TDP-43 that preclude interactions with hnRNP A2/B1 abolish TDP-43-dependent rescue of CGG repeat toxicity. In contrast, suppression of CGG repeat toxicity by hnRNP A2/B1 is not affected by RNAi-mediated knockdown of the fly TDP-43 orthologue, TBPH. Lastly, TDP-43 suppresses CGG repeat-triggered mis-splicing of an hnRNP A2/B1-targeted transcript. These data support a model in which TDP-43 suppresses CGG-mediated toxicity through interactions with hnRNP A2/B1 and suggest a convergence of pathogenic cascades between repeat expansion disorders and RNA-binding proteins implicated in neurodegenerative disease.
Project description:The air-stable title compound, C(12)H(13)N(3)O, which is of inter-est due to its anti-bacterial properties, is an almost planar mol-ecule in which the ten atoms forming the 1,8-naphthyridine ring have an r.m.s. deviation of 0.03?Å from the least-squares plane calculated using the ten atoms. The plane of the acetyl-amino group is slightly inclined [11.7?(2)°] to the plane of the 1,8-naphthyridine ring.
Project description:Transcribed CGG repeat expansions cause neurodegeneration in Fragile X-associated tremor/ataxia syndrome (FXTAS). CGG repeat RNAs sequester RNA-binding proteins (RBPs) into nuclear foci and undergo repeat-associated non-AUG (RAN) translation into toxic peptides. To identify proteins involved in these processes, we employed a CGG repeat RNA-tagging system to capture repeat-associated RBPs by mass spectrometry in mammalian cells. We identified several SR (serine/arginine-rich) proteins that interact selectively with CGG repeats basally and under cellular stress. These proteins modify toxicity in a Drosophila model of FXTAS. Pharmacologic inhibition of serine/arginine protein kinases (SRPKs), which alter SRSF protein phosphorylation, localization, and activity, directly inhibits RAN translation of CGG and GGGGCC repeats (associated with C9orf72 ALS/FTD) and triggers repeat RNA retention in the nucleus. Lowering SRPK expression suppressed toxicity in both FXTAS and C9orf72 ALS/FTD model flies, and SRPK inhibitors suppressed CGG repeat toxicity in rodent neurons. Together, these findings demonstrate roles for CGG repeat RNA binding proteins in RAN translation and repeat toxicity and support further evaluation of SRPK inhibitors in modulating RAN translation associated with repeat expansion disorders.
Project description:Transcribed CGG repeat expansions cause neurodegeneration in Fragile X-associated tremor/ataxia syndrome (FXTAS). CGG repeat RNAs sequester RNA-binding proteins (RBPs) into nuclear foci and undergo repeat-associated non-AUG (RAN) translation into toxic peptides. To identify proteins involved in these processes, we employed a CGG repeat RNA-tagging system to capture repeat associated RBPs by mass spectrometry in mammalian cells. We identified several SR (serine/arginine-rich) proteins that interact selectively with CGG repeats basally and under cellular stress. These proteins modify toxicity in a Drosophila model of FXTAS. Pharmacologic inhibition of serine/arginine protein kinases (SRPKs), which alter SRSF protein phosphorylation, localization and activity, directly inhibits RAN translation of CGG and GGGGCC repeats (associated with C9orf72 ALS/FTD) and triggers repeat RNA retention in the nucleus. Lowering SRPK expression suppressed toxicity in both FXTAS and C9orf72 ALS/FTD model flies and SRPK inhibitors suppressed CGG repeat toxicity in rodent neurons. Together, these findings demonstrate roles for CGG repeat RNA binding proteins in RAN translation and repeat toxicity and support further evaluation of SRPK inhibitors in modulating RAN translation associated with repeat expansion disorders.
Project description:We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)(exp)) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)(exp) toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)(exp) in vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)(exp)'s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2'-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide.
Project description:The asymmetric unit of the title compound, [RuCl(2)(C(13)H(9)N(3))(CO)(2)], consists of four crystallographically independent Ru(II) complexes. Each Ru(II) atom is in a distorted octa-hedral environment coordinated by two carbonyl ligands, two Cl atoms and a chelating 2-(2-pyrid-yl)-1,8-naphthyridine (pynp) ligand. The carbonyl ligands are cis to each other, while the Cl atoms are trans. Relatively short inter-atomic distances (2.60-2.67?Å) between the uncoordinated N atom of pynp and the C atom of the carbonyl imply a donor-acceptor inter-action between the pynp and carbonyl ligands.