Antisense RNA C9orf72 hexanucleotide repeat associated with amyotrophic lateral sclerosis and frontotemporal dementia forms a triplex-like structure and binds small synthetic ligand.

The abnormal expansion of GGGGCC/GGCCCC hexanucleotide repeats (HR) in C9orf72 is associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Structural polymorphisms of HR result in the multifactorial pathomechanism of ALS/FTD. Consequently, many ongoing studies are focused at developing therapies targeting pathogenic HR RNA. One of them involves small molecules blocking sequestration of important proteins, preventing formation of toxic nuclear foci. However, rational design of potential therapeutics is hindered by limited number of structural studies of RNA-ligand complexes. We determined the crystal structure of antisense HR RNA in complex with ANP77 ligand (1.1 Šresolution) and in the free form (0.92 and 1.5 Šresolution). HR RNA folds into a triplex structure composed of four RNA chains. ANP77 interacted with two neighboring single-stranded cytosines to form pseudo-canonical base pairs by adopting sandwich-like conformation and adjusting the position of its naphthyridine units to the helical twist of the RNA. In the unliganded structure, the cytosines formed a peculiar triplex i-motif, assembled by trans C•C+ pair and a third cytosine located at the Hoogsteen edge of the C•C+ pair. These results extend our knowledge of the structural polymorphisms of HR and can be used for rational design of small molecules targeting disease-related RNAs.

2-Amino-1,8-naphthyridine Dimer (ANP77), a High-Affinity Binder to the Internal Loops of C/CC and T/CC Sites in Double-Stranded DNA.

Small molecules targeting DNA regions with structural fluctuation are an important class of molecule as chemical probes for studying the role of these structures in biological systems and the development of neurological disorders. The molecule ANP77 we described here, where a three-atom linker connects two 2-amino-1,8-naphthyridines at the C7 position, was found to form stacked structure with protonation of naphthyridine at low pH, and bound to the internal loop consisting of C/CC and T/CC in double-stranded DNA with affinities of 4.8 and 34.4 nM, respectively. Mass spectrometry and isothermal titration calorimetry analyses determined the stoichiometry for the binding as 1:1, and chemical footprinting with permanganate and NMR structural analysis revealed that the T in the T/CC was forced to flip out toward an extrahelical position upon ANP77 binding. Protonated stacked ANP77 interacted with two adjacent cytosines through hydrogen bonding and occupied the position in the duplex by flipping out the C or T opposite CC. Finally, this study demonstrated the potential of ANP77 for binding to the sequences of biological significance with the TG(T/C)CC repeat of the PIG3 promoter and the telomere repeat CCCTAA.

A small-molecule fluorescence probe ANP77 for sensing RNA internal loop of C, U and A/CC motifs and their binding molecules.

Small-molecules interacting with particular RNAs and modulating their functions are vital tools for RNA-targeting drug discovery. Considering the substantial distribution of the internal loops involving two contiguous cytosines opposite to a single-nucleotide base (Y/CC; Y = C, U or A) within the biologically significant functional RNAs, developing small-molecule probes targeting Y/CC sites should provide profound insight into their functions and roles in biochemical processes. Herein, we report ANP77 as the small-molecule probe for sensing RNA internal loop of Y/CC motifs and molecules binding to the motifs. The Y/CC motifs interact with ANP77 via the formation of a 1:1 complex and quench the fluorescence of ANP77. The flanking sequence-dependent binding to C/CC and U/CC sites was assessed by fluorometric screening, provided the binding heat maps. The quenching phenomena of ANP77 fluorescence was confirmed with intrinsic potential drug target pre-miR-1908. Finally, the binding-dependent fluorescence quenching of ANP77 was utilized in the fluorescence indicator displacement assay to demonstrate the potential of ANP77 as an indicator by using the RNA-binding drugs, risdiplam and branaplam.

Small Molecule-Induced Dimerization of Hairpin RNA Interfered with the Dicer Cleavage Reaction.

MicroRNAs are potential targets for drug development. Small molecules that can inhibit or promote a specific miRNA's biogenesis would be useful for regulating its target genes. Various types of small molecules have been investigated so far for their potential application in modulating miRNA biogenesis. They bind to the target primary or precursor miRNAs and inhibit the processing of these precursors by Drosha or Dicer. However, the binding site that effectively interferes with the Dicer cleavage reaction is still undetermined. Here we report that our designed small molecule restricted naphthyridine dimer (RND) binds to the hairpin loop of a hairpin RNA and induces its dimerization. This study shows that the binding of the RND to the hairpin loop was not effective in interfering with the Dicer cleavage reaction, but dimerization of the hairpin RNA by RND binding effectively interfered with the Dicer cleavage reaction.

Synthesis of Naphthyridine Dimers with Conformational Restriction and Binding to DNA and RNA.

One of the important determinants in the efficiency of a molecular interaction is the necessity for conformational changes in host and/or guest molecules upon binding. In small-molecule interactions with nucleic acids, conformational changes on both molecules are often involved, especially in intercalating binding. Mismatch binding ligands (MBLs) we described here consist of two heterocycles that predominantly exist in one conformation, so it is of interest to determine if such molecules can bind to any DNA and RNA structures. One molecule, 1-NHR, which predominantly exists as the unstacked conformation in aqueous solvent, has been successfully synthesized and characterized. Compound 1-NHR did not efficiently bind to GX/Y DNA and RNA sequences, but the binding pattern is different from that of authentic MBL naphthyridine carbamate dimer. In vitro selection of RNA that specifically binds to 1-NHR was performed from pre-miR-29a loop library RNA, and one RNA, to which 1-NHR bound with high affinity, has been successfully identified. Although it was anticipated that 1-NHR, with a predominantly unstacked conformation, would show entropy-driven binding, isothermal titration calorimetry analysis suggested that the binding of 1-NHR to RNA was enthalpy driven with an apparent Kd of about 100 nm.

One-Pot Access to Benzo[a]carbazoles via Palladium(II)-Catalyzed Hetero- and Carboannulations.

A Pd(II)-catalyzed direct synthesis of benzo[a]carbazoles has been achieved through aminopalladation of alkynes, followed by intramolecular nucleophilic addition of the generated carbon-palladium bond to a tethered cyano/aldehyde group. Compared to literature procedures, this synthetic approach is operationally simple, uses simple substrates, and offers a fast intramolecular assembly resulting in the direct synthesis of benzo[a]carbazoles in which a wide variation of substituents at different sites is well-tolerated, leaving enough opportunity for diversification.

Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway.

BACKGROUND: Current chemotherapeutic agents based on apoptosis induction are lacking in desired efficacy. Therefore, there is continuous effort to bring about new dimension in control and gradual eradication of cancer by means of ever evolving therapeutic strategies. Various forms of PCD are being increasingly implicated in anti-cancer therapy and the complex interplay among them is vital for the ultimate fate of proliferating cells. We elaborated and illustrated the underlying mechanism of the most potent Andrographolide analogue (AG-4) mediated action that involved the induction of dual modes of cell death-apoptosis and autophagy in human leukemic U937 cells. PRINCIPAL FINDINGS: AG-4 induced cytotoxicity was associated with redox imbalance and apoptosis which involved mitochondrial depolarisation, altered apoptotic protein expressions, activation of the caspase cascade leading to cell cycle arrest. Incubation with caspase inhibitor Z-VAD-fmk or Bax siRNA decreased cytotoxic efficacy of AG-4 emphasising critical roles of caspase and Bax. In addition, AG-4 induced autophagy as evident from LC3-II accumulation, increased Atg protein expressions and autophagosome formation. Pre-treatment with 3-MA or Atg 5 siRNA suppressed the cytotoxic effect of AG-4 implying the pro-death role of autophagy. Furthermore, incubation with Z-VAD-fmk or Bax siRNA subdued AG-4 induced autophagy and pre-treatment with 3-MA or Atg 5 siRNA curbed AG-4 induced apoptosis-implying that apoptosis and autophagy acted as partners in the context of AG-4 mediated action. AG-4 also inhibited PI3K/Akt/mTOR pathway. Inhibition of mTOR or Akt augmented AG-4 induced apoptosis and autophagy signifying its crucial role in its mechanism of action. CONCLUSIONS: Thus, these findings prove the dual ability of AG-4 to induce apoptosis and autophagy which provide a new perspective to it as a potential molecule targeting PCD for future cancer therapeutics.

Facile synthesis of 2-arylmethylindoles and 2-vinylic indoles through palladium-catalyzed heteroannulations of 2-(2-propynyl)aniline and 2-(2-propynyl)tosylanilide.

A facile method for the general synthesis of 2-arylmethylindoles has been developed through the reaction of 2-(2-propynyl)aniline or 2-(2-propynyl)tosylanilide with aryl iodides in the presence of Pd(OAc)2, PPh3, and DBU. 2-(2-Propynyl)tosylanilide is found to be reactive also towards electron deficient alkenes in the presence of Pd(OAc)2 and sodium iodide under an oxygen atmosphere, providing easy access to 2-vinylic indoles which possess exclusive E-stereochemistry in the side chain double bond. Operational simplicity, compatibility of the various functional groups, and ease of product formation are the hallmarks of these methods. A mechanism has been proposed to explain the product formation.

Palladium-catalyzed approach for the general synthesis of (E)-2-arylmethylidene-N-tosylindolines and (E)-2-arylmethylidene-N-tosyl/nosyltetrahydroquinolines: access to 2-substituted indoles and quinolines.

A facile and efficient method for the synthesis of (E)-2-arylmethylidene-N-tosylindolines and (E)-2-arylmethylidene-N-tosyl/nosyltetrahydroquinoline variants has been developed through palladium-catalyzed cyclocondensation of aryl iodides with readily available 1-(2-tosylaminophenyl)prop-2-yn-1-ols and their higher homologues, respectively. The proposed reaction mechanism invokes the operation of trans-aminopalladation during cyclization (5/6-exo-dig), which ensures exclusive (E)-stereochemistry in the products. The method is fast, operationally simple, totally regio- and stereoselective, and versatile enough to access a variety of 2-substituted indoles and quinolines. The reactions proceeded efficiently with a wide variety of substrates and afforded the corresponding products in moderate to excellent yields.

Synthesis, cytotoxicity, and structure-activity relationship (SAR) studies of andrographolide analogues as anti-cancer agent.

A series of analogues of andrographolide, prepared through chemo-selective functionalization at C14 hydroxy, have been evaluated for in vitro cytotoxicities against human leukemic cell lines. Two of the analogues (6a, 9b) exhibited significant potency. Preliminary studies on structure-activity relationship (SAR) revealed that the α-alkylidene-γ-butyrolactone moiety of andrographolide played a major role in the activity profile. The structures of the analogues were established through spectroscopic and analytical data.

Expedient and rapid synthesis of 1,2,3-triazolo[5,1-c]morpholines through palladium-copper catalysis.

A one-pot approach using palladium-copper as catalyst has been developed for the synthesis of morpholines fused with 1,2,3-triazole. Good regioselectivity, mild reaction conditions, high yields and short reaction time are the hallmarks of this method.