Chirality matters: stereo-defined phosphorothioate linkages at the termini of small interfering RNAs improve pharmacology in vivo.

A critical challenge for the successful development of RNA interference-based therapeutics therapeutics has been the enhancement of their in vivo metabolic stability. In therapeutically relevant, fully chemically modified small interfering RNAs (siRNAs), modification of the two terminal phosphodiester linkages in each strand of the siRNA duplex with phosphorothioate (PS) is generally sufficient to protect against exonuclease degradation in vivo. Since PS linkages are chiral, we systematically studied the properties of siRNAs containing single chiral PS linkages at each strand terminus. We report an efficient and simple method to introduce chiral PS linkages and demonstrate that Rp diastereomers at the 5' end and Sp diastereomers at the 3' end of the antisense siRNA strand improved pharmacokinetic and pharmacodynamic properties in a mouse model. In silico modeling studies provide mechanistic insights into how the Rp isomer at the 5' end and Sp isomer at the 3' end of the antisense siRNA enhance Argonaute 2 (Ago2) loading and metabolic stability of siRNAs in a concerted manner.

Small circular interfering RNAs (sciRNAs) as a potent therapeutic platform for gene-silencing.

In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using 'click' chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5'-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.

Incorporating a Thiophosphate Modification into a Common RNA Tetraloop Motif Causes an Unanticipated Stability Boost.

GNRA (N = A, C, G, or U; R = A or G) tetraloops are common RNA secondary structural motifs and feature a phosphate stacked atop a nucleobase. The rRNA sarcin/ricin loop (SRL) is capped by GApGA, and the phosphate p stacks on G. We recently found that regiospecific incorporation of a single dithiophosphate (PS2) but not a monothiophosphate (PSO) instead of phosphate in the backbone of RNA aptamers dramatically increases the binding affinity for their targets. In the RNA:thrombin complex, the key contribution to the 1000-fold tighter binding stems from an edge-on contact between PS2 and a phenylalanine ring. Here we investigated the consequences of replacing the SRL phosphate engaged in a face-on interaction with guanine with either PS2 or PSO for stability. We found that PS2···G and Rp-PSO···G contacts stabilize modified SRLs compared to the parent loop to unexpected levels: up to 6.3 °C in melting temperature Tm and -4.7 kcal/mol in ΔΔG°. Crystal structures demonstrate that the vertical distance to guanine for the closest sulfur is just 0.05 Å longer on average compared to that of oxygen despite the larger van der Waals radius of the former (1.80 Å for S vs 1.52 Å for O). The higher stability is enthalpy-based, and the negative charge as assessed by a neutral methylphosphonate modification plays only a minor role. Quantum mechanical/molecular mechanical calculations are supportive of favorable dispersion attraction interactions by sulfur making the dominant contribution. A stacking interaction between phosphate and guanine (SRL) or uracil (U-turn) is also found in newly classified RNA tetraloop families besides GNRA.

Properties of short double-stranded RNAs carrying randomized base pairs: toward better controls for RNAi experiments.

Short interfering RNAs (siRNAs) are mediators of RNA interference (RNAi), a commonly used technique for selective down-regulation of target gene expression. Using an equimolar mixture of A, G, C, and U phosphoramidites during solid-phase synthesis, we introduced degenerate positions into RNA guide and passenger strands so that, when annealed, a large pool of distinct siRNA duplexes with randomized base pairs at defined sites was created. We assessed the randomization efficiency by deep sequencing one of the RNAs. All possible individual sequences were present in the pool with generally an excellent distribution of bases. Melting temperature analyses suggested that pools of randomized guide and passenger strands RNAs with up to eight degenerate positions annealed so that mismatched base-pairing was minimized. Transfections of randomized siRNAs (rnd-siRNAs) into cells led to inhibition of luciferase reporters by a miRNA-like mechanism when the seed regions of rnd-siRNA guide strands were devoid of degenerate positions. Furthermore, the mRNA levels of a select set of genes associated with siRNA off-target effects were measured and indicated that rnd-siRNAs with degenerate positions in the seed likely show typical non-sequence-specific effects, but not miRNA-like off-target effects. In the wake of recent reports showing the preponderance of miRNA-like off-target effects of siRNAs, our findings are of value for the design of a novel class of easily prepared and universally applicable negative siRNA controls.

3-(4-Fluoro-phen-yl)-6-meth-oxy-2-(4-pyrid-yl)quinoxaline.

In the title compound, C(20)H(14)FN(3)O, the quinoxaline system makes dihedral angles of 32.38 (7) and 48.04 (7)° with the 4-fluoro-phenyl and pyridine rings, respectively. The 4-fluoro-phenyl ring makes a dihedral angle of 57.77 (9)° with the pyridine ring. In the crystal, the mol-ecules form dimeric C-H⋯N hydrogen-bonded R(2) (2)(20) ring motifs lying about crystallographic inversion centers. The dimeric units stack via π-π inter-actions between methoxy-phenyl rings and pyridine-fluoro-phenyl rings with centroid-centroid distances of 3.720 (1) and 3.823 (1) Å, respectively. The respective average perpendicular distances are 3.421 and 3.378 Å, with dihedral angles between the rings of 1.31 (9) and 11.64 (9)°.

1-[2-(Benzyl-amino)-4-pyrid-yl]-2-(4-fluoro-phen-yl)ethane-1,2-dione.

The crystal structure of the title compound, C(20)H(15)FN(2)O(2), contains two crystallographically independent mol-ecules, which are related by a pseudo-inversion center and linked into dimers via inter-molecular N-H⋯N hydrogen bonds. The 4-fluoro-phenyl ring of mol-ecule A makes dihedral angles of 17.17 (16) and 62.25 (15)°, respectively, with the phenyl and pyridine rings. The 4-fluoro-phenyl ring of mol-ecule B makes dihedral angles of 8.50 (16) and 64.59 (15)°, respectively, with the phenyl and pyridine rings. The dihedral angle between the pyridine ring and the phenyl ring of mol-ecule A [60.97 (15)°] is bigger than in mol-ecule B [59.49 (15)°]. The dihedral angle between the two pyridine rings is 1.37 (14)° and between the two phenyl rings is 3.64 (16)°.

Carrier-free Gene Silencing by Amphiphilic Nucleic Acid Conjugates in Differentiated Intestinal Cells.

Nucleic acid therapy can be beneficial for the local treatment of gastrointestinal diseases that currently lack appropriate treatments. Indeed, several oligonucleotides (ONs) are currently progressing through clinical trials as potential treatments for inflammatory bowel diseases. However, due to low uptake of carrier-free ONs by mucosal cells, strategies aimed at increasing the potency of orally administered ONs would be highly desirable. In this work, we explored the silencing properties of chemically modified and highly resistant ONs derivatized with hydrophobic alkyl chain on intestinal epithelial cells. We screened a set of lipid-ON conjugates for the silencing of model Bcl-2 mRNA and selected 2'-deoxy-2'-fluoro-arabinonucleic acid modified ON bearing docosanoyl moiety (L-FANA) as the most potent candidate with lowest toxicity. The efficacy of L-FANA conjugate was preserved in simulated intestinal fluids and in the inverted transfection setup. Importantly, L-FANA conjugate was able to downregulate target gene expression at both mRNA and protein levels in a difficult-to-transfect polarized epithelial cell monolayer in the absence of delivery devices and membrane disturbing agents. These findings indicate that lipid-ON conjugates could be promising therapeutics for the treatment of intestinal diseases as well as a valuable tool for the discovery of new therapeutic targets.

Activation of the Lin28/let-7 Axis by Loss of ESE3/EHF Promotes a Tumorigenic and Stem-like Phenotype in Prostate Cancer.

Although cancer stem-like cells (CSC) are thought to be the most tumorigenic, metastatic, and therapy-resistant cell subpopulation within human tumors, current therapies target bulk tumor cells while tending to spare CSC. In seeking to understand mechanisms needed to acquire and maintain a CSC phenotype in prostate cancer, we investigated connections between the ETS transcription factor ESE3/EHF, the Lin28/let-7 microRNA axis, and the CSC subpopulation in this malignancy. In normal cells, we found that ESE3/EHF bound and repressed promoters for the Lin28A and Lin28B genes while activating transcription and maturation of the let-7 microRNAs. In cancer cells, reduced expression of ESE3/EHF upregulated Lin28A and Lin28B and downregulated the let-7 microRNAs. Notably, we found that deregulation of the Lin28/let-7 axis with reduced production of let-7 microRNAs was critical for cell transformation and expansion of prostate CSC. Moreover, targeting Lin28A/Lin28B in cell lines and tumor xenografts mimicked the effects of ESE3/EHF and restrained tumor-initiating and self-renewal properties of prostate CSC both in vitro and in vivo These results establish that tight control by ESE3/EHF over the Lin28/let-7 axis is a critical barrier to malignant transformation, and they also suggest new strategies to antagonize CSC in human prostate cancer for therapeutic purposes. Cancer Res; 76(12); 3629-43. ©2016 AACR.

Stereochemical bias introduced during RNA synthesis modulates the activity of phosphorothioate siRNAs.

An established means of improving the pharmacokinetics properties of oligoribonucleotides (ORNs) is to exchange their phosphodiester linkages for phosphorothioates (PSs). However, this strategy has not been pursued for small interfering RNAs (siRNAs), possibly because of sporadic reports that PS siRNAs show reduced inhibitory activity. The PS group is chiral at phosphorous (Rp/Sp centres), and conventional solid-phase synthesis of PS ORNs produces a population of diastereoisomers. Here we show that the choice of the activating agent for the synthesis of a PS ORN influences the Rp/Sp ratio of PS linkages throughout the strand. Furthermore, PS siRNAs composed of ORNs with a higher fraction of Rp centres show greater resistance to nucleases in serum and are more effective inhibitors in cells than their Sp counterparts. The finding that a stereochemically biased population of ORN diastereoisomers can be synthesized and exploited pharmacologically is important because uniform PS modification of siRNAs may provide a useful compromise of their pharmacokinetics and pharmacodynamics properties in RNAi therapeutics.

Pyridinylquinoxalines and pyridinylpyridopyrazines as lead compounds for novel p38 alpha mitogen-activated protein kinase inhibitors.

Various substituted 2(3)-(4-fluorophenyl)-3(2)-(pyridin-4-yl)quinoxalines and 2(3)-(4-fluorophenyl)-3(2)-(pyridin-4-yl)pyridopyrazines were synthesized as novel p38 alpha MAP kinase inhibitors via different short synthetic strategies with high variation possibilities. The formation of the quinoxaline/pyridopyrazine core was achieved from alpha-diketones and o-phenylenediamines/alpha-diaminopyridines under microwave irradiation. Introduction of an amino moiety at the pyridine C2 position of the 2(3)-(4-fluorophenyl)-3(2)-(pyridin-4-yl)quinoxalines led to compounds showing potent enzyme inhibition down to the double-digit nanomolar range (6f; IC(50) = 81 nM). Replacement of the quinoxaline core with pyrido[2,3-b]pyrazine gave compound 9e with superior p38 alpha MAP kinase inhibition (IC(50) = 38 nM).