Small Interfering RNAs Containing Dioxane- and Morpholino-Derived Nucleotide Analogues Show Improved Off-Target Profiles and Chirality-Dependent In Vivo Knock-Down.

To expand the applicability of recently developed dioxane- and morpholino-based nucleotide analogues, their seed region destabilizing properties in small interfering RNAs (siRNAs) were investigated in order to improve potential off-target profiles. For this purpose, the corresponding adenosine analogues were synthesized in two diastereomeric series as building blocks for the automated oligonucleotide synthesis. The obtained nucleotide precursors were integrated at position 7 of an siRNA antisense strand, targeting transthyretin messenger RNA. Evaluation of the melting temperatures revealed significant differences in the obtained duplex stabilities between the two diastereomeric series, while the influence of the central scaffold was small. All siRNAs containing these novel nucleotide structures showed improved off-target profiles in vitro compared to their parent sequence with the common 2'-OMe-modified adenosine at the same position. In contrast, in vivo potencies were highly dependent on the chirality within the six-membered nucleotide scaffolds and showed high mRNA downregulations for the (2R,6R)-configured diastereomers.

Syntheses of Morpholine-Based Nucleotide Analogs for Hepatic siRNA Targeting and Stabilization.

A morpholine-based nucleotide analog was developed as a building block for hepatic siRNA targeting and stabilization. Attachment of an asialoglycoprotein-binding GalNAc ligand at the morpholine nitrogen was realized with different linkers. The obtained morpholino GalNAc scaffolds were coupled to the sense strand of a transthyretin-targeting siRNA and tested for their knockdown potency in vitro and in vivo. A clear structure-activity relationship was developed with regard to the linker type and length as well as the attachment site of the morpholino GalNAc moieties at the siRNA sense strand. Further, simple alkylation of the morpholine nitrogen led to a nucleotide analog, which increased siRNA stability, when used as a double 3'-overhang at the sense strand sequence. Combination of the best morpholino GalNAc building blocks as targeting nucleotides with an optimized stabilizing alkyl-substituted morpholine as 3'-overhangs resulted in siRNAs without any phosphorothioate stabilization in the sense strand and clearly improved the duration of action in vivo.

Novel Dioxane and Morpholino Nucleotide Analogues: Syntheses and RNA-Hybridization Properties.

A novel class of nucleotide analogues with a dioxane ring as central scaffold has been developed. Synthetic routes in two diastereomeric series were realized, and the final thymidine analogues were synthesized with common functionalities for the automated oligonucleotide synthesis. The chemical space of the initially derived nucleotides was expanded by changing the central dioxane to analogous morpholine derivatives. This opens up the possibility for further derivatization by attaching different substituents at the morpholine nitrogen. The novel nucleotide building blocks were incorporated into double-stranded RNA sequences, and their hybridization properties investigated by melting-temperature analysis. Both scaffolds, dioxanes and morpholines, had an equal impact on double-strand stability, but Tm values differed depending on the chirality in the six-membered ring.

Application of ray-traced tropospheric slant delays to geodetic VLBI analysis.

The correction of tropospheric influences via so-called path delays is critical for the analysis of observations from space geodetic techniques like the very long baseline interferometry (VLBI). In standard VLBI analysis, the a priori slant path delays are determined using the concept of zenith delays, mapping functions and gradients. The a priori use of ray-traced delays, i.e., tropospheric slant path delays determined with the technique of ray-tracing through the meteorological data of numerical weather models (NWM), serves as an alternative way of correcting the influences of the troposphere on the VLBI observations within the analysis. In the presented research, the application of ray-traced delays to the VLBI analysis of sessions in a time span of 16.5 years is investigated. Ray-traced delays have been determined with program RADIATE (see Hofmeister in Ph.D. thesis, Department of Geodesy and Geophysics, Faculty of Mathematics and Geoinformation, Technische Universität Wien. http://resolver.obvsg.at/urn:nbn:at:at-ubtuw:1-3444, 2016) utilizing meteorological data provided by NWM of the European Centre for Medium-Range Weather Forecasts (ECMWF). In comparison with a standard VLBI analysis, which includes the tropospheric gradient estimation, the application of the ray-traced delays to an analysis, which uses the same parameterization except for the a priori slant path delay handling and the used wet mapping factors for the zenith wet delay (ZWD) estimation, improves the baseline length repeatability (BLR) at 55.9% of the baselines at sub-mm level. If no tropospheric gradients are estimated within the compared analyses, 90.6% of all baselines benefit from the application of the ray-traced delays, which leads to an average improvement of the BLR of 1 mm. The effects of the ray-traced delays on the terrestrial reference frame are also investigated. A separate assessment of the RADIATE ray-traced delays is carried out by comparison to the ray-traced delays from the National Aeronautics and Space Administration Goddard Space Flight Center (NASA GSFC) (Eriksson and MacMillan in http://lacerta.gsfc.nasa.gov/tropodelays, 2016) with respect to the analysis performances in terms of BLR results. If tropospheric gradient estimation is included in the analysis, 51.3% of the baselines benefit from the RADIATE ray-traced delays at sub-mm difference level. If no tropospheric gradients are estimated within the analysis, the RADIATE ray-traced delays deliver a better BLR at 63% of the baselines compared to the NASA GSFC ray-traced delays.

Discovery of N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active and Selective SGK1 Inhibitors.

From a virtual screening starting point, inhibitors of the serum and glucocorticoid regulated kinase 1 were developed through a combination of classical medicinal chemistry and library approaches. This resulted in highly active small molecules with nanomolar activity and a good overall in vitro and ADME profile. Furthermore, the compounds exhibited unusually high kinase and off-target selectivity due to their rigid structure.

End-organ protection in hypertension by the novel and selective Rho-kinase inhibitor, SAR407899.

AIM: To compare the therapeutic efficacy of SAR407899 with the current standard treatment for hypertension [an angiotensin converting enzyme (ACE)-inhibitor and a calcium channel blocker] and compare the frequency and severity of the hypertension-related end-organ damage. METHODS: Long-term pharmacological characte-rization of SAR407899 has been performed in two animal models of hypertension, of which one is sensitive to ACE-inhibition (LNAME) and the other is insensitive [deoxycorticosterone acetate (DOCA)]. SAR407899 efficiently lowered high blood pressure and significantly reduced late-stage end organ damage as indicated by improved heart, kidney and endothelial function and reduced heart and kidney fibrosis in both models of chronic hypertension. RESULTS: Long term treatment with SAR407899 has been well tolerated and dose-dependently reduced elevated blood pressure in both models with no signs of tachyphylaxia. Blood pressure lowering effects and protective effects on hypertension related end organ damage of SAR407899 were superior to ramipril and amlodipine in the DOCA rat. Typical end-organ damage was significantly reduced in the SAR407899-treated animals. Chronic administration of SAR407899 significantly reduced albuminuria in both models. The beneficial effect of SAR407899 was associated with a reduction in leukocyte/macrophage tissue infiltration. The overall protective effect of SAR407899 was superior or comparable to that of ACE-inhibition or calcium channel blockade. Chronic application of SAR407899 protects against hypertension and hypertension-induced end organ damage, regardless of the pathophysiological mechanism of hypertension. CONCLUSION: Rho-kinases-inhibition by the SAR407899 represents a new therapeutic option for the treatment of hypertension and its complications.

Antihypertensive and laxative effects by pharmacological inhibition of sodium-proton-exchanger subtype 3-mediated sodium absorption in the gut.

High intestinal sodium absorption is one mechanism of hypertension and constipation. The sodium-proton-exchanger subtype 3 (NHE3) is an important mediator of sodium absorption in the gut. SAR218034 (SAR) is an orally nonabsorbable specific NHE3 inhibitor. The effect of SAR (1 mg/kg per day in chow) on feces sodium excretion, systolic blood pressure via tail cuff, and gene expression of NHE3 in the gut were studied in senescent lean hypertensive rats (spontaneously hypertensive rats-lean, loaded with NaCl 0.7% in drinking water) and in hypertensive, obese, and hyperinsulinemic rats (spontaneously hypertensive rats-obese, not loaded with NaCl). In spontaneously hypertensive rats-lean, inhibition of intestinal NHE3 by SAR increased feces sodium excretion and reduced urinary sodium excretion, whereas absolute sodium balance and serum sodium concentration were not changed. This suggests reduced intestinal sodium absorption in SAR-treated animals and was associated with increased feces water content (58% versus 42% in placebo treated animals; P=0.0001) and reduction in systolic blood pressure from 222 ± 7 to 198 ± 2 mm Hg (P=0.0001). Angiotensin-converting enzyme inhibition by ramipril plus NHE3 inhibition resulted in an additive blood pressure-lowering effect. In spontaneously hypertensive rats-obese, SAR lowered systolic blood pressure but did not modify serum insulin or cholesterol levels. Gene expression of NHE3 was upregulated in the ileum and colon but not in the jejunum of SAR-treated rats. Reduction of intestinal sodium absorption by selective NHE3 inhibition in the gut reduces high blood pressure and increases feces water excretion. Intestinal NHE3 blockade could be a new treatment strategy for elderly patients suffering from high blood pressure and constipation.

Pharmacological characterization of SAR407899, a novel rho-kinase inhibitor.

Recent advances in basic and clinical research have identified Rho kinase as an important target potentially implicated in a variety of cardiovascular diseases. Rho kinase is a downstream mediator of RhoA that leads to stress fiber formation, membrane ruffling, smooth muscle contraction, and cell motility. Increased Rho-kinase activity is associated with vasoconstriction and elevated blood pressure. We identified a novel inhibitor of Rho kinase (SAR407899) and characterized its effects in biochemical, cellular, tissue-based, and in vivo assays. SAR407899 is an ATP-competitive Rho-kinase inhibitor, equipotent against human and rat-derived Rho-kinase 2 with inhibition constant values of 36 nM and 41 nM, respectively. It is highly selective in panel of 117 receptor and enzyme targets. SAR407899 is approximately 8-fold more active than fasudil. In vitro, SAR407899 demonstrated concentration-dependent inhibition of Rho-kinase-mediated phosphorylation of myosin phosphatase, thrombin-induced stress fiber formation, platelet-derived growth factor-induced proliferation, and monocyte chemotactic protein-1-stimulated chemotaxis. SAR407899 potently (mean IC(50) values: 122 to 280 nM) and species-independently relaxed precontracted isolated arteries of different species and different vascular beds. In vivo, over the dose range 3 to 30 mg/kg PO, SAR407899 lowered blood pressure in a variety of rodent models of arterial hypertension. The antihypertensive effect of SAR407899 was superior to that of fasudil and Y-27632. In conclusion, SAR407899 is a novel and potent selective Rho-kinase inhibitor with promising antihypertensive activity.