Human genetic evidence suggests a protective role of loss-of-function variants in 17-beta hydroxysteroid dehydrogenase 13 (HSD17B13) for liver fibrotic diseases. Although there is limited preclinical experimental data on Hsd17b13 antisense oligonucleotide (ASO) or siRNA in a fibrosis model, several ASO and siRNA approaches are being tested clinically as potential therapies for nonalcoholic steatohepatitis (NASH). The aim of this study was to assess the therapeutic potential of Hsd17b13 ASO in a preclinical advanced NASH-like hepatic fibrosis in vivo model. In vitro testing on primary hepatocytes demonstrated that Hsd17b13 ASO exhibited strong efficacy and specificity for knockdown of the Hsd17b13 gene. In choline-deficient, L-amino acid-defined, HFD (CDAHFD)-induced steatotic and fibrotic mice, therapeutic administration of Hsd17b13 ASO resulted in a significant and dose-dependent reduction of hepatic Hsd17b13 gene expression. The CDAHFD group exhibited considerably elevated liver enzyme levels, hepatic steatosis score, hepatic fibrosis, and increased fibrotic and inflammatory gene expression, indicating an advanced NASH-like hepatic fibrosis phenotype. Although Hsd17b13 ASO therapy significantly affected hepatic steatosis, it had no effect on hepatic fibrosis. Our findings demonstrate, for the first time, that Hsd17b13 ASO effectively suppressed Hsd17b13 gene expression both in vitro and in vivo, and had a modulatory effect on hepatic steatosis in mice, but did not affect fibrosis in the CDAHFD mouse model of NASH.

Tau is a microtubule-associated protein (MAPT, tau) implicated in the pathogenesis of tauopathies, a spectrum of neuro-degenerative disorders characterized by accumulation of hyper-phosphorylated, aggregated tau. Because tau pathology can be distinct across diseases, a pragmatic therapeutic approach may be to intervene at the level of the tau transcript, as it makes no assumptions to mechanisms of tau toxicity. Here we per-formed a large library screen of locked-nucleic-acid (LNA)-modified antisense oligonucleotides (ASOs), where careful tiling of the MAPT locus resulted in the identification of hot spots for activity in the 30 UTR. Further modifications to the LNA design resulted in the generation of ASO-001933, which selectively and potently reduces tau in primary cultures from hTau mice, monkey, and human neurons. ASO-001933 was well tolerated and produced a robust, long-lasting reduction in tau protein in both mouse and cynomolgus monkey brain. In monkey, tau protein reduction was maintained in brain for 20 weeks post injection and corresponded with tau protein reduction in the cerebrospinal fluid (CSF). Our results demonstrate that LNA-ASOs exhibit excellent drug-like properties and sustained efficacy likely translating to infrequent, intrathecal dosing in patients. These data further support the development of LNA-ASOs against tau for the treatment of tauopathies.

Antisense oligonucleotides are a relatively new therapeutic modality and safety evaluation is still a developing area of research. We have observed that some oligonucleotides can produce acute, nonhybridization dependent, neurobehavioral side effects after intracerebroventricular (ICV) dosing in mice. In this study, we use a combination of in vitro, in vivo, and bioinformatics approaches to identify a sequence design algorithm, which can reduce the number of acutely toxic molecules synthesized and tested in mice. We find a cellular assay measuring spontaneous calcium oscillations in neuronal cells can predict the behavioral side effects after ICV dosing, and may provide a mechanistic explanation for these observations. We identify sequence features that are overrepresented or underrepresented among oligonucleotides causing these reductions in calcium oscillations. A weighted linear combination of the five most informative sequence features predicts the outcome of ICV dosing with >80% accuracy. From this, we develop a bioinformatics tool that allows oligonucleotide designs with acceptable acute neurotoxic potential to be identified, thereby reducing the number of toxic molecules entering drug discovery pipelines. The informative sequence features we identified also suggest areas in which to focus future medicinal chemistry efforts.

Calcitonin gene-related peptide (CGRP) receptor antagonists have been shown clinically to be effective treatments for migraine. Zavegepant (BHV-3500, BMS-742413) is a high affinity antagonist of the CGRP receptor (hCGRP K-i = 0.023 nM) that has demonstrated efficacy in the acute treatment of migraine with intranasal delivery in a Phase 2/3 trial, despite showing low oral bioavailability in rats (F-PO = 1.7%). Using zavegepant as a template, we sought to improve oral bioavailability through a series of azepinones which were designed in an attempt to reduce the number of rotatable bonds. These efforts led to the discovery of compound 21 which was able to mostly maintain high affinity binding (hCGRP K-i = 0.100 nM) and in vivo efficacy in the marmoset facial blood flow assay, while greatly improving oral bioavailability (rat F-PO = 17%).

The early promise of gene-based therapies is currently being realized at an accelerated pace with over 155 active clinical trials for antisense compounds and multiple FDA-approved oligonucleotide therapeutics. Fundamental advances in this area are vital and present an unprecedented opportunity to both address disease states that have been resistant to other common modalities and improve the significant sustainability challenges associated with production of these complex molecules on a commercial scale. The advent of phosphoramidite coupling chemistry and solid-phase synthesis 40 years ago democratized oligonucleotide synthesis to the scientific community, paving the way for many of these stunning developments. The reliability and generality of this approach for the preparation of native phosphate-diesters is attributed to the high reactivity of phosphorus when in the P(III)-oxidation state versus the desired P(V), as it enables rapid P-heteroatom bond formation. However, the growing demand for more diverse phosphorus-based linkages has challenged the limits of this technology. For example, the phosphorothioate (PS) linkage, which stabilizes oligonucleotides towards nuclease cleavage, is universally employed in current oligonucleotide therapeutics but is generally incorporated in racemic form. Stereodefined PS oligonucleotides may have desirable biological and physical properties but are accessed with difficulty using phosphoramidite chemistry. Here we report a flexible and efficient [P(V)]-based platform that can install a wide variety of phosphate linkages at will into oligonucleotides. This approach uses readily accessible reagents and can efficiently install not only stereodefined or racemic thiophosphates, but can install any combination of (S, R or rac)-PS with native phosphodiester (PO2) and phosphorodithioate (PS2) linkages into DNA and other modified nucleotides. Importantly this platform easily accesses this diversity under a standardized coupling protocol with sustainably prepared, stable, P(V) reagents.

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Phosphorothioate nucleotides have emerged as powerful pharmacological substitutes of their native phosphodiester analogs with important translational applications in antisense oligonucleotide (ASO) therapeutics and cyclic dinucleotide (CDN) synthesis. Stereocontrolled installation of this chiral motif has long been hampered by the systemic use of phosphorus(III) [P(III)]-based reagent systems as the sole practical means of oligonucleotide assembly. A fundamentally different approach is described herein: the invention of a P(V)-based reagent platform for programmable, traceless, diastereoselective phosphorus-sulfur incorporation. The power of this reagent system is demonstrated through the robust and stereocontrolled synthesis of various nucleotidic architectures, including ASOs and CDNs, via an efficient, inexpensive, and operationally simple protocol.

Key words cyclic dinucleotides - oligonucleotides - phosphorus(V) reagents - phosphorothioates

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Mirk kinase is a gene upregulated and sometimes amplified in pancreatic cancers and in ovarian cancers, but expressed at very low levels in most normal diploid cells except for skeletal muscle. The muscle cell function of Mirk kinase selected for by cancer cells is unknown. It is now shown that Mirk protein is expressed at low levels and is largely nuclear in cycling skeletal muscle C2C12 myoblasts, but is translocated to the cytoplasm and upregulated when myoblasts initiate differentiation, as shown by immunofluorescence staining and by cell fractionation. Either Mirk depletion or Mirk kinase inhibition increased ROS levels in cycling C2C12 myoblasts. However, Mirk protein is localized in the cytoplasm of mature muscle fibers, specifically in the fast twitch fibers of human skeletal muscle where toxic ROS levels are generated by muscle contraction. C2C12 myoblasts at high density in differentiation media fuse to form differentiated postmitotic myotubes that can contract. A Mirk kinase inhibitor induced a dose-dependent increase in ROS in this model for fast twitch fibers of human skeletal muscle. Efficient Mirk depletion in SU86.86 pancreatic cancer cells by an inducible shRNA decreased expression of eight antioxidant genes. Thus both cancer cells and differentiated myotubes utilize Mirk kinase to relieve oxidative stress.

Calcitonin gene-related peptide (CGRP) receptor antagonists have been shown to be efficacious as abortive migraine therapeutics with the absence of cardiovascular liabilities that are associated with triptans. Herein, we report the discovery of a highly potent CGRP receptor antagonist, BMS-742413, with the potential to provide rapid onset of action through intranasal delivery. The compound displays excellent aqueous solubility, oxidative stability, and toxicological profile. BMS-742413 has good intranasal bioavailability in the rabbit and shows a robust, dose-dependent inhibition of CGRP-induced increases in marmoset facial blood flow.

Calcitonin gene-related peptide (CGRP) receptor antagonists have been clinically shown to be effective in the treatment of migraine, but identification of potent and orally bioavailable compounds has been challenging. Herein, we describe the conceptualization, synthesis, and preclinical characterization of a potent, orally active CGRP receptor antagonist 5 (BMS-846372). Compound 5 has good oral bioavailability in rat, dog, and cynomolgus monkeys and overall attractive preclinical properties including strong (>50% inhibition) exposure-dependent in vivo efficacy in a marmoset migraine model.

Constrained phenylalanine derivatives containing hydrophobic groups and hydrogen bond acceptor and/or donor functionalities were synthesized through a tandem palladium-mediated Heck reaction followed by a rhodium(II)-catalyzed asymmetric hydrogenation. Aryl bromides were found to be better substrates in providing products with higher purity and in good yield. The cesium carbonate-mediated cyclization proceeded smoothly in good yield and optical purity. Aryl iodides reacted selectively over bromides under Jeffery-type conditions (Pd(OAc)(2), Bu(4)NCl, Et(3)N) providing an opportunity for further metal-mediated functionalization. (C) 2010 Elsevier Ltd. All rights reserved.

The calcitonin gene-related peptide (CGRP) receptor has been implicated in the pathogenesis of migraine. A class of urethanamide derivatives has been identified as potent inhibitors of the CGRP receptor. Compound 20 was found to be among the most potent (IC(50)=17pM). It was shown to retain excellent aqueous solubility (>50mg/mL, pH 7) while dramatically improving solution stability as compared to our previously disclosed development candidate, BMS-694153 (1).

A practical and expedient synthesis of the title compounds is described. They were prepared by Stille reaction of nitro halopyridines 4 or nitro fluro-halobenzenes 10, followed by Michael addition of tertbutyl 4-aminopiperidine-1-carboxylate to the resulting activated vinyl compounds 5 and 11, hydrogenation (-NO(2)->-NH(2)), cyclic urea formation, Boc removal, and HCl salt formation. However, N3 and F1 analogs could not be made by this general strategy. Activated vinyl compounds 5a and 5d when reacted with tert-butyl 4-aminopiperidine-1-carboxylate did not stop at the desired Michael addition stage; but proceeded to produce azaindolines 8 and 9. Michael addition did not occur to compound 11d; instead, the fluorine atom was displaced. (c) 2008 Elsevier Ltd. All rights reserved.

The role of the intestine in the elimination of (2R)-2-{(3R)-3-amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide (DPC 333), a potent inhibitor of tissue necrosis factor α–converting enzyme, was investigated in mice and rats in vivo and in vitro. In Madine-Darby canine kidney cells stably transfected with P-glycoprotein (P-gp) and DPC 333, the transport from B→A reservoirs exceeded the transport from A→B by approximately 7-fold. In Caco-2 monolayers and isolated rat ileal mucosa, DPC 333 was transported from basolateral to apical reservoirs in a concentration-dependent, saturable manner, and transport was blocked by N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), confirming the contribution of P-gp/breast cancer resistance protein in B→A efflux of DPC 333. In quantitative whole body autoradiography studies with [14C]DPC 333 in mice and rats, radioactivity was distributed throughout the small intestine in both species. In GF120918-pretreated bile duct–cannulated rats, radioactivity in feces was reduced 60%. Using the in situ perfused rat intestine model, ∼20% of an i.v. dose of [14C]DPC 333 was measured in the intestinal lumen within 3 h postdose, 12% as parent. Kinetic analysis of data suggested that excreted DPC 333 may be further metabolized in the gut. Intestinal clearance was 0.2 to 0.35 l/h/kg. The above data suggest that in the rodent the intestine serves as an organ of DPC 333 excretion, mediated in part by the transporter P-gp.

Enantioselective synthesis of constrained trans-amino benzazepi none utilizing palladium-mediated Jeffery-Heck reaction and rhodium(II) catalyzed asymmetric hydrogenation as key steps are described. Diverse functional groups such as alkyl, aryl, basic or amino acid moieties were introduced with minimal racemization. (c) 2007 Elsevier Ltd. All rights reserved.