In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization.

With many safety and technical limitations partly mitigated through chemical modifications, antisense oligonucleotides (ASOs) are gaining recognition as therapeutic entities. The increase in potency realized by 'third generation chemistries' may, however, simultaneously increase affinity to unintended targets with partial sequence complementarity. However, putative hybridization-dependent off-target effects (OTEs), a risk historically regarded as low, are not being adequately investigated. Here we show an unexpectedly high OTEs confirmation rate during screening of fully phosphorothioated (PS)-LNA gapmer ASOs designed against the BACH1 transcript. We demonstrate in vitro mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. Furthermore, with RNase H1 activity residing within the nucleus, hybridization predicted against intronic regions of pre-mRNAs was tested and confirmed. This dramatically increased ASO-binding landscape together with relatively high potency of such interactions translates into a considerable safety concern. We show here that with base pairing-driven target recognition it is possible to predict the putative off-targets and address the liability during lead design and optimization phases. Moreover, in silico analysis performed against both primary as well as spliced transcripts will be invaluable in elucidating the mechanism behind the hepatoxicity observed with some LNA-modified gapmers.

Development of an insect-cell-based assay for detection of kinase inhibition using NF-kappaB-inducing kinase as a paradigm.

Identification of small-molecule inhibitors by high-throughput screening necessitates the development of robust, reproducible and cost-effective assays. The assay approach adopted may utilize isolated proteins or whole cells containing the target of interest. To enable protein-based assays, the baculovirus expression system is commonly used for generation and isolation of recombinant proteins. We have applied the baculovirus system into a cell-based assay format using NIK [NF-kappaB (nuclear factor kappaB)-inducing kinase] as a paradigm. We illustrate the use of the insect-cell-based assay in monitoring the activity of NIK against its physiological downstream substrate IkappaB (inhibitor of NF-kappaB) kinase-1. The assay was robust, yielding a signal/background ratio of 2:1 and an average Z' value of >0.65 when used to screen a focused compound set. Using secondary assays to validate a selection of the hits, we identified a compound that (i) was non-cytotoxic, (ii) interacted directly with NIK, and (iii) inhibited lymphotoxin-induced NF-kappaB p52 translocation to the nucleus. The insect cell assay represents a novel approach to monitoring kinase inhibition, with major advantages over other cell-based systems including ease of use, amenability to scale-up, protein expression levels and the flexibility to express a number of proteins by infecting with numerous baculoviruses.

Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness.

BACKGROUND: Practical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF). METHODS: We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 per protocol). Doses of ultrapure LPS (1, 10, 30 or 100µg/100µl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge. RESULTS: Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1ß, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100µg LPS). At 100µg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10µg and 30µg LPS). CONCLUSIONS: Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa. KEY MESSAGES: Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1ß, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS. TRIAL REGISTRATION: Clinical Trials.gov NCT02284074.

The discovery of 2-amino-3,5-diarylbenzamide inhibitors of IKK-alpha and IKK-beta kinases.

A potent and selective series of 2-amino-3,5-diarylbenzamide inhibitors of IKK-alpha and IKK-beta is described. The most potent compounds are 8h, 8r and 8v, with IKK-beta inhibitory potencies of pIC(50) 7.0, 6.8 and 6.8, respectively. The series has excellent selectivity, both within the IKK family over IKK-epsilon, and across a wide variety of kinase assays. The potency of 8h in the IKK-beta enzyme assay translates to significant cellular activity (pIC(50) 5.7-6.1) in assays of functional and mechanistic relevance.