Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides.

The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.

A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling.

Evaluation and optimization of physicochemical and metabolic properties of compounds are a crucial component of the drug development process. Continuous access to this information during the design-make-test-analysis cycle enables identification of chemical entities with suitable properties for efficient project progression. In this study, we describe an integrated and automated assay panel (DMPK Wave 1) that informs weekly on lipophilicity, solubility, human plasma protein binding, and metabolic stability in rat hepatocytes and human liver microsomes. All assays are running in 96-well format with ultraperformance liquid chromatography-mass spectrometry (MS)/MS as read-out. A streamlined overall workflow has been developed by optimizing all parts of the process, including shipping of compounds between sites, use of fit-for-purpose equipment and information systems, and technology for compound requesting, data analysis, and reporting. As a result, lead times can be achieved that well match project demands across sites independently of where compounds are synthesized. This robust screening strategy is run on a weekly basis and enables optimization of structure-activity relationships in parallel with DMPK properties to allow efficient and informed decision making.

Secretome-Based Screening in Target Discovery.

Secreted proteins and their cognate plasma membrane receptors regulate human physiology by transducing signals from the extracellular environment into cells resulting in different cellular phenotypes. Systematic use of secretome proteins in assays enables discovery of novel biology and signaling pathways. Several secretome-based phenotypic screening platforms have been described in the literature and shown to facilitate target identification in drug discovery. In this review, we summarize the current status of secretome-based screening. This includes annotation, production, quality control, and sample management of secretome libraries, as well as how secretome libraries have been applied to discover novel target biology using different disease-relevant cell-based assays. A workflow for secretome-based screening is shared based on the AstraZeneca experience. The secretome library offers several advantages compared with other libraries used for target discovery: (1) screening using a secretome library directly identifies the active protein and, in many cases, its cognate receptor, enabling a rapid understanding of the disease pathway and subsequent formation of target hypotheses for drug discovery; (2) the secretome library covers significant areas of biological signaling space, although the size of this library is small; (3) secretome proteins can be added directly to cells without additional manipulation. These factors make the secretome library ideal for testing in physiologically relevant cell types, and therefore it represents an attractive approach to phenotypic target discovery.

Phenotypic Screen with the Human Secretome Identifies FGF16 as Inducing Proliferation of iPSC-Derived Cardiac Progenitor Cells.

Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count. From this screen, we identified FGF1, FGF4, FGF9, FGF16, FGF18, and seven additional proteins that induce proliferation of cardiac progenitor cells. FGF9 and FGF16 belong to the same FGF subfamily, share high sequence identity, and are described to have similar receptor preferences. Interestingly, FGF16 was shown to be specific for proliferation of cardiac progenitor cells, whereas FGF9 also proliferated human cardiac fibroblasts. Biosensor analysis of receptor preferences and quantification of receptor abundances suggested that FGF16 and FGF9 bind to different FGF receptors on the cardiac progenitor cells and cardiac fibroblasts. FGF16 also proliferated naïve cardiac progenitor cells isolated from mouse heart and human cardiomyocytes derived from induced pluripotent cells. Taken together, the data suggest that FGF16 could be a suitable paracrine factor to induce cardiac regeneration and repair.

Communication between the ERRalpha homodimer interface and the PGC-1alpha binding surface via the helix 8-9 loop.

Although structural studies on the ligand-binding domain (LBD) have established the general mode of nuclear receptor (NR)/coactivator interaction, determinants of binding specificity are only partially understood. The LBD of estrogen receptor-alpha (ERalpha), for example, interacts only with a region of peroxisome proliferator-activated receptor coactivator (PGC)-1alpha, which contains the canonical LXXLL motif (NR box2), whereas the LBD of estrogen-related receptor-alpha (ERRalpha) also binds efficiently an untypical, LXXYL-containing region (NR box3) of PGC-1alpha. Surprisingly, in a previous structural study, the ERalpha LBD has been observed to bind NR box3 of transcriptional intermediary factor (TIF)-2 untypically via LXXYL, whereas the ERRalpha LBD binds this region of TIF-2 only poorly. Here we present a new crystal structure of the ERRalpha LBD in complex with a PGC-1alpha box3 peptide. In this structure, residues N-terminal of the PGC-1alpha LXXYL motif formed contacts with helix 4, the loop connecting helices 8 and 9, and with the C terminus of the ERRalpha LBD. Interaction studies using wild-type and mutant PGC-1alpha and ERRalpha showed that these contacts are functionally relevant and are required for efficient ERRalpha/PGC-1alpha interaction. Furthermore, a structure comparison between ERRalpha and ERalpha and mutation analyses provided evidence that the helix 8-9 loop, which differs significantly in both nuclear receptors, is a major determinant of coactivator binding specificity. Finally, our results revealed that in ERRalpha the helix 8-9 loop allosterically links the LBD homodimer interface with the coactivator cleft, thus providing a plausible explanation for distinct PGC-1alpha binding to ERRalpha monomers and homodimers.

Expression of genes involved in oxidative stress responses in airway epithelial cells of smokers with chronic obstructive pulmonary disease.

RATIONALE: The molecular mechanisms involved in airway oxidative stress responses reported in healthy smokers and in those with chronic obstructive pulmonary disease (COPD) are poorly understood. OBJECTIVES: To assess the expression of genes involved in oxidative stress responses in the bronchial epithelium of smokers with or without COPD and in relation to disease severity. METHODS: Global gene expression was assessed in bronchial brushings in 38 subjects with COPD, 14 healthy nonsmokers, and 18 healthy smokers. RESULTS: Gene expression analysis using Affymetrix arrays revealed mRNAs representing 341 out of 642 oxidative stress genes from two predefined gene sets to be differentially expressed in healthy nonsmokers when compared with healthy smokers, and 200 differentially expressed oxidative genes in subjects with COPD when compared with healthy smokers. Gene set enrichment analysis showed that pathways involved in oxidant/antioxidant responses were among the most differentially expressed gene pathways in smoking individuals, with further differences seen in COPD. Distinct, nonlinear gene expression patterns were identified across the severity spectrum of COPD, which correlated with the presence of certain transcription factor binding sites in their promoters. Significant changes in oxidant response genes observed in vivo were reproduced in vitro using primary bronchial epithelial cells from the same donors cultured at an air-liquid interface and exposed to cigarette smoke extract. CONCLUSIONS: Cigarette smoke induces significant changes in oxidant defense responses; some of these are further amplified, but not in a linear fashion, in individuals who develop COPD.

Identification of the human ApoAV gene as a novel RORalpha target gene.

Retinoic acid receptor-related orphan receptor-alpha (RORalpha) (NR1F1) is an orphan nuclear receptor with a potential role in metabolism. Previous studies have shown that RORalpha regulates transcription of the murine Apolipoprotein AI gene and human Apolipoprotein CIII genes. In the present study, we present evidence that RORalpha also induces transcription of the human Apolipoprotein AV gene, a recently identified apolipoprotein associated with triglyceride levels. Adenovirus-mediated overexpression of RORalpha increased the endogenous expression of ApoAV in HepG2 cells and RORalpha also enhanced the activity of an ApoAV promoter construct in transiently transfected HepG2 cells. Deletion and mutation studies identified three AGGTCA motifs in the ApoAV promoter that mediate RORalpha transactivation, one of which overlaps with a previously identified binding site for PPARalpha. Together, these results suggest a novel mechanism whereby RORalpha modulates lipid metabolism and implies RORalpha as a potential target for the treatment of dyslipidemia and atherosclerosis.