Study of CRTC2 Pharmacology Using Antisense Oligonuceotides Robert Dullea, 1 Christopher Salatto, 1 Simone Sciabola, 2 Tracy Chen, 2 Debra DiMattia, 1 Harmeet Gandhok, 1 John Kreeger, 3 Yan Weng, 4 Tracey Clark, 4 Chandra Vage, 4 and Robert Stanton 2 The cAMP response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is a key component of the transcription complex regulating glucagon driven hepatic glucose production and previous evidence suggests that ‘‘inhibition’’ of CRTC2 improves glucose homeostasis in multiple rodent models of type 2 diabetes. Here we describe a process of identifying potential therapeutic antisense oligonucleotides (ASOs) directed against CRTC2. These ASOs were designed as locked nucleic acid (LNA) gapmers and a panel of approximately 400 sequences were first screened in vitro within both human and mouse liver cell lines. A group of active and selective compounds were then profiled in acute studies in mice to determine the level of CRTC2 mRNA reduction in liver as well as to obtain a preliminary indication of safety and tolerability. The compounds with the best activity and safety profiles were then evaluated in subchronic efficacy studies using the diet induced obese (DIO) mouse model of type 2 diabetes and primary human hepatocytes. Efficacy findings broadly confirmed the beneficial effect of reducing CRTC2 mRNA levels towards improving glucose control and other markers of metabolic function. Additionally, for the first time, translation to human cells has been established with demonstration of a reduction in glucagon-mediated glucose production in primary human hepatocytes and a potential clinical biomarker source identified to assess modulation of CRTC2 mRNA following ASO treatment. While the compounds identified herein did not demonstrate a therapeutic index sufficient for further devel- opment, this study should facilitate more efficient prosecution of compounds within an in vivo setting. Introduction H yperglycemia driven by increased hepatic glucose output is a prevalent pathophysiology associated with insulin resistance ultimately contributing to the development of type 2 diabetes mellitus (T2DM) (Consoli et al., 1989). The pancreas-derived glucagon hormone is a key mediator ini- tiating the gluconeogenic program in the liver where upon binding to its receptor activates a transcription complex con- taining the cAMP response element binding protein (CREB)- regulated transcription coactivator 2 (CRTC2). Activation of CRTC2 is then responsible for the increased expression of genes involved in the conversion of three carbon precursors to glucose, principally phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) (Hoeffler et al., 1988; Gonzalez and Montminy, 1989; Habener, 1990). CRTC2 activity is tightly regulated downstream of nutrient sensing signals by multiple post-translation modifications including phosphorylation and acetylation. In the fed state, it is seques- tered in a phosphorylation-dependent inactive form within the cytoplasm while after fasting and subsequent dephos- phorylation translocates to the nucleus where it promotes tar- get gene transcription (Screaton et al., 2004; Canettieri et al., 2005; Koo et al., 2005). Interestingly, phosphorylation deficient (i.e., constitutively active) CRTC2 protein due to O-linked glycosylation at the inhibitory serine-171 phosphorylation site in the liver has been described in insulin-resistance states (Dentin et al., 2008).Through physical interaction, active CRTC2 appears to increase occupancy of the CREB for its target promoters. This association is likely the critical event for pro- moting glucagon-mediated gluconeogenesis as deletion of the CREB binding domain in CRTC2 lowers blood glucose and improves insulin sensitivity in diet induced obese (DIO) mice (Wang et al., 2009). In addition to the ability of CRTC2 to enhance the expres- sion of genes associated with hepatic glucose output, it has been demonstrated that CREB/CRTC2 activity modulates transcriptional response in adipose tissue to enhance whole 1 Cardiovascular and Metabolic Disease Research Unit and 2 Oligonucleotide Therapeutic Unit, Pfizer, Cambridge, Massachusetts. 3 Safety Science, Groton, Connecticut. 4 Pharmacokinetics, Dynamics and Metabolism, Groton, Connecticut. NUCLEIC ACID THERAPEUTICS Volume 24, Number 2, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/nat.2013.0456 127