AMP-activated protein kinase (AMPK) is a key sensor and regulator of intracellular and whole-body energy metabolism. We have identified a thienopyridone family of AMPK activators. A-769662 directly stimulated partially purified rat liver AMPK (EC50 = 0.8 microM) and inhibited fatty acid synthesis in primary rat hepatocytes (IC50 = 3.2 microM). Short-term treatment of normal Sprague Dawley rats with A-769662 decreased liver malonyl CoA levels and the respiratory exchange ratio, VCO2/VO2, indicating an increased rate of whole-body fatty acid oxidation. Treatment of ob/ob mice with 30 mg/kg b.i.d. A-769662 decreased hepatic expression of PEPCK, G6Pase, and FAS, lowered plasma glucose by 40%, reduced body weight gain and significantly decreased both plasma and liver triglyceride levels. These results demonstrate that small molecule-mediated activation of AMPK in vivo is feasible and represents a promising approach for the treatment of type 2 diabetes and the metabolic syndrome.

Adipocyte differentiation is regulated both positively and negatively by external growth factors such as insulin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF). A key component of the adipocyte differentiation process is PPAR␥, peroxisomal proliferator-activated receptor ␥. To determine the relationship between PPAR␥ activation and growth factor stimulation in adipogenesis, we investigated the effects of PDGF and EGF on PPAR␥1 activity. PDGF treatment decreased ligand-activated PPAR␥1 transcriptional activity in a transient reporter assay. In vivo [ 32 P]orthophosphate labeling experiments demonstrated that PPAR␥1 is a phosphoprotein that undergoes EGF-stimulated MEK/mitogen-activated protein (MAP) kinase-dependent phosphorylation. Purified PPAR␥1 protein was phosphorylated in vitro by recombinant activated MAP kinase. Examination of the PPAR␥1 sequence revealed a single MAP kinase consensus recognition site at Ser 82 . Mutation of Ser 82 to Ala inhibited both in vitro and in vivo phosphorylation and growth factor-mediated transcriptional repression. Therefore, phosphorylation of PPAR␥1 by MAP kinase contributes to the reduction of PPAR␥1 transcriptional activity by growth factor treatment.Peroxisome proliferator-activated receptors (PPARs) 1 are members of the nuclear hormone receptor superfamily (1). These receptors heterodimerize with retinoic acid-like receptor, RXR, and become transcriptionally active when bound to ligand. The three PPAR isoforms (␣, ␦, and ␥) differ in their C-terminal ligand binding domains, and each appears to bind and respond to a specific subset of agents including hypolipidemic drugs, long chain fatty acids, aracadonic acid metabolites, and antidiabetic thiazolidinediones (2-4). PPAR␥ is expressed predominantly in mouse white and brown fat, with lower levels in liver, whereas PPAR␣ is present in heart, kidney, and liver (5, 6). PPAR␦ expression is ubiquitous (7,8).Ectopic expression of either PPAR␣ or PPAR␥ in NIH-3T3 cells is sufficient to induce adipocyte differentiation in the presence of PPAR␥ activators (9, 10). The rapid induction of PPAR␥ during adipocyte differentiation and its enriched expression in adipose tissues suggest that PPAR␥ is responsible for the initiation and maintenance of the adipocyte phenotype in vivo (9). Previously two isotypes of PPAR␥ (PPAR␥1 and PPAR␥2) have been identified in 3T3-L1 adipocytes (11). Zhu et al. (12) have demonstrated that these two isotypes are derived from a single PPAR␥ gene by alternative promoter usage and RNA splicing. However, thus far, no functional difference has been found between the two isotypes.Adipogenesis is a complex process; multiple hormones and factors regulate the conversion of progenitor cells to adipocytes. Insulin and/or insulin-like growth factor enhance the ability of PPAR ligand to induce differentiation of both 3T3-L1-and PPAR␥-overexpressing cell lines (9, 13). In contrast, growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and fibroblast gro...

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To determine functional differences between the two splice variants of PPAR␥ (␥1 and ␥2), we sought to selectively repress ␥2 expression by targeting engineered zinc finger repressor proteins (ZFPs) to the ␥2-specific promoter, P2. In 3T3-L1 cells, expression of ZFP55 resulted in >50% reduction in ␥2 expression but had no effect on ␥1, whereas adipogenesis was similarly reduced by 50%. However, ZFP54 virtually abolished both ␥2 and ␥1 expression, and completely blocked adipogenesis. Overexpression of exogenous ␥2 in the ZFP54-expressing cells completely restored adipogenesis, whereas overexpression of ␥1 had no effect. This finding clearly identifies a unique role for the PPAR␥2 isoform. The nuclear hormone receptor PPAR␥ is essential for cellular differentiation and lipid accumulation during adipogenesis (Barak et al. 1999;Kubota et al. 1999;Rosen et al. 1999). The adipocyte-specific ␥2 isoform differs from the more widely expressed ␥1 in that it contains additionally 30 amino acid residues at the amino terminus (Kliewer et al. 1994;Tontonoz et al. 1994a;Zhu et al. 1995). Evidence suggests these residues contribute to a constitutive transcription activation function that is 5-10-fold greater than in ␥1 (Werman et al. 1997). PPAR␥2 is selectively expressed in adipose tissue (Fajas et al. 1997) and is strongly up-regulated during adipogenesis (Tontonoz et al. 1994b;Wu et al. 1998), suggesting a specific role for this isoform in fat cell differentiation. Nevertheless, a specific role for ␥2 that could not be substituted by ␥1 has not been clearly determined.The ability to selectively knock out or knock down the expression of a specific gene provides a powerful approach for understanding its biological function. The targeting of individual mRNA splice variants offers an even greater level of selective control and understanding of differential isoform function. Rationally engineered transcription factors potentially provide a powerful tool for targeted regulation of endogenous genes by combining a functional transcription regulatory domain with a customized DNA binding domain that can bind to a specific sequence within the target gene. C2H2 zinc finger proteins (ZFPs) can be engineered to bind with high specificity to wide a diversity of DNA sequences (Desjarlais and Berg 1992;Choo and Klug 1994;Jamieson et al. 1994;Rebar and Pabo 1994;Greisman and Pabo 1997). Previous studies have demonstrated the utility of both engineered activator-and repressor-ZFPs in the regulation of endogenous chromosomal loci (Bartsevich and Juliano 2000;Beerli et al. 2000;Zhang et al. 2000;Liu et al. 2001). Our goal for this study was to selectively inhibit expression of the PPAR␥2 isoform in the adipogenic mouse 3T3-L1 cell line by utilizing engineered zinc finger repressor proteins. Results and DiscussionThe mouse PPAR␥ gene spans >105 kb (Zhu et al. 1995). Coding exons 1 to 6 are conserved between the ␥1 and ␥2 isoforms (Fig. 1A) and transcription of these is driven by an upstream promoter (P1) that also drives expression of two untrans...

This is a repository copy of Upadacitinib as monotherapy in patients with active rheumatoid arthritis and inadequate response to methotrexate (SELECT-MONOTHERAPY): a randomised, placebo-controlled, double-blind phase 3 study.

Background Anti-cytokine therapies such as adalimumab, tocilizumab, and the small molecule JAK inhibitor tofacitinib have proven that cytokines and their subsequent downstream signaling processes are important in the pathogenesis of rheumatoid arthritis. Tofacitinib, a pan-JAK inhibitor, is the first approved JAK inhibitor for the treatment of RA and has been shown to be effective in managing disease. However, in phase 2 dose-ranging studies tofacitinib was associated with dose-limiting tolerability and safety issues such as anemia. Upadacitinib (ABT-494) is a selective JAK1 inhibitor that was engineered to address the hypothesis that greater JAK1 selectivity over other JAK family members will translate into a more favorable benefit:risk profile. Upadacitinib selectively targets JAK1 dependent disease drivers such as IL-6 and IFNγ, while reducing effects on reticulocytes and natural killer (NK) cells, which potentially contributed to the tolerability issues of tofacitinib. Methods Structure-based hypotheses were used to design the JAK1 selective inhibitor upadacitinib. JAK family selectivity was defined with in vitro assays including biochemical assessments, engineered cell lines, and cytokine stimulation. In vivo selectivity was defined by the efficacy of upadacitinib and tofacitinib in a rat adjuvant induced arthritis model, activity on reticulocyte deployment, and effect on circulating NK cells. The translation of the preclinical JAK1 selectivity was assessed in healthy volunteers using ex vivo stimulation with JAK-dependent cytokines. Results Here, we show the structural basis for the JAK1 selectivity of upadacitinib, along with the in vitro JAK family selectivity profile and subsequent in vivo physiological consequences. Upadacitinib is ~ 60 fold selective for JAK1 over JAK2, and > 100 fold selective over JAK3 in cellular assays. While both upadacitinib and tofacitinib demonstrated efficacy in a rat model of arthritis, the increased selectivity of upadacitinib for JAK1 resulted in a reduced effect on reticulocyte deployment and NK cell depletion relative to efficacy. Ex vivo pharmacodynamic data obtained from Phase I healthy volunteers confirmed the JAK1 selectivity of upadactinib in a clinical setting. Conclusions The data presented here highlight the JAK1 selectivity of upadacinitinib and supports its use as an effective therapy for the treatment of RA with the potential for an improved benefit:risk profile. Electronic supplementary material The online version of this article (10.1186/s41927-018-0031-x) contains supplementary material, which is available to authorized users.

ObjectiveTo evaluate the efficacy and safety of ABT‐494, a selective JAK‐1 inhibitor, in patients with moderate‐to‐severe rheumatoid arthritis (RA) and an inadequate response to methotrexate (MTX).MethodsThree hundred RA patients receiving stable doses of MTX were randomly assigned equally to receive immediate‐release ABT‐494 at 3, 6, 12, or 18 mg twice daily, 24 mg once daily, or placebo for 12 weeks. The primary efficacy end point was the proportion of patients meeting the American College of Rheumatology 20% improvement criteria (achieving an ACR20 response) at week 12, as determined using the last observation carried forward method.ResultsAt week 12, the proportion of ACR20 responses was higher with ABT‐494 (62%, 68%, 80%, 64%, and 76% for the 3, 6, 12, 18, and 24 mg doses, respectively) than with placebo (46%) (using nonresponder imputation) (P < 0.05 for the 6, 12, and 24 mg doses). There was a significant dose‐response relationship among all ABT‐494 doses (P < 0.001). The proportions of patients achieving ACR50 and ACR70 responses were significantly higher for all ABT‐494 doses (except the 12 mg dose for the ACR70 response) than for placebo, as were changes in the Disease Activity Score in 28 joints using the C‐reactive protein level (DAS28‐CRP). Rapid improvement was demonstrated by significant differences in ACR20 response rates and changes in the DAS28‐CRP for all doses compared with placebo at week 2 (the first postbaseline visit). The incidence of adverse events was similar across groups; most were mild, and infections were the most frequent. One serious infection (community‐acquired pneumonia) occurred with ABT‐494 at 12 mg. There were dose‐dependent increases in high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL) cholesterol, but the LDL cholesterol:HDL cholesterol ratios were unchanged through week 12. Mean hemoglobin levels remained stable at lower doses, but decreases were observed at higher doses.ConclusionThis study evaluated a broad range of doses of ABT‐494 in RA patients with an inadequate response to MTX. ABT‐494 demonstrated efficacy, with a safety and tolerability profile similar to that of other JAK inhibitors.

ClinicalTrials.gov ( https://clinicaltrials.gov/ ) identifier: NCT01741493.