Altered desensitization and internalization patterns of rodent versus human glucose-dependent insulinotropic polypeptide (GIP) receptors. An important drug discovery challenge.

BACKGROUND AND PURPOSE: The gut hormone glucose-dependent insulinotropic polypeptide (GIP) signals via the GIP receptor (GIPR), resulting in postprandial potentiation of glucose-stimulated insulin secretion. The translation of results from rodent studies to human studies has been challenged by the unexpected effects of GIPR-targeting compounds. We, therefore, investigated the variation between species, focusing on GIPR desensitization and the role of the receptor C-terminus. EXPERIMENTAL APPROACH: The GIPR from humans, mice, rats, pigs, dogs and cats was studied in vitro for cognate ligand affinity, G protein activation (cAMP accumulation), recruitment of beta-arrestin and internalization. Variants of the mouse, rat and human GIPRs with swapped C-terminal tails were studied in parallel. KEY RESULTS: The human GIPR is more prone to internalization than rodent GIPRs. Despite similar agonist affinities and potencies for Gαs activation, especially, the mouse GIPR shows reduced receptor desensitization, internalization and beta-arrestin recruitment. Using an enzyme-stabilized, long-acting GIP analogue, the species differences were even more pronounced. 'Tail-swapped' human, rat and mouse GIPRs were all fully functional in their Gαs coupling, and the mouse GIPR regained internalization and beta-arrestin 2 recruitment properties with the human tail. The human GIPR lost the ability to recruit beta-arrestin 2 when its own C-terminus was replaced by the rat or mouse tail. CONCLUSIONS AND IMPLICATIONS: Desensitization of the human GIPR is dependent on the C-terminal tail. The species-dependent functionality of the C-terminal tail and the different species-dependent internalization patterns, especially between human and mouse GIPRs, are important factors influencing the preclinical evaluation of GIPR-targeting therapeutic compounds.

Structure-Guided Design of a Highly Potent Partial RXR Agonist with Superior Physicochemical Properties.

Retinoid X receptors (RXRs, NR2B1-3) hold therapeutic potential in oncology, neurodegeneration, and metabolic diseases, but traditional RXR agonists mimicking the natural ligand 9-cis retinoic acid exhibit poor physicochemical properties, pharmacokinetics, and safety profiles. Improved RXR ligands are needed to exploit RXR modulation as a promising therapeutic concept in various indications beyond its current role in second-line cancer treatment. Here, we report the co-crystal structure of RXR in complex with a novel pyrimidine-based ligand and the structure-informed optimization of this scaffold to highly potent and highly soluble RXR agonists. Focused structure-activity relationship elucidation and rigidization resulted in a substantially optimized partial RXR agonist with low nanomolar potency, no cytotoxic activity, and very favorable physicochemical properties highlighting this promising scaffold for the development of next-generation RXR targeting drugs.

Development of Light-Activated LXR Agonists.

Activation of the oxysterol-sensing transcription factor liver X receptor (LXR) has been studied as a therapeutic strategy in metabolic diseases and cancer but is compromised by the side effects of LXR agonists. Local LXR activation in cancer treatment may offer an opportunity to overcome this issue suggesting potential uses of photopharmacology. We report the computer-aided development of photoswitchable LXR agonists based on the T0901317 scaffold, which is a known LXR agonist. Azologization and structure-guided structure-activity relationship evaluation enabled the design of an LXR agonist, which activated LXR with low micromolar potency in its light-induced (Z)-state and was inactive as (E)-isomer. This tool sensitized human lung cancer cells to chemotherapeutic treatment in a light-dependent manner supporting potential of locally activated LXR agonists as adjuvant cancer treatment.

Structure-Based Design of Dual Partial Peroxisome Proliferator-Activated Receptor γ Agonists/Soluble Epoxide Hydrolase Inhibitors.

Polypharmaceutical regimens often impair treatment of patients with metabolic syndrome (MetS), a complex disease cluster, including obesity, hypertension, heart disease, and type II diabetes. Simultaneous targeting of soluble epoxide hydrolase (sEH) and peroxisome proliferator-activated receptor γ (PPARγ) synergistically counteracted MetS in various in vivo models, and dual sEH inhibitors/PPARγ agonists hold great potential to reduce the problems associated with polypharmacy in the context of MetS. However, full activation of PPARγ leads to fluid retention associated with edema and weight gain, while partial PPARγ agonists do not have these drawbacks. In this study, we designed a dual partial PPARγ agonist/sEH inhibitor using a structure-guided approach. Exhaustive structure-activity relationship studies lead to the successful optimization of the designed lead. Crystal structures of one representative compound with both targets revealed potential points for optimization. The optimized compounds exhibited favorable metabolic stability, toxicity, selectivity, and desirable activity in adipocytes and macrophages.

Development and Profiling of Inverse Agonist Tools for the Neuroprotective Transcription Factor Nurr1.

The ligand-sensing transcription factor nuclear receptor related 1 (Nurr1) evolves as an appealing target to treat neurodegenerative diseases. Despite its therapeutic potential observed in various rodent models, potent modulators for Nurr1 are lacking as pharmacological tools. Here, we report the structure-activity relationship and systematic optimization of indole-based inverse Nurr1 agonists. Optimized analogues decreased the receptor's intrinsic transcriptional activity by up to more than 90% and revealed preference for inhibiting Nurr1 monomer activity. In orthogonal cell-free settings, we detected displacement of NCoRs and disruption of the Nurr1 homodimer as molecular modes of action. The inverse Nurr1 agonists reduced the expression of Nurr1-regulated genes in T98G cells, and treatment with an inverse Nurr1 agonist mimicked the effect of Nurr1 silencing on interleukin-6 release from LPS-stimulated human astrocytes. The indole-based inverse Nurr1 agonists valuably extend the toolbox of Nurr1 modulators to further probe the role of Nurr1 in neuroinflammation, cancer, and beyond.

Propranolol Activates the Orphan Nuclear Receptor TLX to Counteract Proliferation and Migration of Glioblastoma Cells.

The ligand-sensing transcription factor tailless homologue (TLX, NR2E1) is an essential regulator of neuronal stem cell homeostasis with appealing therapeutic potential in neurodegenerative diseases and central nervous system tumors. However, knowledge on TLX ligands is scarce, providing an obstacle to target validation and medicinal chemistry. To discover TLX ligands, we have profiled a drug fragment collection for TLX modulation and identified several structurally diverse agonists and inverse agonists of the nuclear receptor. Propranolol evolved as the strongest TLX agonist and promoted TLX-regulated gene expression in human glioblastoma cells. Structure-activity relationship elucidation of propranolol as a TLX ligand yielded a structurally related negative control compound. In functional cellular experiments, we observed an ability of propranolol to counteract glioblastoma cell proliferation and migration, while the negative control had no effect. Our results provide a collection of TLX modulators as initial chemical tools and set of lead compounds and support therapeutic potential of TLX modulation in glioblastoma.

Endogenous vitamin E metabolites mediate allosteric PPARγ activation with unprecedented co-regulatory interactions.

Vitamin E exhibits pharmacological effects beyond established antioxidant activity suggesting involvement of unidentified mechanisms. Here, we characterize endogenously formed tocopherol carboxylates and the vitamin E mimetic garcinoic acid (GA) as activators of the peroxisome proliferator-activated receptor gamma (PPARγ). Co-stimulation of PPARγ with GA and the orthosteric agonist pioglitazone resulted in additive transcriptional activity. In line with this, the PPARγ-GA complex adopted a fully active conformation and interestingly contained two bound GA molecules with one at an allosteric site. A co-regulator interaction scan demonstrated an unanticipated co-factor recruitment profile for GA-bound PPARγ compared with canonical PPARγ agonists and gene expression analysis revealed different effects of GA and pioglitazone on PPAR signaling in hepatocytes. These observations reveal allosteric mechanisms of PPARγ modulation as an alternative avenue to PPARγ targeting and suggest contributions of PPARγ activation by α-13-tocopherolcarboxylate to the pharmacological effects of vitamin E.

Chemical Starting Matter for HNF4α Ligand Discovery and Chemogenomics.

Hepatocyte nuclear factor 4α (HNF4α) is a ligand-sensing transcription factor and presents as a potential drug target in metabolic diseases and cancer. In humans, mutations in the HNF4α gene cause maturity-onset diabetes of the young (MODY), and the elevated activity of this protein has been associated with gastrointestinal cancers. Despite the high therapeutic potential, available ligands and structure-activity relationship knowledge for this nuclear receptor are scarce. Here, we disclose a chemically diverse collection of orthogonally validated fragment-like activators as well as inverse agonists, which modulate HNF4α activity in a low micromolar range. These compounds demonstrate the druggability of HNF4α and thus provide a starting point for medicinal chemistry as well as an early tool for chemogenomics.

Photohormones Enable Optical Control of the Peroxisome Proliferator-Activated Receptor γ (PPARγ).

Photopharmacology aims at the optical control of protein activity using synthetic photoswitches. This approach has been recently expanded to nuclear hormone receptors with the introduction of "photohormones" for the retinoic acid receptor, farnesoid X receptor, and estrogen receptor. Herein, we report the development and profiling of photoswitchable agonists for peroxisome proliferator-activated receptor γ (PPARγ). Based on known PPARγ ligands (MDG548, GW1929, and rosiglitazone), we have designed and synthesized azobenzene derivatives, termed AzoGW1929 and AzoRosi, which were confirmed to be active in cell-based assays. Subsequent computer-aided optimization of AzoRosi resulted in the photohormone AzoRosi-4, which bound and activated PPARγ preferentially in its light-activated cis-configuration.

Boosting Anti-Inflammatory Potency of Zafirlukast by Designed Polypharmacology.

Multitarget design offers access to bioactive small molecules with potentially superior efficacy and safety. Particularly multifactorial chronic inflammatory diseases demand multiple pharmacological interventions for stable treatment. By minor structural changes, we have developed a close analogue of the cysteinyl-leukotriene receptor antagonist zafirlukast that simultaneously inhibits soluble epoxide hydrolase and activates peroxisome proliferator-activated receptor γ. The triple modulator exhibits robust anti-inflammatory activity in vivo and highlights the therapeutic potential of designed multitarget agents.