Discovery and Structure-Guided Optimization of Diarylmethanesulfonamide Disrupters of Glucokinase-Glucokinase Regulatory Protein (GK-GKRP) Binding: Strategic Use of a N → S (nN → σ*S-X) Interaction for Conformational Constraint.

The HTS-based discovery and structure-guided optimization of a novel series of GKRP-selective GK-GKRP disrupters are revealed. Diarylmethanesulfonamide hit 6 (hGK-hGKRP IC50 = 1.2 µM) was optimized to lead compound 32 (AMG-0696; hGK-hGKRP IC50 = 0.0038 µM). A stabilizing interaction between a nitrogen atom lone pair and an aromatic sulfur system (nN → σ*S-X) in 32 was exploited to conformationally constrain a biaryl linkage and allow contact with key residues in GKRP. Lead compound 32 was shown to induce GK translocation from the nucleus to the cytoplasm in rats (IHC score = 0; 10 mg/kg po, 6 h) and blood glucose reduction in mice (POC = -45%; 100 mg/kg po, 3 h). X-ray analyses of 32 and several precursors bound to GKRP were also obtained. This novel disrupter of GK-GKRP binding enables further exploration of GKRP as a potential therapeutic target for type II diabetes and highlights the value of exploiting unconventional nonbonded interactions in drug design.

Small Molecule Disruptors of the Glucokinase-Glucokinase Regulatory Protein Interaction: 5. A Novel Aryl Sulfone Series, Optimization Through Conformational Analysis.

The glucokinase-glucokinase regulatory protein (GK-GKRP) complex plays an important role in controlling glucose homeostasis in the liver. We have recently disclosed a series of arylpiperazines as in vitro and in vivo disruptors of the GK-GKRP complex with efficacy in rodent models of type 2 diabetes mellitus (T2DM). Herein, we describe a new class of aryl sulfones as disruptors of the GK-GKRP complex, where the central piperazine scaffold has been replaced by an aromatic group. Conformational analysis and exploration of the structure-activity relationships of this new class of compounds led to the identification of potent GK-GKRP disruptors. Further optimization of this novel series delivered thiazole sulfone 93, which was able to disrupt the GK-GKRP interaction in vitro and in vivo and, by doing so, increases cytoplasmic levels of unbound GK.

Small molecule disruptors of the glucokinase-glucokinase regulatory protein interaction: 4. Exploration of a novel binding pocket.

Structure-activity relationship investigations conducted at the 5-position of the N-pyridine ring of a series of N-arylsulfonyl-N'-2-pyridinyl-piperazines led to the identification of a novel bis-pyridinyl piperazine sulfonamide (51) that was a potent disruptor of the glucokinase-glucokinase regulatory protein (GK-GKRP) interaction. Analysis of the X-ray cocrystal of compound 51 bound to hGKRP revealed that the 3-pyridine ring moiety occupied a previously unexplored binding pocket within the protein. Key features of this new binding mode included forming favorable contacts with the top face of the Ala27-Val28-Pro29 ("shelf region") as well as an edge-to-face interaction with the Tyr24 side chain. Compound 51 was potent in both biochemical and cellular assays (IC50=0.005 µM and EC50=0.205 µM, respectively) and exhibited acceptable pharmacokinetic properties for in vivo evaluation. When administered to db/db mice (100 mg/kg, po), compound 51 demonstrated a robust pharmacodynamic effect and significantly reduced blood glucose levels up to 6 h postdose.

Small molecule disruptors of the glucokinase-glucokinase regulatory protein interaction: 3. Structure-activity relationships within the aryl carbinol region of the N-arylsulfonamido-N'-arylpiperazine series.

We have recently reported a novel approach to increase cytosolic glucokinase (GK) levels through the binding of a small molecule to its endogenous inhibitor, glucokinase regulatory protein (GKRP). These initial investigations culminated in the identification of 2-(4-((2S)-4-((6-amino-3-pyridinyl)sulfonyl)-2-(1-propyn-1-yl)-1-piperazinyl)phenyl)-1,1,1,3,3,3-hexafluoro-2-propanol (1, AMG-3969), a compound that effectively enhanced GK translocation and reduced blood glucose levels in diabetic animals. Herein we report the results of our expanded SAR investigations that focused on modifications to the aryl carbinol group of this series. Guided by the X-ray cocrystal structure of compound 1 bound to hGKRP, we identified several potent GK-GKRP disruptors bearing a diverse set of functionalities in the aryl carbinol region. Among them, sulfoximine and pyridinyl derivatives 24 and 29 possessed excellent potency as well as favorable PK properties. When dosed orally in db/db mice, both compounds significantly lowered fed blood glucose levels (up to 58%).

Small molecule disruptors of the glucokinase-glucokinase regulatory protein interaction: 2. Leveraging structure-based drug design to identify analogues with improved pharmacokinetic profiles.

In the previous report , we described the discovery and optimization of novel small molecule disruptors of the GK-GKRP interaction culminating in the identification of 1 (AMG-1694). Although this analogue possessed excellent in vitro potency and was a useful tool compound in initial proof-of-concept experiments, high metabolic turnover limited its advancement. Guided by a combination of metabolite identification and structure-based design, we have successfully discovered a potent and metabolically stable GK-GKRP disruptor (27, AMG-3969). When administered to db/db mice, this compound demonstrated a robust pharmacodynamic response (GK translocation) as well as statistically significant dose-dependent reductions in fed blood glucose levels.

Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors.

Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic ß-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

Hydroxyethylamine-based inhibitors of BACE1: P1-P3 macrocyclization can improve potency, selectivity, and cell activity.

We describe a systematic study of how macrocyclization in the P1-P3 region of hydroxyethylamine-based inhibitors of ß-site amyloid precursor protein (APP)-cleaving enzyme (BACE1) modulates in vitro activity. This study reveals that in a number of instances macrocyclization of bis-terminal dienes leads to improved potency toward BACE1 and selectivity against cathepsin D (CatD), as well as greater amyloid ß-peptide (Aß)-lowering activity in HEK293T cells stably expressing APPSW. However, for several closely related analogs the benefits of macrocyclization are attenuated by the effects of other structural features in different regions of the molecules. X-ray crystal structures of three of these novel macrocyclic inhibitors bound to BACE1 revealed their binding conformations and interactions with the enzyme.

A hybrid structural approach to analyze ligand binding by the serotonin type 4 receptor (5-HT4).

Hybrid structural methods have been used in recent years to understand protein-protein or protein-ligand interactions where high resolution crystallography or NMR data on the protein of interest has been limited. For G protein-coupled receptors (GPCRs), high resolution structures of native structural forms other than rhodopsin have not yet been achieved; gaps in our knowledge have been filled by creative crystallography studies that have developed stable forms of receptors by multiple means. The neurotransmitter serotonin (5-hydroxytryptamine) is a key GPCR-based signaling molecule affecting many physiological manifestations in humans ranging from mood and anxiety to bowel function. However, a high resolution structure of any of the serotonin receptors has not yet been solved. Here, we used structural mass spectrometry along with theoretical computations, modeling, and other biochemical methods to develop a structured model for human serotonin receptor subtype 4(b) in the presence and absence of its antagonist GR125487. Our data confirmed the overall structure predicted by the model and revealed a highly conserved motif in the ligand-binding pocket of serotonin receptors as an important participant in ligand binding. In addition, identification of waters in the transmembrane region provided clues as to likely paths mediating intramolecular signaling. Overall, this study reveals the potential of hybrid structural methods, including mass spectrometry, to probe physiological and functional GPCR-ligand interactions with purified native protein.

2-amino-1,3-thiazol-4(5H)-ones as potent and selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitors: enzyme-ligand co-crystal structure and demonstration of pharmacodynamic effects in C57Bl/6 mice.

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) has attracted considerable attention during the past few years as a potential target for the treatment of diseases associated with metabolic syndrome. In our ongoing work on 11beta-HSD1 inhibitors, a series of new 2-amino-1,3-thiazol-4(5 H)-ones were explored. By inserting various cycloalkylamines at the 2-position and alkyl groups or spirocycloalkyl groups at the 5-position of the thiazolone, several potent 11beta-HSD1 inhibitors were identified. An X-ray cocrystal structure of human 11beta-HSD1 with compound 6d (Ki=28 nM) revealed a large lipophilic pocket accessible by substitution off the 2-position of the thiazolone. To increase potency, analogues were prepared with larger lipophilic groups at this position. One of these compounds, the 3-noradamantyl analogue 8b, was a potent inhibitor of human 11beta-HSD1 (Ki=3 nM) and also inhibited 11beta-HSD1 activity in lean C57Bl/6 mice when evaluated in an ex vivo adipose and liver cortisone to cortisol conversion assay.

Crystal structure of murine 11 beta-hydroxysteroid dehydrogenase 1: an important therapeutic target for diabetes.

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of 11-dehydrocorticosterone to its active form corticosterone in rodents (or cortisone to cortisol in humans). The reductive reaction of the 11-keto to 11-hydroxyl is the pivotal switch in the activation of glucocorticoids. An excess of active glucocorticoids has been shown to play a key role in metabolic disorders such as diabetes and obesity. Therefore, 11beta-HSD1 represents an important therapeutic target for the treatment of these diseases. To facilitate the iterative design of inhibitors, we have crystallized and determined the three-dimensional structures of a binary complex of murine 11beta-HSD1 with NADP(H) to a resolution of 2.3 A and of a ternary complex with corticosterone and NADP(H) to a resolution of 3.0 A by X-ray crystallography. The enzyme forms a homodimer in the crystal and has a fold similar to those of other members of the family of short chain steroid dehydrogenases/reductases (SDRs). The structure shows a novel folding feature at the C-terminus of the enzyme. The C-terminal helix insertions provide additional dimer contacts, exert an influence on the conformations of the substrate binding loops, and present hydrophobic regions for potential membrane attachment. The structure also reveals how 11beta-HSD1 achieves its selectivity for its substrate.