Structure-Based Design of Active-Site-Directed, Highly Potent, Selective, and Orally Bioavailable Low-Molecular-Weight Protein Tyrosine Phosphatase Inhibitors.

Protein tyrosine phosphatases constitute an important class of drug targets whose potential has been limited by the paucity of drug-like small-molecule inhibitors. We recently described a class of active-site-directed, moderately selective, and potent inhibitors of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP). Here, we report our extensive structure-based design and optimization effort that afforded inhibitors with vastly improved potency and specificity. The leading compound inhibits LMW-PTP potently and selectively (Ki = 1.2 nM, >8000-fold selectivity). Many compounds exhibit favorable drug-like properties, such as low molecular weight, weak cytochrome P450 inhibition, high metabolic stability, moderate to high cell permeability (Papp > 0.2 nm/s), and moderate to good oral bioavailability (% F from 23 to 50% in mice), and therefore can be used as in vivo chemical probes to further dissect the complex biological as well as pathophysiological roles of LMW-PTP and for the development of therapeutics targeting LMW-PTP.

Activation of the human insulin receptor by non-insulin-related peptides.

The human insulin receptor signalling system plays a critical role in glucose homeostasis. Insulin binding brings about extensive conformational change in the receptor extracellular region that in turn effects trans-activation of the intracellular tyrosine kinase domains and downstream signalling. Of particular therapeutic interest is whether insulin receptor signalling can be replicated by molecules other than insulin. Here, we present single-particle cryoEM structures that show how a 33-mer polypeptide unrelated to insulin can cross-link two sites on the receptor surface and direct the receptor into a signalling-active conformation. The 33-mer polypeptide engages the receptor by two helical binding motifs that are each potentially mimicable by small molecules. The resultant conformation of the receptor is distinct from-but related to-those in extant three-dimensional structures of the insulin-complexed receptor. Our findings thus illuminate unexplored pathways for controlling the signalling of the insulin receptor as well as opportunities for development of insulin mimetics.

A High-Throughput Assay for the Pancreatic Islet Beta-Cell Potassium Channel: Use in the Pharmacological Characterization of Insulin Secretagogues Identified from Phenotypic Screening.

Phenotypic screening is a neoclassical approach for drug discovery. We conducted phenotypic screening for insulin secretion enhancing agents using INS-1E insulinoma cells as a model system for pancreatic beta-cells. A principal regulator of insulin secretion in beta-cells is the metabolically regulated potassium channel Kir6.2/SUR1 complex. To characterize hit compounds, we developed an assay to quantify endogenous potassium channel activity in INS-1E cells. We quantified ligand-regulated potassium channel activity in INS-1E cells using fluorescence imaging and thallium flux. Potassium channel activity was metabolically regulated and coupled to insulin secretion. The pharmacology of channel opening agents (diazoxide) and closing agents (sulfonylureas) was used to validate the applicability of the assay. A precise high-throughput assay was enabled, and phenotypic screening hits were triaged to enable a higher likelihood of discovering chemical matter with novel and useful mechanisms of action.

Regulation of Endogenous (Male) Rodent GLP-1 Secretion and Human Islet Insulin Secretion by Antagonism of Somatostatin Receptor 5.

Incretin and insulin responses to nutrient loads are suppressed in persons with diabetes, resulting in decreased glycemic control. Agents including sulfonylureas and dipeptidyl peptidase-4 inhibitors (DPP4i) partially reverse these effects and provide therapeutic benefit; however, their modes of action limit efficacy. Because somatostatin (SST) has been shown to suppress insulin and glucagonlike peptide-1 (GLP-1) secretion through the Gi-coupled SST receptor 5 (SSTR5) isoform in vitro, antagonism of SSTR5 may improve glycemic control via intervention in both pathways. Here, we show that a potent and selective SSTR5 antagonist reverses the blunting effects of SST on insulin secretion from isolated human islets, and demonstrate that SSTR5 antagonism affords increased levels of systemic GLP-1 in vivo. Knocking out Sstr5 in mice provided a similar increase in systemic GLP-1 levels, which were not increased further by treatment with the antagonist. Treatment of mice with the SSTR5 antagonist in combination with a DPP4i resulted in increases in systemic GLP-1 levels that were more than additive and resulted in greater glycemic control compared with either agent alone. In isolated human islets, the SSTR5 antagonist completely reversed the inhibitory effect of exogenous SST-14 on insulin secretion. Taken together, these data suggest that SSTR5 antagonism should increase circulating GLP-1 levels and stimulate insulin secretion (directly and via GLP-1) in humans, improving glycemic control in patients with diabetes.

Building machines that learn and think for themselves.

We agree with Lake and colleagues on their list of "key ingredients" for building human-like intelligence, including the idea that model-based reasoning is essential. However, we favor an approach that centers on one additional ingredient: autonomy. In particular, we aim toward agents that can both build and exploit their own internal models, with minimal human hand engineering. We believe an approach centered on autonomous learning has the greatest chance of success as we scale toward real-world complexity, tackling domains for which ready-made formal models are not available. Here, we survey several important examples of the progress that has been made toward building autonomous agents with human-like abilities, and highlight some outstanding challenges.

The insulin secretory action of novel polycyclic guanidines: discovery through open innovation phenotypic screening, and exploration of structure-activity relationships.

We report the discovery of the glucose-dependent insulin secretogogue activity of a novel class of polycyclic guanidines through phenotypic screening as part of the Lilly Open Innovation Drug Discovery platform. Three compounds from the University of California, Irvine, 1-3, having the 3-arylhexahydropyrrolo[1,2-c]pyrimidin-1-amine scaffold acted as insulin secretagogues under high, but not low, glucose conditions. Exploration of the structure-activity relationship around the scaffold demonstrated the key role of the guanidine moiety, as well as the importance of two lipophilic regions, and led to the identification of 9h, which stimulated insulin secretion in isolated rat pancreatic islets in a glucose-dependent manner.

Ectonucleotidase NTPDase3 is abundant in pancreatic ß-cells and regulates glucose-induced insulin secretion.

Extracellular ATP released from pancreatic ß-cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide-metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. Therefore, we investigated the expression and role of ectonucleotidases in pancreatic ß-cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding the NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulin-secreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose-dependent insulin secretion. Downregulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated the effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic ß-cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors.

Activation of prostaglandin E receptor 4 triggers secretion of gut hormone peptides GLP-1, GLP-2, and PYY.

Prostaglandins E1 and E2 are synthesized in the intestine and mediate a range of gastrointestinal functions via activation of the prostanoid E type (EP) family of receptors. We examined the potential role of EP receptors in the regulation of gut hormone secretion from L cells. Analysis of mRNA expression in mouse enteroendocrine GLUTag cells demonstrated the abundant expression of EP4 receptor, whereas expression of other EP receptors was much lower. Prostaglandin E1 and E2, nonselective agonists for all EP receptor subtypes, triggered glucagon like peptide 1 (GLP-1) secretion from GLUTag cells, as did the EP4-selective agonists CAY10580 and TCS2510. The effect of EP4 agonists on GLP-1 secretion was blocked by incubation of cells with the EP4-selective antagonist L161,982 or by down-regulating EP4 expression with specific small interfering RNA. Regulation of gut hormone secretion with EP4 agonists was further studied in mice. Administration of EP4 agonists to mice produced a significant elevation of plasma levels of GLP-1, glucagon like peptide 2 (GLP-2) and peptide YY (PYY), whereas gastric inhibitory peptide (GIP) levels were not increased. Thus, our data demonstrate that activation of the EP4 receptor in enteroendocrine L cells triggers secretion of gut hormones.

Regulation of GPR119 receptor activity with endocannabinoid-like lipids.

The GPR119 receptor plays an important role in the secretion of incretin hormones in response to nutrient consumption. We have studied the ability of an array of naturally occurring endocannabinoid-like lipids to activate GPR119 and have identified several lipid receptor agonists. The most potent receptor agonists identified were three N-acylethanolamines: oleoylethanolamine (OEA), palmitoleoylethanolamine, and linoleylethanolamine (LEA), all of which displayed similar potency in activating GPR119. Another lipid, 2-oleoylglycerol (2-OG), also activated GPR119 receptor but with significantly lower potency. Endogenous levels of endocannabinoid-like lipids were measured in intestine in fasted and refed mice. Of the lipid GPR119 agonists studied, the intestinal levels of only OEA, LEA, and 2-OG increased significantly upon refeeding. Intestinal levels of OEA and LEA in the fasted mice were low. In the fed state, OEA levels only moderately increased, whereas LEA levels rose drastically. 2-OG was the most abundant of the three GPR119 agonists in intestine, and its levels were radically elevated in fed mice. Our data suggest that, in lean mice, 2-OG and LEA may serve as physiologically relevant endogenous GPR119 agonists that mediate receptor activation upon nutrient uptake.

Sparse coding of birdsong and receptive field structure in songbirds.

Auditory neurons can be characterized by a spectro-temporal receptive field, the kernel of a linear filter model describing the neuronal response to a stimulus. With a view to better understanding the tuning properties of these cells, the receptive fields of neurons in the zebra finch auditory fore-brain are compared to a set of artificial kernels generated under the assumption of sparseness; that is, the assumption that in the sensory pathway only a small number of neurons need be highly active at any time. The sparse kernels are calculated by finding a sparse basis for a corpus of zebra-finch songs. This calculation is complicated by the highly-structured nature of the songs and requires regularization. The sparse kernels and the receptive fields, though differing in some respects, display several significant similarities, which are described by computing quantative properties such as the seperability index and Q-factor. By comparison, an identical calculation performed on human speech recordings yields a set of kernels which exhibit widely different tuning. These findings imply that Field L neurons are specifically adapted to sparsely encode birdsong and supports the idea that sparsification may be an important element of early sensory processing.

Acyclic, orally bioavailable ketone-based cathepsin K inhibitors.

Starting from a potent ketone-based inhibitor with poor drug properties, incorporation of P(2)-P(3) elements from a ketoamide-based inhibitor led to the identification of a hybrid series of ketone-based cathepsin K inhibitors with better oral bioavailability than the starting ketone.

Novel, potent P2-P3 pyrrolidine derivatives of ketoamide-based cathepsin K inhibitors.

Starting from a potent pantolactone ketoamide cathepsin K inhibitor discovered from structural screening, conversion of the lactone scaffold to a pyrrolidine scaffold allowed exploration of the S(3) subsite of cathepsin K. Manipulation of P3 and P1' groups afforded potent inhibitors with drug-like properties.

Semicarbazone-based inhibitors of cathepsin K, are they prodrugs for aldehyde inhibitors?

Starting from potent aldehyde inhibitors with poor drug properties, derivatization to semicarbazones led to the identification of a series of semicarbazone-based cathepsin K inhibitors with greater solubility and better pharmacokinetic profiles than their parent aldehydes. Furthermore, a representative semicarbazone inhibitor attenuated bone resorption in an ex vivo rat calvarial bone resorption model. However, based on enzyme inhibition comparisons at neutral pH, semicarbazone hydrolysis rates, and 13C NMR experiments, these semicarbazones probably function as prodrugs of aldehydes.

P2-P3 conformationally constrained ketoamide-based inhibitors of cathepsin K.

An orally bioavailable series of ketoamide-based cathepsin K inhibitors with good pharmacokinetic properties has been identified. Starting from a potent inhibitor endowed with poor drug properties, conformational constraint of the P(2)-P(3) linker and modifications to P(1') elements led to an enhancement in potency, solubility, clearance, and bioavailability. These optimized inhibitors attenuated bone resorption in a rat TPTX hypocalcemic bone resorption model.

A novel glucokinase activator modulates pancreatic islet and hepatocyte function.

The glucose-sensing enzyme glucokinase (GK) plays a key role in glucose metabolism. We report here the effects of a novel glucokinase activator, LY2121260. The activator enhanced GK activity via binding to the allosteric site located in the hinge region of the enzyme. LY2121260 stimulated insulin secretion in a glucose-dependent manner in pancreatic beta-cells and increased glucose use in rat hepatocytes. In addition, incubation of beta-cells with the GK activator resulted in increased GK protein levels, suggesting that enhanced insulin secretion on chronic treatment with a GK activator may be due to not only changed enzyme kinetics but also elevated enzyme levels. Animals treated with LY2121260 showed an improved glucose tolerance after oral glucose challenge. These results support the concept that GK activators represent a new class of compounds that increase both insulin secretion and hepatic glucose use and in doing so may prove to be effective agents for the control of blood glucose levels in patients with type 2 diabetes.

Acyclic cyanamide-based inhibitors of cathepsin K.

Conversion of the proline-derived cyanamide lead to an acyclic cyanamide capable of forming an additional hydrogen bond with cathepsin K resulted in a large increase in inhibitory activity. An X-ray structure of a co-crystal of a cyanamide with cathepsin K confirmed the enzyme interaction. Furthermore, a representative acyclic cyanamide inhibitor 6r was able to attenuate bone resorption in the rat calvarial model.

A structural screening approach to ketoamide-based inhibitors of cathepsin K.

Several novel ketoamide-based inhibitors of cathepsin K have been identified. Starting from a modestly potent inhibitor, structural screening of P2 elements led to 100-fold enhancements in inhibitory activity. Modifications to one of these leads resulted in an orally bioavailable cathepsin K inhibitor.

Potent and selective P2-P3 ketoamide inhibitors of cathepsin K with good pharmacokinetic properties via favorable P1', P1, and/or P3 substitutions.

A series of ketoamides were synthesized and evaluated for inhibitory activity against cathepsin K. Exploration of the interactions between achiral P(2) substituents and the cysteine protease based on molecular modelling suggestions resulted in potent cathepsin K inhibitors that demonstrated high selectivity versus cathepsins B, H, and L. Subsequent modifications of the P(3), P(1), and P(1') moieties afforded orally bioavailable inhibitors.

Orally bioavailable small molecule ketoamide-based inhibitors of cathepsin K.

An orally available series of ketoamide-based inhibitors of cathepsin K has been identified. Starting from a potent inhibitor with poor oral bioavailability, modifications to P1 and P1' elements led to enhancements in solubility and permeability. These improvements resulted in orally available cathepsin K inhibitors.

Expression, preparation, and high-throughput screening of caspase-8: discovery of redox-based and steroid diacid inhibition.

Because of the intimate role of caspase-8 in apoptosis signaling pathways from FAS, TNFR1, and other death receptors, the enzyme is a potentially important therapeutic target. We have generated an Escherichia coli expression construct for caspase-8 in which a His-tag sequence is inserted ahead of codon 217 of caspase-8. The strain produced a significant amount of soluble His-tagged 31-kDa inactive single-chain enzyme precursor. This 31-kDa protein could be purified to 98% purity. Hydroxyapatite resolved the enzyme into two species, one with the appropriate 31,090 relative mass and the other with 178 units additional mass. The latter proved to result from E. coli-based modification of the His-tag with one equivalent of glucono-1,5-lactone. The purified proteins could be activated by autoproteolysis to the appropriate 19- plus 11-kDa enzyme by the addition of dithiothreitol in appropriate buffer conditions. This yielded an enzyme with specific activity of 4-5 units/mg against 200 microM Ac-IETD-pNA at 25 degrees C. The fully active protein was used in a high-throughput screen for inhibitors of caspase-8. A preliminary robustness screen demonstrated that caspase-8 is susceptible to reactive oxygen-based inactivation in the presence of dithiothreitol (DTT) but not in the presence of cysteine. Investigation into the mechanism of this inactivation showed that quinone-like compounds were reduced by DTT establishing a reactive oxygen generating redox cycle the products of which (likely H(2)O(2)) inactivated the enzyme. A new class of caspase-8 inhibitors, steroid-derived diacids, with affinity in the low micromolar range were discovered in the refined screen. Structure--activity investigation of the inhibitors showed that both the steroid template and the acid moieties were required for activity.