Development of novel dual binders as potent, selective, and orally bioavailable tankyrase inhibitors.

Tankyrases (TNKS1 and TNKS2) are proteins in the poly ADP-ribose polymerase (PARP) family. They have been shown to directly bind to axin proteins, which negatively regulate the Wnt pathway by promoting ß-catenin degradation. Inhibition of tankyrases may offer a novel approach to the treatment of APC-mutant colorectal cancer. Hit compound 8 was identified as an inhibitor of tankyrases through a combination of substructure searching of the Amgen compound collection based on a minimal binding pharmacophore hypothesis and high-throughput screening. Herein we report the structure- and property-based optimization of compound 8 leading to the identification of more potent and selective tankyrase inhibitors 22 and 49 with improved pharmacokinetic properties in rodents, which are well suited as tool compounds for further in vivo validation studies.

Discovery of novel, induced-pocket binding oxazolidinones as potent, selective, and orally bioavailable tankyrase inhibitors.

Tankyrase (TNKS) is a poly-ADP-ribosylating protein (PARP) whose activity suppresses cellular axin protein levels and elevates ß-catenin concentrations, resulting in increased oncogene expression. The inhibition of tankyrase (TNKS1 and 2) may reduce the levels of ß-catenin-mediated transcription and inhibit tumorigenesis. Compound 1 is a previously described moderately potent tankyrase inhibitor that suffers from poor pharmacokinetic properties. Herein, we describe the utilization of structure-based design and molecular modeling toward novel, potent, and selective tankyrase inhibitors with improved pharmacokinetic properties (39, 40).

Rational design and binding mode duality of MDM2-p53 inhibitors.

Structural analysis of both the MDM2-p53 protein-protein interaction and several small molecules bound to MDM2 led to the design and synthesis of tetrasubstituted morpholinone 10, an MDM2 inhibitor with a biochemical IC50 of 1.0 µM. The cocrystal structure of 10 with MDM2 inspired two independent optimization strategies and resulted in the discovery of morpholinones 16 and 27 possessing distinct binding modes. Both analogues were potent MDM2 inhibitors in biochemical and cellular assays, and morpholinone 27 (IC50 = 0.10 µM) also displayed suitable PK profile for in vivo animal experiments. A pharmacodynamic (PD) experiment in mice implanted with human SJSA-1 tumors showed p21(WAF1) mRNA induction (2.7-fold over vehicle) upon oral dosing of 27 at 300 mg/kg.

Discovery of a class of novel tankyrase inhibitors that bind to both the nicotinamide pocket and the induced pocket.

Potent and selective inhibitors of tankyrases have recently been characterized to bind to an induced pocket. Here we report the identification of a novel potent and selective tankyrase inhibitor that binds to both the nicotinamide pocket and the induced pocket. The crystal structure of human TNKS1 in complex with this "dual-binder" provides a molecular basis for their strong and specific interactions and suggests clues for the further development of tankyrase inhibitors.

Structure-based design of 2-aminopyridine oxazolidinones as potent and selective tankyrase inhibitors.

Aberrant activation of the Wnt pathway has been implicated in the development and formation of many cancers. TNKS inhibition has been shown to antagonize Wnt signaling via Axin stabilization in APC mutant colon cancer cell lines. We employed structure-based design to identify a series of 2-aminopyridine oxazolidinones as potent and selective TNKS inhibitors. These compounds exhibited good enzyme and cell potency as well as selectivity over other PARP isoforms. Co-crystal structures of these 2-aminopyridine oxazolidinones complexed to TNKS reveal an induced-pocket binding mode that does not involve interactions with the nicotinamide binding pocket. Oral dosing of lead compounds 3 and 4 resulted in significant effects on several Wnt-pathway biomarkers in a three day DLD-1 mouse tumor PD model.

Structure-based design of novel inhibitors of the MDM2-p53 interaction.

Structure-based rational design led to the discovery of novel inhibitors of the MDM2-p53 protein-protein interaction. The affinity of these compounds for MDM2 was improved through conformational control of both the piperidinone ring and the appended N-alkyl substituent. Optimization afforded 29 (AM-8553), a potent and selective MDM2 inhibitor with excellent pharmacokinetic properties and in vivo efficacy.

Structure-Based Design of Potent and Selective CK1γ Inhibitors.

Aberrant activation of the Wnt pathway is believed to drive the development and growth of some cancers. The central role of CK1γ in Wnt signal transduction makes it an attractive target for the treatment of Wnt-pathway dependent cancers. We describe a structure-based approach that led to the discovery of a series of pyridyl pyrrolopyridinones as potent and selective CK1γ inhibitors. These compounds exhibited good enzyme and cell potency, as well as selectivity against other CK1 isoforms. A single oral dose of compound 13 resulted in significant inhibition of LRP6 phosphorylation in a mouse tumor PD model.

Identification of substrates of SMURF1 ubiquitin ligase activity utilizing protein microarrays.

The ubiquitin proteasome pathway (UPP) has been implicated in a number of pathogenic diseases: cancer, inflammation, metabolic disorders, and viral infection. The human genome contains well over 500 genes encoding proteins involved in the UPP. Ubiquitin ligases (E3s) comprise the largest subset of these genes, and together with an E2 partner, provide the substrate selectivity required for regulating cellular proteins through the covalent attachment of ubiquitin. Many ligases that have been identified in critical cellular pathways have no known substrates. Even those E3s with known substrates may have a yet unidentified role in the pathways on which they lie and as such may have additional substrates. It is critical to identify these substrates for discovery of selective small molecule inhibitors aimed at therapeutic intervention. Other methods, such as mass spectrometry, have been utilized for identifying ligase substrates, but these are labor-intensive and require a significant investment. In this study, we utilized protein microarrays for the identification of substrates of the HECT domain E3, Smurf1. Smurf1 is a critical regulator of TGF-beta and bone morphogenic protein signaling, and has been demonstrated to play a role in regulating cell polarity through the degradation of RhoA. We set out to identify novel Smurf1 substrates involved in the regulation of the aforementioned pathways. Proof-of-principle experiments with known Smurf1 substrates demonstrated efficient ubiquitination thereby validating this approach. Assaying a human protein microarray for ubiquitination with Smurf1 and the partner E2 ubiquitin ligase Ubch5 or Ubch7 identified 89 potential substrates of the Smurf1 E3 activity, which spanned a number of different biological pathways. Substrates identified utilizing protein microarray technology have been validated in vitro. Here we demonstrate the utility of this approach for identifying substrates of particular E2/E3 complexes.

Exercise in the treatment of type 2 diabetes

Unlike the type 1, type 2 diabetes benefits from an improvement in glycaemic control, because of the effect of exercise on insulin sensitivity. The level of improvement is comparable to that achieved with pharmacological agents. This is significant where cost is a major consideration. These positive effects of exercise are maintained only if exercise is sustained. The effect of a single bout of exercise lasts from 12-48 hours, so exercise should be performed at least every other day or preferably every day. Improvement in glycaemic control with exercise is not related to level of fitness so everyone can benefit, as long as glycogen depletion is achieved, with results from moderately intense exercise for a reasonable duration of time. Even more important than glycaemic control is the benefit of reducing the risk of developing coronary events. Cardiovascular abnormalities are commonly seen in diabetes especially in those persons with type 2 and this is all related to the state of insulin resistance. Exercise also reduces the progression of metabolic abnormalities from mild to more severe forms of diabetes mellitus. The exercise prescription must consider socioeconomic and personal factors in addition to scientific criteria of exercise intensity. The current trend is to recommend a combination of modest, high-volume resistance training and aerobic training. Diabetes have a lower VO2max than normal individuals and so should not be placed on the same programmes. Flexibility is advised. For example, although the exercise period may be an hour long, little rest periods at intervals will not undo the metabolic benefits.(AU)

Exercise in the treatment of type 2 diabetes

Unlike the type 1, type 2 diabetes benefits from an improvement in glycaemic control, because of the effect of exercise on insulin sensitivity. The level of improvement is comparable to that achieved with pharmacological agents. This is significant where cost is a major consideration. These positive effects of exercise are maintained only if exercise is sustained. The effect of a single bout of exercise lasts from 12-48 hours, so exercise should be performed at least every other day or preferably every day. Improvement in glycaemic control with exercise is not related to level of fitness so everyone can benefit, as long as glycogen depletion is achieved, with results from moderately intense exercise for a reasonable duration of time. Even more important than glycaemic control is the benefit of reducing the risk of developing coronary events. Cardiovascular abnormalities are commonly seen in diabetes especially in those persons with type 2 and this is all related to the state of insulin resistance. Exercise also reduces the progression of metabolic abnormalities from mild to more severe forms of diabetes mellitus. The exercise prescription must consider socioeconomic and personal factors in addition to scientific criteria of exercise intensity. The current trend is to recommend a combination of modest, high-volume resistance training and aerobic training. Diabetes have a lower VO2max than normal individuals and so should not be placed on the same programmes. Flexibility is advised. For example, although the exercise period may be an hour long, little rest periods at intervals will not undo the metabolic benefits.