Identification of Small Molecule Inhibitors of PTPσ through an Integrative Virtual and Biochemical Approach

PTPσ is a dual-domain receptor type protein tyrosine phosphatase (PTP) with physiologically important functions which render this enzyme an attractive biological target. Specifically, loss of PTPσ has been shown to elicit a number of cellular phenotypes including enhanced nerve regeneration following spinal cord injury (SCI), chemoresistance in cultured cancer cells, and hyperactive autophagy, a process critical to cell survival and the clearance of pathological aggregates in neurodegenerative diseases. Owing to these functions, modulation of PTPσ may provide therapeutic value in a variety of contexts. Furthermore, a small molecule inhibitor would provide utility in discerning the cellular functions and substrates of PTPσ. To develop such molecules, we combined in silico modeling with in vitro phosphatase assays to identify compounds which effectively inhibit the enzymatic activity of PTPσ. Importantly, we observed that PTPσ inhibition was frequently mediated by oxidative species generated by compounds in solution, and we further optimized screening conditions to eliminate this effect. We identified a compound that inhibits PTPσ with an IC₅₀ of 10 µM in a manner that is primarily oxidation-independent. This compound favorably binds the D1 active site of PTPσ in silico, suggesting it functions as a competitive inhibitor. This compound will serve as a scaffold structure for future studies designed to build selectivity for PTPσ over related PTPs.     

Cofractionation of HMGB proteins with histone dimers

An effective and flexible method is presented that can be used to investigate cofractionation of groups of nuclear proteins. The method was used to analyze chromatin-related proteins, of which high-mobility group B (HMGB) proteins consistently cofractionated by cation-exchange chromatography with the histone dimer (H2A–H2B). This led to the hypothesis that the two form a complex, further suggested by gel filtration, in which the HMGBs with core histones eluted as a defined high-molecular-weight peak. A necessary requirement for further studying protein interactions is that the constituents are of the highest possible purity and the pure histone dimers and tetramers used in this study were derived from pure histone octamers with their native marks. There is a growing interest in protein–protein interactions and an increasing focus on protein-interaction domains: most frequently, pull-down assays are used to examine these. The technology presented here can provide an effective system that complements pull-down assays.     

Inference of Low and High-Grade Glioma Gene Regulatory Networks Delineates the Role of Rnd3 in Establishing Multiple Hallmarks of Cancer

Gliomas are a highly heterogeneous group of brain tumours that are refractory to treatment, highly invasive and pro-angiogenic. Glioblastoma patients have an average survival time of less than 15 months. Understanding the molecular basis of different grades of glioma, from well differentiated, low-grade tumours to high-grade tumours, is a key step in defining new therapeutic targets. Here we use a data-driven approach to learn the structure of gene regulatory networks from observational data and use the resulting models to formulate hypothesis on the molecular determinants of glioma stage. Remarkably, integration of available knowledge with functional genomics datasets representing clinical and pre-clinical studies reveals important properties within the regulatory circuits controlling low and high-grade glioma. Our analyses first show that low and high-grade gliomas are characterised by a switch in activity of two subsets of Rho GTPases. The first one is involved in maintaining normal glial cell function, while the second is linked to the establishment of multiple hallmarks of cancer. Next, the development and application of a novel data integration methodology reveals novel functions of RND3 in controlling glioma cell migration, invasion, proliferation, angiogenesis and clinical outcome.     

Specialization for underwater hearing by the tympanic middle ear of the turtle, Trachemys scripta elegans

Turtles, like other amphibious animals, face a trade-off between terrestrial and aquatic hearing. We used laser vibrometry and auditory brainstem responses to measure their sensitivity to vibration stimuli and to airborne versus underwater sound. Turtles are most sensitive to sound underwater, and their sensitivity depends on the large middle ear, which has a compliant tympanic disc attached to the columella. Behind the disc, the middle ear is a large air-filled cavity with a volume of approximately 0.5 ml and a resonance frequency of approximately 500 Hz underwater. Laser vibrometry measurements underwater showed peak vibrations at 500-600 Hz with a maximum of 300 μm s(-1) Pa(-1), approximately 100 times more than the surrounding water. In air, the auditory brainstem response audiogram showed a best sensitivity to sound of 300-500 Hz. Audiograms before and after removing the skin covering reveal that the cartilaginous tympanic disc shows unchanged sensitivity, indicating that the tympanic disc, and not the overlying skin, is the key sound receiver. If air and water thresholds are compared in terms of sound intensity, thresholds in water are approximately 20-30 dB lower than in air. Therefore, this tympanic ear is specialized for underwater hearing, most probably because sound-induced pulsations of the air in the middle ear cavity drive the tympanic disc.     

Striatal dopamine release is triggered by synchronized activity in cholinergic interneurons

Striatal dopamine plays key roles in our normal and pathological goal-directed actions. To understand dopamine function, much attention has focused on how midbrain dopamine neurons modulate their firing patterns. However, we identify a presynaptic mechanism that triggers dopamine release directly, bypassing activity in dopamine neurons. We paired electrophysiological recordings of striatal channelrhodopsin2-expressing cholinergic interneurons with simultaneous detection of dopamine release at carbon-fiber microelectrodes in striatal slices. We reveal that activation of cholinergic interneurons by light flashes that cause only single action potentials in neurons from a small population triggers dopamine release via activation of nicotinic receptors on dopamine axons. This event overrides ascending activity from dopamine neurons and, furthermore, is reproduced by activating ChR2-expressing thalamostriatal inputs, which synchronize cholinergic interneurons in vivo. These findings indicate that synchronized activity in cholinergic interneurons directly generates striatal dopamine signals whose functions will extend beyond those encoded by dopamine neuron activity.     

SAR studies around a series of triazolopyridines as potent and selective PI3Kγ inhibitors

Herein we describe the SAR of a novel series of 6-aryl-2-amino-triazolopyridines as potent and selective PI3Kγ inhibitors. The 6-aryl-triazolopyridine core was identified by chemoproteomic screening of a kinase focused library. Rapid chemical expansion around a bi-functional core identified the key features required for PI3Kγ activity and selectivity. The series was optimized to afford 43 (CZC19945), a potent PI3Kγ inhibitor with high oral bioavailability and selectivity over PI3Kα and PI3Kδ. Modification to the core afforded 53 (CZC24832) which showed increased selectivity over the entire kinome in particular over PI3Kβ.     

Finger-loop inhibitors of the HCV NS5b polymerase. Part 1: Discovery and optimization of novel 1,6- and 2,6-macrocyclic indole series

Novel conformationaly constrained 1,6- and 2,6-macrocyclic HCV NS5b polymerase inhibitors, in which either the nitrogen or the phenyl ring in the C2 position of the central indole core is tethered to an acylsulfamide acid bioisostere, have been designed and tested for their anti-HCV potency. This transformational route toward non-zwitterionic finger loop-directed inhibitors led to the discovery of derivatives with improved cell potency and pharmacokinetic profile.     

The design and synthesis of potent, selective benzodiazepine sulfonamide bombesin receptor subtype 3 (BRS-3) agonists with an increased barrier of atropisomerization

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor expressed primarily in the hypothalamus which plays a role in the onset of both diabetes and obesity. We report herein our progress made towards identifying a potent, selective bombesin receptor subtype-3 (BRS-3) agonist related to the previously described MK-7725(1) Chobanian et al. (2012) that would prevent atropisomerization through the increase of steric bulk at the C-2 position. This would thereby make clinical development of this class of compounds more cost effective by inhibiting racemization which can occur over long periods of time at room/elevated temperature.