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Isolating tumor exosomes (TEX) secreted by cancer cells can provide valuable information about the state of a tumor. Here, we present a method to rapidly isolate TEX using magnetic nanowires (MNWs). Specifically, two sets of Fe/Au segmented MNWs were used to isolate TEX released by canine osteosarcoma cell lines (OSCA 8, 32, and 40). These MNWs were prepared by electrodeposition showcasing similar length (2.2(1) μm) and diameter (36(3) nm) but different Fe/Au segment thickness: 120(20)/30(6) nm (sample A) and 28(7)/3(1) nm (sample B). Magnetic measurements indicate that we can effectively tune the magnetic response of the MNWs by changing their segment thickness, obtaining a more anisotropic behavior for sample A. The internalization of these MNWs by OSCA cells as a function of their concentration has been followed by fluorescence microscopy, and a concentration around 25 μg of Fe/Au MNWs per 3 × 105 cells has been defined as optimal. Electron microscopy images have revealed that, once internalized, these MNWs end up residing within lysosomes inside the cancer cells, where they tend to be degraded (especially the Fe segments) and fragmented into smaller pieces. Lower degradation for sample B has been observed and related to differences in the synthesis/functionalization process of both samples. We have hypothesized that these fragments of Fe/Au MNWs are packaged into TEX released to the medium which can then be isolated via a magnetic stand. This has been tested by carrying out TEX isolation experiments on the OSCA cell and comparing the magnetically isolated TEX with those isolated by using conventional methods based on centrifugation. Nanoparticle tracking analysis (NanoSight) has confirmed that the TEX isolated with MNWs have a comparable size distribution and yield to those obtained by using conventional methods, indicating that our magnetic isolation method can consistently provide relatively high TEX yields in a low-cost and fast way. [ABSTRACT FROM AUTHOR]

The decarboxylative nucleophilic addition reactions of malonic acid half thioesters (MAHTs) and malonic acid half oxyesters (MAHOs) with ketone and aldehyde electrophiles in the presence of stoichiometric or catalytic quantities of triethylamine at room temperature are described. The mechanistic insights gained through the studies could be employed in the development of a new decarboxylative coumarin synthesis.

Wehave recently reported a series of tetrahydroquinazoline (THQ)mTOR inhibitors that produced a clinical candidate 1(GDC-0349).Through insightful design, we hoped to discover and synthesize a newseries of small molecule inhibitors that could attenuate CYP3A4 time-dependentinhibition commonly observed with the THQ scaffold, maintain or improveaqueous solubility and oral absorption, reduce free drug clearance,and selectively increase mTOR potency. Through key in vitro and invivo studies, we demonstrate that a pyrimidoaminotropane based corewas able to address each of these goals. This effort culminated inthe discovery of 20(GNE-555), a highly potent, selective,metabolically stable, and efficacious mTOR inhibitor. [ABSTRACT FROM AUTHOR]

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed in sensory neurons where it functions as an irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and respiratory disease. To study its function in various disease contexts, we sought to identify novel, potent, and selective small-molecule TRPA1 antagonists. Herein we describe the evolution of an N-isopropylglycine sulfonamide lead (1) to a novel and potent (4 R,5 S)-4-fluoro-5-methylproline sulfonamide series of inhibitors. Molecular modeling was utilized to derive low-energy three-dimensional conformations to guide ligand design. This effort led to compound 20, which possessed a balanced combination of potency and metabolic stability but poor solubility that ultimately limited in vivo exposure. To improve solubility and in vivo exposure, we developed methylene phosphate prodrug 22, which demonstrated superior oral exposure and robust in vivo target engagement in a rat model of AITC-induced pain.

Leucine-rich repeat kinase 2 (LRRK2) has drawn significant interest in the neuroscience research community because it is one of the most compelling targets for a potential disease-modifying Parkinson's disease therapy. Herein, we disclose structurally diverse small molecule inhibitors suitable for assessing the implications of sustained in vivo LRRK2 inhibition. Using previously reported aminopyrazole 2 as a lead molecule, we were able to engineer structural modifications in the solvent-exposed region of the ATP-binding site that significantly improve human hepatocyte stability, rat free brain exposure, and CYP inhibition and induction liabilities. Disciplined application of established optimal CNS design parameters culminated in the rapid identification of GNE-0877 (11) and GNE-9605 (20) as highly potent and selective LRRK2 inhibitors. The demonstrated metabolic stability, brain penetration across multiple species, and selectivity of these inhibitors support their use in preclinical efficacy and safety studies.

There is a high demand for potent, selective, and brain-penetrant small molecule inhibitors of leucine-rich repeat kinase 2 (LRRK2) to test whether inhibition of LRRK2 kinase activity is a potentially viable treatment option for Parkinson's disease patients. Herein we disclose the use of property and structure-based drug design for the optimization of highly ligand efficient aminopyrimidine lead compounds. High throughput in vivo rodent cassette pharmacokinetic studies enabled rapid validation of in vitro-in vivo correlations. Guided by this data, optimal design parameters were established. Effective incorporation of these guidelines into our molecular design process resulted in the discovery of small molecule inhibitors such as GNE-7915 (18) and 19, which possess an ideal balance of LRRK2 cellular potency, broad kinase selectivity, metabolic stability, and brain penetration across multiple species. Advancement of GNE-7915 into rodent and higher species toxicity studies enabled risk assessment for early development.

The modulation of LRRK2 kinase activity by a selective small molecule inhibitor has been proposed as a potentially viable treatment for Parkinson's disease. By using aminopyrazoles as aniline bioisosteres, we discovered a novel series of LRRK2 inhibitors. Herein, we describe our optimization effort that resulted in the identification of a highly potent, brain-penetrant aminopyrazole LRRK2 inhibitor (18) that addressed the liabilities (e.g., poor solubility and metabolic soft spots) of our previously disclosed anilino-aminopyrimidine inhibitors. In in vivo rodent PKPD studies, 18 demonstrated good brain exposure and engendered significant reduction in brain pLRRK2 levels post-ip administration. The strategies of bioisosteric substitution of aminopyrazoles for anilines and attenuation of CYP1A2 inhibition described herein have potential applications to other drug discovery programs.

Mutations in the genetic sequence of leucine-rich repeat kinase 2 (LRRK2) have been linked to increased LRRK2 activity and risk for the development of Parkinson's disease (PD). Potent and selective small molecules capable of inhibiting the kinase activity of LRRK2 will be important tools for establishing a link between the kinase activity of LRRK2 and PD. In the absence of LRRK2 kinase domain crystal structures, a LRRK2 homology model was developed that provided robust guidance in the hit-to-lead optimization of small molecule LRRK2 inhibitors. Through a combination of molecular modeling, sequence analysis, and matched molecular pair (MMP) activity cliff analysis, a potent and selective lead inhibitor was discovered. The selectivity of this compound could be understood using the LRRK2 homology model, and application of this learning to a series of 2,4-diaminopyrimidine inhibitors in a scaffold hopping exercise led to the identification of highly potent and selective LRRK2 inhibitors that were also brain penetrable.

New reaction conditions are described that enable the direct arylation of pentafluorobenzene with sterically encumbered aryl bromides and aryl chlorides. These reactions occur in high yield and under mild conditions. Notably, the reactions can be performed at 80 degrees C in isopropyl acetate with a catalyst generated by the in situ mixing of Pd(OAc)(2) and S-Phos. The enhanced scope of these transformations should further reduce the need to use pentafluorophenylboronic acid in the construction of perfluoroarenes. [reaction: see text]