Bioapplications of Magnetic Nanowires: Barcodes, Biocomposites, Heaters.

Magnetic nanowires (MNWs) can have their moments reversed via several mechanisms that are controlled using the composition, length, diameter, and density of nanowires in arrays as-synthesized or as individual nanoparticles in assays or gels. This tailoring of magnetic reversal leads to unique properties that can be used as a signature for reading out the type of MNW for applications as nano-barcodes. When synthesized inside track-etched polycarbonate membranes, the resulting MNW-embedded membranes can be used as biocompatible bandaids for detection without contact or optical sighting. When etched out of the growth template, free-floating MNWs are internalized by cells at 37 °C such that cells and/or exosomes can be collected and detected. In applications of cryopreservation, MNWs can be suspended in cryopreservation agents (CPAs) for injection into the blood vessels of tissues and organs as they are vitrified to -200 °C. Using an alternating magnetic field, the MNWs can then be nanowarmed rapidly to prevent crystallization and uniformly to prevent cracking of specimens, for example, as grafts or transplants. This invited paper is a review of recent progress in the specific bioapplications of MNWs to barcodes, biocomposites, and nanowarmers.

Discovery of potent azaindazole leucine-rich repeat kinase 2 (LRRK2) inhibitors possessing a key intramolecular hydrogen bond - Part 2.

The discovery of disease-modifying therapies for Parkinson's Disease (PD) represents a critical need in neurodegenerative medicine. Genetic mutations in LRRK2 are risk factors for the development of PD, and some of these mutations have been linked to increased LRRK2 kinase activity and neuronal toxicity in cellular and animal models. As such, research towards brain-permeable kinase inhibitors of LRRK2 has received much attention. In the course of a program to identify structurally diverse inhibitors of LRRK2 kinase activity, a 5-azaindazole series was optimized for potency, metabolic stability and brain penetration. A key design element involved the incorporation of an intramolecular hydrogen bond to increase permeability and potency against LRRK2. This communication will outline the structure-activity relationships of this matched pair series including the challenge of obtaining a desirable balance between metabolic stability and brain penetration.

Galfenol Thin Films and Nanowires.

Galfenol (Fe1-xGax, 10 < x < 40) may be the only smart material that can be made by electrochemical deposition which enables thick film and nanowire structures. This article reviews the deposition, characterization, and applications of Galfenol thin films and nanowires. Galfenol films have been made by sputter deposition as well as by electrochemical deposition, which can be difficult due to the insolubility of gallium. However, a stable process has been developed, using citrate complexing, a rotating disk electrode, Cu seed layers, and pulsed deposition. Galfenol thin films and nanowires have been characterized for crystal structures and magnetostriction both by our group and by collaborators. Films and nanowires have been shown to be largely polycrystalline, with magnetostrictions that are on the same order of magnitude as textured bulk Galfenol. Electrodeposited Galfenol films were made with epitaxial texture on GaAs. Galfenol nanowires have been made by electrodeposition into anodic aluminum oxide templates using similar parameters defined for films. Segmented nanowires of Galfenol/Cu have been made to provide engineered magnetic properties. Applications of Galfenol and other magnetic nanowires include microfluidic sensors, magnetic separation, cellular radio-frequency identification (RFID) tags, magnetic resonance imaging (MRI) contrast, and hyperthermia.

α-Aryl pyrrolidine sulfonamides as TRPA1 antagonists.

A series of α-aryl pyrrolidine sulfonamide TRPA1 antagonists were advanced from an HTS hit to compounds that were stable in liver microsomes with retention of TRPA1 potency. Metabolite identification studies and physicochemical properties were utilized as a strategy for compound design. These compounds serve as starting points for further compound optimization.

Inducing cells to disperse nickel nanowires via integrin-mediated responses.

We present non-cytotoxic, magnetic, Arg-Gly-Asp (RGD)-functionalized nickel nanowires (RGD-nanowires) that trigger specific cellular responses via integrin transmembrane receptors, resulting in dispersal of the nanowires. Time-lapse fluorescence and phase contrast microscopy showed that dispersal of 3 µm long nanowire increased by a factor of 1.54 with functionalization by RGD, compared to polyethylene glycol (PEG), through integrin-specific binding, internalization and proliferation in osteosarcoma cells. Further, a 35.5% increase in cell density was observed in the presence of RGD-nanowires, compared to an increase of only 15.6% with PEG-nanowires. These results promise to advance applications of magnetic nanoparticles in drug delivery, hyperthermia, and cell separation where uniformity and high efficiency in cell targeting is desirable.

Discovery of TRPA1 Antagonist GDC-6599: Derisking Preclinical Toxicity and Aldehyde Oxidase Metabolism with a Potential First-in-Class Therapy for Respiratory Disease.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium ion channel highly expressed in the primary sensory neurons, functioning as a polymodal sensor for exogenous and endogenous stimuli, and has been implicated in neuropathic pain and respiratory disease. Herein, we describe the optimization of potent, selective, and orally bioavailable TRPA1 small molecule antagonists with strong in vivo target engagement in rodent models. Several lead molecules in preclinical single- and short-term repeat-dose toxicity studies exhibited profound prolongation of coagulation parameters. Based on a thorough investigative toxicology and clinical pathology analysis, anticoagulation effects in vivo are hypothesized to be manifested by a metabolite─generated by aldehyde oxidase (AO)─possessing a similar pharmacophore to known anticoagulants (i.e., coumarins, indandiones). Further optimization to block AO-mediated metabolism yielded compounds that ameliorated coagulation effects in vivo, resulting in the discovery and advancement of clinical candidate GDC-6599, currently in Phase II clinical trials for respiratory indications.

Tetrahydrofuran-Based Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonists: Ligand-Based Discovery, Activity in a Rodent Asthma Model, and Mechanism-of-Action via Cryogenic Electron Microscopy.

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.

A TRPA1 inhibitor suppresses neurogenic inflammation and airway contraction for asthma treatment.

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.

Decarboxylative ketone aldol reactions: development and mechanistic evaluation under metal-free conditions.

Malonic acid half thioesters (MAHTs) and malonic acid half oxyesters (MAHOs) are shown to undergo decarboxylative nucleophilic addition reactions with ketone and aldehyde electrophiles in the presence of stoichiometric or catalytic quantities of triethylamine at room temperature. The ability to perform these reactions under metal-free conditions has enabled a detailed mechanistic analysis of the reaction pathway leading to the (1)H NMR spectroscopic characterization of a postnucleophilic addition/predecarboxylation intermediate and experimental evidence for a reversible formation of this intermediate followed by an irreversible decarboxylation. Rate constants for each of the bond forming/bond breaking steps in the reaction pathway were also determined, casting light on the differing reactivity between MAHO and MAHT nucleophiles in these processes. Finally, the mechanistic insights gained through these studies have been employed in the development of a new decarboxylative coumarin synthesis.

Nanowarming using Au-tipped Co35Fe65 ferromagnetic nanowires.

Ferromagnetic Co35Fe65, Fe, Co, and Ni nanowires have high saturation magnetizations (Ms) and magnetic anisotropies, making them ideal for magnetic heating in an alternating magnetic field (AMF). Here, Au-tipped nanowires were coated with polyethylene glycol (PEG) and specific absorption rates (SAR) were measured in glycerol. SAR increased when using metals with increasing Ms (Co35Fe65 > Fe > Co > Ni), reaching 1610 ± 20 W g-1 metal at 1 mg metal per ml glycerol for Co35Fe65 nanowires using 190 kHz and 20 kA m-1. Aligning these nanowires parallel to the AMF increased SAR up to 2010 W g-1 Co35Fe65. Next, Co35Fe65 nanowires were used to nanowarm vitrified VS55, a common cryoprotective agent (CPA).Nanowarming rates up to 1000 °C min-1 (5 mg Co35Fe65 per ml VS55) were achieved, which is 20× faster than the critical warming rate (50 °C min-1) for VS55 and other common CPAs. Human dermal fibroblast cells exposed to VS55, and Co35Fe65 nanowire concentrations of 0, 1 and 2.5 mg Fe per ml all showed similar cell viability, indicating that the nanowires had minimal cytotoxicity. With the ability to provide rapid and uniform heating, ferromagnetic nanowires have excellent potential for nanowarming cryopreserved tissues.

Enrichment and Quantification of Epitope-specific CD4+ T Lymphocytes using Ferromagnetic Iron-gold and Nickel Nanowires.

Epitope-specific CD4+ T lymphocytes were magnetically enriched using ferromagnetic Ni and Fe-Au nanowires coated with a monomer containing a major histocompatibility complex class II-bound peptide epitope (pMHCII). The enriched lymphocytes were subsequently quantified using fluorescence-activated cell sorting (FACS). This was the first use of magnetic nanowires for cell sorting using FACS, and improvements in both specificity and fluorescent signal strength were predicted due to higher particle moments and lengths than conventional paramagnetic beads. Three different types of nanowires (Ni, Fe with Au tip and Fe-Au multilayers) were made by electrodeposition. Ni nanowires separated fewer T cells than Au tipped Fe nanowires, likely because Ni has a lower magnetic moment than Fe. Fe-Au multilayer nanowires separated more T cells than Au-tipped Fe nanowires because there was more monomer per nanowire. Also, increasing the amount of monomer increased the number of CD4+ cells separated. Compared to conventional paramagnetic beads, the nanowires had lower specificity for CD4+ T cells, but had stronger fluorescent signals due to more fluorophores per particle. This results in broader FACS baseline separation between the positive and negative cells, which is useful to detect T cells, even those with lower binding affinity for pMHCII ligands.

Discovery of a Potent (4 R,5 S)-4-Fluoro-5-methylproline Sulfonamide Transient Receptor Potential Ankyrin 1 Antagonist and Its Methylene Phosphate Prodrug Guided by Molecular Modeling.

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.

Mild and general conditions for the cross-coupling of aryl halides with pentafluorobenzene and other perfluoroaromatics.

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]

Electrodeposited Fe and Fe-Au nanowires as MRI contrast agents.

Electrodeposited Fe and Fe-Au nanowires were studied for potential as MRI contrast agents, especially for T2-weighted imaging. Transverse relaxivities up to r2 = 77.1 mM Fe-1 s-1 (at 1.5 T) were achieved when Fe-Au nanowires were coated with thiol and carboxylic acid functionalized poly(ethyleneglycol). T2-Weighted images (9 T) verified successful contrast.

Discovery of highly potent, selective, and brain-penetrant aminopyrazole leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors.

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.

Pyrimidoaminotropanes as potent, selective, and efficacious small molecule kinase inhibitors of the mammalian target of rapamycin (mTOR).

We have 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 new series of small molecule inhibitors that could attenuate CYP3A4 time-dependent inhibition commonly observed with the THQ scaffold, maintain or improve aqueous solubility and oral absorption, reduce free drug clearance, and selectively increase mTOR potency. Through key in vitro and in vivo studies, we demonstrate that a pyrimidoaminotropane based core was able to address each of these goals. This effort culminated in the discovery of 20 (GNE-555), a highly potent, selective, metabolically stable, and efficacious mTOR inhibitor.

Discovery of a Highly Selective, Brain-Penetrant Aminopyrazole LRRK2 Inhibitor.

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.

Discovery of selective LRRK2 inhibitors guided by computational analysis and molecular modeling.

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.

Discovery of highly potent, selective, and brain-penetrable leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors.

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.