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Inhibitors of leucine-rich repeat kinase 2 (LRRK2) and mutants, such as G2019S, have potential utility in Parkinson's disease treatment. Fragment hit-derived pyrrolo[2,3-d]pyrimidines underwent optimization using X-ray structures of LRRK2 kinase domain surrogates, based on checkpoint kinase 1 (CHK1) and a CHK1 10-point mutant. (2R)-2-Methylpyrrolidin-1-yl derivative 18 (LRRK2 G2019S cKi 0.7 nM, LE 0.66) was identified, with increased potency consistent with an X-ray structure of 18/CHK1 10-pt. mutant showing the 2-methyl substituent proximal to Ala147 (Ala2016 in LRRK2). Further structure-guided elaboration of 18 gave the 2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino] derivative 32. Optimization of 32 afforded diastereomeric oxolan-3-yl derivatives 44 and 45, which demonstrated a favorable in vitro PK profile, although they displayed species disconnects in the in vivo PK profile, and a propensity for P-gp- and/or BCRP-mediated efflux in a mouse model. Compounds 44 and 45 demonstrated high potency and exquisite selectivity for LRRK2 and utility as chemical probes for the study of LRRK2 inhibition.

We report a non-cytotoxic resin compatible with and designed for use in custom high-resolution 3D printers that follow the design approach described in Gong et al., Lab Chip 17, 2899 (2017). The non-cytotoxic resin is based on a poly(ethylene glycol) diacrylate (PEGDA) monomer with avobenzone as the UV absorber instead of 2-nitrophenyl phenyl sulfide (NPS). Both NPS-PEGDA and avobenzone-PEGDA (A-PEGDA) resins were evaluated for cytotoxicity and cell adhesion. We show that NPS-PEGDA can be made effectively non-cytotoxic with a post-print 12-hour ethanol wash, and that A-PEGDA, as-printed, is effectively non-cytotoxic. 3D prints made with either resin do not support strong cell adhesion in their as-printed state; however, cell adhesion increases dramatically with a short plasma treatment. Using A-PEGDA, we demonstrate spheroid formation in ultra-low adhesion 3D printed wells, and cell migration from spheroids on plasma-treated adherent surfaces. Given that A-PEGDA can be 3D printed with high resolution, it has significant promise for a wide variety of cell-based applications using 3D printed microfluidic structures.

We previously showed that a small molecule of natural origin, 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose (PGG), binds to capillary morphogenesis gene 2 (CMG2) with a submicromolar IC and also has antiangiogenic activity in vitro and in vivo. In this work, we synthetized derivatives of PGG with different sugar cores and phenolic substituents and tested these as angiogenesis inhibitors. In a high-throughput Förster resonant energy transfer-based binding assay, we found that one of our synthetic analogues (1,2,3,4,6-penta-O-galloyl-β-d-mannopyranose (PGM)), with mannose as central core and galloyl substituents, exhibit higher (up to 10×) affinity for CMG2 than the natural glucose prototype PGG and proved to be a potent angiogenesis inhibitor. These findings demonstrate that biochemical CMG2 binding in vitro predicts inhibition of endothelial cell migration ex vivo and antiangiogenic activity in vivo. The molecules herein described, and in particular PGM, might be useful prototypes for the development of novel agents for angiogenesis-dependent diseases, including blinding eye disease and cancer., This work was supported by the Spanish MINECO [Project SAF2015-64629-C2-1-R (MINECO/FEDER)], the Spanish Agencia Estatal Consejo Superior de Investigaciones Cientif́ - icas (CSIC, Project CSIC201680E079), and NIH R01 EY018829. The Spanish MINECO is also acknowledged for a grant to B.M.-G.

Trypanosoma brucei, which causes human African typanosomiasis (HAT), derives cellular ATP from glucose metabolism while in the mammalian host. Targeting glucose uptake or regulation in the parasite has been proposed as a potential therapeutic strategy. However, few methods have been described to identify and characterize potential inhibitors of glucose uptake and regulation. Here, we report development of a screening assay that identifies small molecule disrupters of glucose levels in the cytosol and glycosomes. Using an endogenously expressed fluorescent protein glucose sensor expressed in cytosol or glycosomes, we monitored intracellular glucose depletion in the different cellular compartments. Two ([JM1]) glucose level disrupters were identified, one of which only exhibited inhibition of glycosomal glucose and did not affect cytosolic levels. In addition to inhibiting glucose uptake with relatively high potency (EC(50) = 700 nM), the compound also showed modest bloodstream form parasite killing activity. Expanding this assay will allow for identification of candidate compounds that disrupt parasite glucose metabolism.

Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson’s disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LR...

A unique class of aromatic ether polymers containing perfluorocyclopentenyl (PFCP) enchainment was prepared from the simple step growth polycondensation of commercial bisphenols and octafluorocyclopentene (OFCP) in the presence of triethylamine. Model studies indicate that the second addition/elimination on OFCP is fast and polycondensation results in linear homopolymers and copolymers without side products. The synthesis of bis(heptafluorocyclopentenyl) aryl ether monomers and their condensation with bisphenols further led to PFCP copolymers with alternating structures. This new class of semifluorinated polymers exhibit surprisingly high crystallinity in some cases and excellent thermal stability.

Silver nanobelts were synthesized without any external seeds or templates using a facile hydrothermal route via citrate reduction in the presence of hydrochloric acid. Both H+ and Cl– ions in HCl are important for the generation of nanobelts. The morphology can be controlled by varying the concentration of HCl and citrate. The obtained nanobelts have 3D orientation-dependent optical properties. By tracking the transportation and rotation of the nanobelts in cells or other biosystems, useful information related to the physical or chemical surroundings may be obtained.

Conjugated polymer nanoparticles are formed by precipitation of highly fluorescent conjugated polymers to form small nanoparticles with extremely bright fluorescence. We characterized cellular uptake and cytotoxicity of 18 ± 5 nm PFBT conjugated polymer nanoparticles in J774A.1 cells. Significant nanoparticle uptake was observed, indicating efficient nanoparticle entry into cells, even for short (1 h) incubations. The high fluorescence of these nanoparticles allows extremely low loading concentrations; PFBT nanoparticle fluorescence in cells could be detected with loading concentrations of 155 pM (270 ppb). Cellular uptake slows at low temperature, consistent with endocytic entry. Nanoparticles colocalize with Texas Red dextran and are trafficked to lysosomes, as demonstrated by the location of nanoparticle fluorescence in perinuclear organelles that also stain with an anti-LAMP-1 antibody. Inhibition of uptake by phosphoinositide 3-kinase inhibitors implicates macropinocytosis as the operative endocytic mechanism. No significant cytotoxic or inflammatory effects could be observed, making PFBT nanoparticles attractive probes for live cell imaging.

It was reported previously that monosaccharide-functionalized single-walled carbon nanotubes (SWNTs) could interact with Bacillus anthracis (Sterne) spores with the mediation of a divalent cation such as Ca(2+) to result in significant spore aggregation and reduction in colony forming units. In this work a more systematic investigation was performed on interactions of the SWNTs functionalized with individual mannose and galactose moieties and their various dendritic configurations with B. anthracis and B. subtilis spores in the presence and absence of a divalent cation. Significant differences and selectivity between the Bacillus spores and between different sugars and their configurations were observed. The relevant results are presented, and their mechanistic implications are discussed.

Highly fluorescent conjugated polymer dots were developed for demanding applications such as fluorescence imaging in live cells. These nanoparticles exhibit small particle diameters, extraordinary fluorescence brightness, and excellent photostability. Single particle fluorescence imaging and kinetic studies indicate much higher emission rates (approximately 10(8) s(-1)) and little or no blinking of the nanoparticles as compared to typical results for single dye molecules and quantum dots. Analysis of single particle photobleaching trajectories reveals excellent photostabilityas many as 10(9) or more photons emitted per nanoparticle prior to irreversible photobleaching. The superior figures of merit of these new fluorescent probes, together with the demonstration of cellular imaging, indicate their enormous potential for demanding fluorescence-based imaging and sensing applications such as high speed super-resolution single molecule/particle tracking and highly sensitive assays.

The behavior of colloidal gels under pressure-driven flow in square microchannels is quantified by microscopic particle image velocimetry (muPIV) and compared to predictions of available rheological models. The gels consist of hydrophobically modified silica microspheres (phi = 0.15-0.33) suspended in a refractive index-matched fluid along with fluorescent tracers to aid visualization. Measured velocity flow profiles show a transition from plug flow to more fluid-like behavior with increasing volumetric flow rate (Q) at all phi. This transition is not captured by theoretical predictions of the flow profile based on the Herschel-Bulkley model. Rather, a model that accounts for gel breakup into a suspension of clusters at elevated shear rates by assuming a finite viscosity at infinite shear is needed to accurately predict the flow behavior of colloidal gels at large Q.

A method for monitoring DPA release from a single germinating Bacillus subtilis endospore is reported. High S/N ratio SERS spectra were obtained with excitation power 3 mW at 647.1 nm and 1 min spectral collection times. The method is proof-of-principle for the SERS detection limit at the single spore level. This represents a 100- to 1000-fold improvement over previously reported detection limits for SERS-based measurements of DPA in endospores.