Inclusion and ethics statementAll required permissions were obtained from institutional review boards.Experimental model and subject detailsCell LinesAll cell culture reagents and plates were purchased from Corning (Corning, NY, USA). Cells were maintained at 8.8% CO2 at 37 °C. HEK 293 T cells were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FCS and Pen/Strep and supplied from ATCC (CRL-3216). Neuro2A cells were cultured in high glucose DMEM with 10% FCS and Pen/Strep and supplied from ATCC (CCL-131). U-373 cells were grown in Minimum Essential Media with 10% FCS and Pen/Strep and supplied from ATCC (HTB-17). Non immortalized HDF cells were cultured in high glucose DMEM with 10% FCS plus supplemented L-glutamine and Pen/Strep and supplied from the UCSF MAC. Mouse embryonic fibroblasts were generated as previously described23 and were cultured in high glucose DMEM with 10% FCS plus supplemented L-glutamine and Pen/Strep.Generation of Grn mutant mice and animal husbandryThe Grn mutant mice were originally obtained from the Farese lab and were propagated for at least 50 generations in the Herz lab. Their generation is described in reference 4040. Animals were maintained on a mixed 129SvEv Bradley; C57BL/6 J background by heterozygous intercrossing. Wild type (Grn+/+) control mice were obtained from the same crossings. Mice were genotyped by PCR using ear genomic DNA with primers (S1 5′-agtggggctggccacttct-3′, S2 5′-aagattcctcgctgggacatg-3′ and AS1 5′-gaatgctggtgtcagagggcc-3′)40. Animals were maintained on a 12 h light/12-h dark cycle and fed a standard rodent chow diet (Diet 7001; Harlan Teklad, Madison, WI) and water ad libitum. No sexual dimorphism of phenotype was observed. All procedures were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center (IACUC APN 2015-101088-G).CompoundsAll resupplied from the 200,000 small molecule screen were purchased as powder from Chembridge (San Diego, California, USA) C15 (5185123); C40 (6328802); C41 (6161539); and Chemdiv (San Diego, California, USA) C19 (8009-7313); C66 (6143-0066); C105 (C276-0473); C107 (6131-0076); C127 (3864-1015). Analogs were synthesized in house.High throughput screeningThe current UT Southwestern compound file is composed of 75,000 compounds purchased from ChemBridge Corporation, 100,000 compounds purchased from Chemical Diversity Labs and 22,000 compounds purchased from ComGenex, 1200 purchased from TimTek, 1100 from Prestwick and the 450 compounds of the NIH Clinical Collection. This library also includes approximately 30,000 natural products isolated from unique marine bacteria by Dr. John MacMillan (UC Santa Cruz). The TimTek compounds are “natural product-like” synthetic compounds and the Prestwick compounds are off-patent drugs.In the screening assay, neuronally derived Neuro-2a (N2A) cells stably transfected with a luciferase reporter under the control of the GRN promoter were used to assess the effects of treatment on GRN activation (see Cenik et al. 201184 for methods and plasmid information). Cells were plated in 384 well plates using a BioTek MicroFlow™ (Agilent, Inc.). Cells were treated with the chemical library 4 h after plating and incubated for 24 h at 37 °C with 5% CO2. Of importance, the compounds on each plate are plated to be in random order and not chemically similar. Compound concentration was set at 2.5 µM and final DMSO concentration in media was 1%. Column 1 contained the positive control sodium butyrate, and columns 2 and 23 contained the DMSO (internal) control. BrightGlo™ (Promega, Inc. E2620) was used to assay for luciferase levels. Proprietary pattern detection algorithms from Genedata (Screener™ v10 analysis suite) were used to perform quality control and normalize raw data for each plate. Raw data values for each plate were normalized using the equation:$$Normalized \; values= 100*\, (Raw\; Values – Median \; of \; Test \; Population) \\ /Median \; of \; Test \; Population$$
(1)
Where the Test Population consists of library compounds in columns 3–22. The Robust Z-score for each compound was generated from the normalized, corrected data using the equation:$$Robust \; Z \; Score= (Normalized \; Activity – Median \; of \; Neutral \; Controls) \\ /(Robust \; Stdev \; of \; Neutral \; Controls)$$
(2)
Robust Z Score was used to select primary hits. Any compound with a score of 3 or higher was considered a primary hit. To choose suitable compounds to pursue from our primary hits, a two-point activity (normalized activity to assay controls) was used with endogenous levels (DMSO treated) set at 0 and the positive control set at 100. Sodium butyrate was used for the positive control at a concentration of 9 µM and induced a two-fold increase in luciferase activation compared to DMSO treated cells. Compound toxicity was assessed against their activity in 12 NSCLC cell lines. CellTiterGlo™ (Promega, Inc., G7572) was used to identify changes in ATP levels compared to DMSO control due to accelerated cell growth or death.In vitro toxicity assayAll cells were grown in 96 well plates with 4 replicates for each treatment. CellTiterGlo™ (Promega, Inc., G7572), which uses ATP as an indicator of metabolically active cells and estimates cell viability based on ATP levels in cells, was utilized to assess toxicity. The assay consumes ATP to produce a luciferase signal which is proportional to the amount of ATP in the cultured cells.In vitro compound treatmentNeuro2A, and U-373 cells were grown in 6 well plates for harvesting. HDF cells were grown in 100 mm plates for harvesting. All cells were treated at a final concentration of 1% DMSO with or without compound over a specific time period before harvest as noted in text.
Secretion Assay
N2A cells were plated, and media was replaced with serum free media when cells were ~ 90% confluent. Cells were then treated with 0 µM, 10 µM, or 30 µM of either A21 or A41 for 24 h before conditioned media was saved for analysis and cells were lysed with 150 µL of lysis buffer. 500 µL of media was concentrated to 100 µl (i.e., 5x concentration) using Amicon ultracentrifugation concentration tubes with a 3 kDa cutoff (EMD Millipore, UFC500308). Lysate and concentrated conditioned media were then analyzed by western blot.
ICV cannulationAll compounds were dissolved in DMSO at a concentration of 100 µM and this solution was further diluted in artificial cerebrospinal fluid (ACSF) (128 mM NaCl, 2.5 mM KCl, 0.95 mM CaCl2, 1.99 mM MgCl2). The final concentration of all compounds, when added to the pump, but pre-infusion, was 10 µM in 10% DMSO and 90% ACSF. The day before surgery, 1007D osmotic pumps (Alzet, Cupertino, CA, USA, 0000290) (flow rate: 0.5 µL/h) were filled with the compound solution, connected via tubing to a cannula [tubing and cannula provided from brain infusion kit 3 (Alzet, Cupertino, CA, USA, 0008851)], placed in 0.9% sodium chloride irrigation, and primed overnight in a water bath at 37 °C.Animals were anesthetized and placed in a stereotaxic apparatus. Cannula and pump were implanted as described for ICV implantation in the manufacturer’s surgical procedures (Alzet) at the coordinates 0.3 mm posterior and 1.25 mm lateral of bregma at a depth of 3.5 mm. 4/0 blue monofilament nylon non-absorbable sutures were used for wound closure. Carprofen was given as needed for pain management during and after cannulation. Implantation was conducted on 72 three-month-old (± 5 days), male Grn wild-type or mutant mice on a mixed 129SvEv Bradley; C57BL/6 J background.Animals were sacrificed and harvested 7 days after implantation. Mice were deeply anesthetized with isoflurane and transcardially perfused with phosphate buffered saline (PBS). The whole brain was removed from the skull, hemispheres were separated and both hemispheres were snap frozen in liquid nitrogen and stored at −80 °C.I.P. injections66 three-month-old (± 5 days), male Grn wild-type or mutant mice were injected with the following compounds in their respective vehicles. C40 was delivered at 10 mg/kg, formulated as 10% DMSO, 5% Tween 80, 85% of a 30% solution of HPβCD. A21 was delivered at 50 mg/kg formulated in straight DMSO. A39, A40, A41 and SAHA were delivered at 50 mg/kg in 5% DMSO, 45% PEG, 50% HPβCD (600 mg/ml). C40 and A41 treatments were delivered at 0.2 mL per 25 grams of mouse. A21 was delivered at 0.04 mL per injection. Mice were sacrificed and tissue harvested as described in ICV methods. Time points for harvest as noted in data sets.ImmunoblottingAll cells were rinsed with PBS then harvested in RIPA buffer (10 mM Tris-HCL pH 8.0, 140 mM NaCl, 0.1% sodium deoxycholate, 0.1% SDS, 1% triton X-100, 1 mM EDTA, 0.5 mM EGTA). The lysate was then cleared by centrifugation at 21,000 × g for 15 min at 4 °C. Brain hemispheres were lysed in RIPA buffer (50 mM Tris-HCL pH 7.4, 150 mM NaCl, 0.25% sodium deoxycholate, 1% NP-40, 1 mM EDTA) and homogenized for 20 s in a 1 mL glass homogenizer. The lysate was cleared by centrifugation at 21,000 × g for 20 min at 4 °C. Protein concentrations were conducted on all sample sets using DC protein assay kit (Bio-Rad, Hercules, CA, USA). Cells were denatured in a sample buffer of 50 mM TCEP, 0.4% Orange G, 40% glycerol, 8% LDS, 125 mM Tris-HCl (pH 6.8) while tissue used Tris-glycine SDS 2x sample buffer (Life Technologies, Carlsbad, CA, USA, LC2676) plus 5% β-mercaptoethanol. All samples were loaded at 25 µg per well on a 10% Tris-glycine gel and separated by SDS-PAGE. Cell samples were transferred to nitrocellulose membranes using Trans-Blot Turbo Transfer System (Bio-Rad) while tissue samples were transferred to polyvinylidene difluoride membranes (PVDF). Both were blocked for 1 h in 5% BSA in TBS with 0.1% Tween 20 for 1 h. Membranes were incubated with proper primary antibody solutions on a rocking shaker overnight at 4 °C. Nitrocellulose membranes were incubated in fluorescent secondary for 1 h and visualized using the Odyssey Imaging System (Li-cor, Lincoln, NE, USA). PVDF membranes were incubated for 2 h on a rocking shaker with ECL-conjugated secondary antibodies. The antibodies were visualized by chemiluminescence and the membranes were exposed to blue x-ray film for 15–180 s at room temperature. All band signals were quantified and normalized to loading control. Primary antibodies: Anti-GRN 1:1,500 dilution, (sigma HPA008763, Lot 000043984); Anti-granulin 1:3,000 (Abcam ab252834, Lot GR334867-1); Anti-beta actin 1:5,000–1:20,000 dilution, (Abcam ab8227, GR342990-1); Anti-HA 1:1000 dilution (Cell Signaling 3724 S, Lot 10); Anti-mouse CD107a 1:1000 dilution (BD Biosciences 553792, Lot 0000055197); Anti-EEA1 1:1000 dilution (Cell Signaling 3288 S, Lot 8); Anti-Rab7 1:1000 dilution (Cell Signaling 9367 S, Lot 3); Anti-GM130 1:1000 dilution (Thermo Fisher MA5-35107, Lot XD3555932); Anti-COX IV 1:1000 dilution (Cell Signaling 4850 S, Lot 10); Anti-Lamin A + C 1:1000 dilution (Abcam ab133256, Lot 7); Anti-Calnexin 1:12,000 dilution (Cell Signaling 2679 S, Lot 03011836); Anti-TPP1 1:100 dilution (Santa Cruz sc-393961, Lot L1621); Secondary antibodies: HRP Anti-rabbit IgG 1:2000-1:20,000 dilution (Cytivia NA9341ML, Lot 18111932); HRP anti-rat IgG 1:5,000 dilution (Biolegend 405405, Lot B364032); IRDye 800CW anti-rabbit IgG 1:20,000 dilution (Li-cor 926-32213, Lot D30627-15); IRDye 800CW donkey anti-mouse IgG 1:20,000 dilution (Li-Cor 926-32212, Lot C61116-02); IRDye 680RD donkey anti-rabbit IgG 1:20,000 dilution (Li-Cor 926-68073, Lot D30627-15); IRDye 680RD goat anti-rat IgG 1:20,000 dilution (Li-Cor 926-68076, Lot AB_10956590).Murine microsome S9 fractionsCompound was incubated with Murine S9 (Lot VLH) fraction and Phase I (NADPH Regenerating System) cofactors for 0–240 min. Reactions were quenched with 0.5 mL (1:1) of methanol containing 0.2% formic acid and 50 ng/ml IS (IS final conc. = 25 ng/ml). Samples were vortexed for 15 s, incubated at RT for 10 min and spun for 5 min at 500 × g. Supernatant (1 mL) was then transferred to an Eppendorf tube and spun in a tabletop, chilled centrifuge for 5 min at 16,000 × g. Supernatant (800 µL) was transferred to an HPLC vial (w/out insert). Analyzed by Qtrap 4000 mass spectrometer.MS parameters (4000 Qtrap:)Method: compound pos + NB IS 110916Ion Source/Gas Parameters: CUR = 45 CAD = med, IS = 5000, TEM = 700, GS1 = 70, GS2 = 70.Buffer A: Water + 0.1% formic acid; Buffer B: Acetonitrile + 0.1% formic acid; flow rate 1.5 ml/min; column Agilent C18 XDB column, 5 micron packing 50 × 4.6 mm size; 0–1.0 min 3% B, 1.0–1.5 min gradient to 100% B, 1.5–3.0 min 100% B, 3.0–3.1 min gradient to 3% B, 3.1–4.0 3% B; IS: N-benzyl-benzamide (Sigma-Aldrich, transition 269.1 to 169.9); Compound transition 335.137/251.0.Plasma stabilityCompound (2 µM final concentration) was incubated with murine plasma and saline for 0-1440 min. Reactions were quenched with 200 µl (1:1) of methanol containing 0.2% formic acid and 50 ng/ml IS. Samples were vortexed for 15 s, incubated at RT for 10 min and spun for 5 min at 16,000 × g. Supernatant was then transferred to an Eppendorf tube and spun in a tabletop, chilled centrifuge for 5 min at 16,000 × g. Supernatant was transferred to an HPLC vial (w/ insert). Analyzed by Qtrap 4000 mass spectrometer.MS parameters (4000 Qtrap:)Method: C40 pos + NB IS 110916Ion Source/Gas Parameters: CUR = 45 CAD = med, IS = 5000, TEM = 700, GS1 = 70, GS2 = 70.Buffer A: Water + 0.1% formic acid; Buffer B: Acetonitrile + 0.1% formic acid; flow rate 1.5 ml/min; column Agilent C18 XDB column, 5 micron packing 50 × 4.6 mm size; 0–1.0 min 3% B, 1.0–1.5 min gradient to 100% B, 1.5–3.0 min 100% B, 3.0–3.1 min gradient to 3% B, 3.1–4.0 3% B; IS: N-benzyl-benzamide (sigma -aldrich, transition 269.1 to 169.9); Compound transition 305.159/237.1.Caco2 assayCaco2-cell monolayer was cultured for 26 days. Monolayer was rinsed with HBSS 0.5% FBS 3 times before 10 µM compound in HBSS 0.5% FBS solution (0.5 mL to apical side for A to B transport and 1.5 ml to basolateral side for B to A transport) was added. 1.5 ml of plain HBSS 0.5%FBS was added to basolateral side for A to B transport and 0.5 ml of plain HBSS0.5% FBS was added to apical side for B to A transport.At 30, 60, 90, 120 min post compound addition, 1000 µl was removed from basolateral side of A to B sample well; 300 µl was removed from apical side of B to A analysis well. Removed media was replaced with blank HBSS 0.5% FBS to ensure sink conditions.200 µl MeOH with 15 ng/ml benzylbenzamide as an internal standard (IS) and 2 mM Ammonium acetate, 0.15% formic acid was added to 100 µl sample from each time point. Sample was vortexed for 15 s, incubated 10 min RT, spun 16,000 × g for 5 min, and supernatant was analyzed by LC-MS/MS using Shimadzu Prominence LC and AB Sciex 4000Trap.Caco2 Controls Prop Nal Quin Cim 031916.dam for control compoundMS parameters (4000 Qtrap:)Control Compounds: Ion Source/Gas Parameters: CUR = 45, CAD = Medium, IS = 5500, TEM = 400, GS1 = 50, GS2 = 50.Buffer A: dH2O + 2 mM ammonium acetate + 0.1% formic acid; Buffer B: MeOH + 2 mM ammonium acetate + 0.1% formic acid; flow rate 1.5 ml/min; column Agilent C18 XDB column, 5 micron packing 50 ×4.6 mm size; 0–1.5 min 97% A, 1.5–2.5 min gradient to 100% B, 2.5–3.5 min 100% B, 3.5–3.6 min gradient to 97% A, 3.6–4.5 min 97% A; IS: n-benzylbenzamide (sigma -aldrich, lot #02914LH, transition 212.1 to 91.1). Compound transitions – see information above.In vivo I.P.Twenty-one six week old female CD-1 mice were dosed i.p. with compound. C40 was delivered at 10 mg/kg, 0.2 ml/mouse formulated as 10% DMSO/5% Tween 80/85% of a 30% solution of HPβCD. C127 was delivered at 10 mg/kg C127, 0.2 ml/mouse formulated as 10% DMSO/10% Cremophor EL/80% D5W (5% dextrose in dH20, pH 7.4). A21 was delivered at 30 mg/kg, 0.04 lmL/mouse formulated in straight DMSO. A41 was delivered at 25 mg/kg, 0.2 ml/mouse formulated as 5% DMSO, 45% PEG, 50% HPβCD (600 mg/ml). Whole blood and brains were harvested. Plasma was processed from whole blood by centrifugation of the ACD treated blood for 10′ at 10,000 × g in a standard centrifuge. Brains were weighed and snap frozen in liquid nitrogen.MS parameters (3200 Qtrap:)Method: Compound + pos IS 113016Ion Source/Gas Parameters: CUR = 45 CAD = med, IS = 5000, TEM = 700, GS1 = 70, GS2 = 70.Buffer A: Water + 0.1% formic acid; Buffer B: Acetonitrile + 0.1% formic acid; flow rate 1.5 ml/min; column Agilent C18 XDB column, 5 micron packing 50 × 4.6 mm size; 0–1.0 min 3% B, 1.0–1.5 min gradient to 100% B, 1.5–3.0 min 100% B, 3.0–3.1 min gradient to 3% B, 3.1–4.0 3% B; IS: N-benzyl-benzamide (sigma -aldrich, transition 269.1 to 169.9); Compound transition 305.159/237.1.In vivo P.O.Twenty-one six week old female CD-1 mice were dosed p.o. with 10 mg/kg C40, 0.2 ml/mouse formulated as 10% DMSO/5% Tween 80/85% of a 30% solution of HPbCD. Whole blood and brains were harvested. Plasma was processed from whole blood by centrifugation of the ACD-treated blood for 10’ at 10,000 × g in a standard centrifuge. Brains were weighed and snap frozen in liquid nitrogen.MS parameters (3200 Qtrap:)Method: C40 +pos IS 113016Ion Source/Gas Parameters: CUR = 45 CAD = med, IS = 5000, TEM = 700, GS1 = 70, GS2 = 70.Buffer A: Water + 0.1% formic acid; Buffer B: Acetonitrile + 0.1% formic acid; flow rate 1.5 ml/min; column Agilent C18 XDB column, 5 micron packing 50 × 4.6 mm size; 0–1.0 min 3% B, 1.0–1.5 min gradient to 100% B, 1.5–3.0 min 100% B, 3.0–3.1 min gradient to 3% B, 3.1–4.0 3% B; IS: N-benzyl-benzamide (Sigma-Aldrich, transition 269.1 to 169.9); Compound transition 305.159/237.1.Mouse complete blood countWhole mouse blood was collected in K3-citrate tubes and inverted several times. Samples were kept at room temperature and measured on IDEXX Procyte Analyzer with tube adapter.Quantitative real-time PCRTotal cellular RNA was isolated from cells using Trizol (Invitrogen, Waltham, MA, USA). and diluted to 100 ng/10 µl then reverse-transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit. Real-Time PCR was performed using PowerUp SYBR Green Master Mix, and analysis was conducted on a ViiA 7 (ThermoFisher, Waltham, MA, USA). Cyclophilin and h36B4 were used for normalization.TFEB activation assay293 T cells stably expressing TFEB-GFP were plated on coverslips and treated for 24 h with 0, 10, 50, 100, or 200 µM A41 in 1% (v/v) DMSO. For the positive control, cells were treated with DMSO only for 24 h and media was replaced by Earle’s balanced salt solution for 4 h. Cells were fixed with 4% paraformaldehyde for 10 min room temperature, washed three times for 5 min with PBS (second wash included 1:10,000 DAPI), and mounted on microscope slides with vectamount. Images were taken on an Axioplan 2 microscope, and images were analyzed by ImageJ.Lentivirus productionFor lentivirus production, 70% confluent 15 cm plates of HEK 293 T cells were co-transfected with 6.75 μg psPAX2, 2.25 μg pMD2.g, and 9 μg the individual construct encoding plasmids. Plasmids were added in a 1:3 ratio of Fugene 6 in 900 μl OptiMEM media. The media was replaced after 12–16 h. Viral particle-containing media was collected, and cell debris spun down. The viral particles were frozen at -80oC. Cells were infected with conditioned media, and media was replaced after 24 h.LysoIPMEFs and HDFs were grown to ~ 80% confluency in 15 cm plates and infected with lentivirus expressing either TMEM192-3xHA-IRES-mCherry or IRES-mCherry alone. Media was replaced after 24 h, and 48 h post-infection, cells underwent LysoIP. Briefly, cells were washed with cold PBS and scraped into 15 ml conical Falcon tubes in 10 mL KPBS (136 mM KCl and 10 mM KH2PO4, pH 7.2). HDFs were pooled 3-4 plates/sample. Cells were spun down at 200 × g 4 °C for 5 min. Liquid was decanted, and cells were resuspended in 1 ml KPBS supplemented with protease and phosphatase inhibitors (Roche). All further steps were performed in a cold room. Cells were “vacuum homogenized” by pulling them into a 5 ml syringe, expelling all air, covering the syringe tip with parafilm, pulling the plunger to create a vacuum inside the syringe, and releasing the plunger to strike the cells against a hard surface. This was repeated 6-8 times/sample. Samples were then spun at 2000 × g 4 °C for 2 min, and the supernatant was added to 50 µl anti-HA Dynabeads (ThermoFisher, Waltham, MA, USA, 88837) pre-washed in KPBS. Samples were incubated with the beads for 10 min on a rotator at 4 °C. Beads were washed 3x with KPBS+protease and phosphatase inhibitors, being moved to fresh tube on third wash. Proteins were eluted from beads by incubation with 100 µl triton-X lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (vol/vol) Triton X-100, protease and phosphatase inhibitors) on ice for 10 min. Beads were removed from solution, and debris was spun down at 17,000 × g 4 °C for 10 min. All steps were performed with low retention tubes (ThermoFisher, Waltham, MA, USA, 3451).Tandem mass tag mass spectrometrySamples were dried for 30 min in a SpeedVac, after which 40 µl of 5% SDS was added to each. Samples were then reduced with TCEP and alkylated with iodoacetamide in the dark. Each sample was loaded onto an S-Trap Micro (Protifi), following which 2 µg of trypsin (Pierce) was added and allowed to digest overnight at 37 °C. Peptides were eluted and dried in a SpeedVac, then were reconstituted in 21 µl of 50 mM TEAB buffer. Samples were then each labeled with 4 µl of TMTpro 16plex reagent (ThermoFisher, Waltham, MA, USA, A44520) and quenched with 2 µl of 5% hydroxylamine and combined in equal peptide amount based on NanoDrop A205 reading. These mixtures were dried in a SpeedVac and reconstituted in 2% acetonitrile, 0.1% TFA buffer.The TMTpro 16plex sample was injected into an Orbitrap Fusion Lumos mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. The sample was injected onto a 75 µm i.d., 75 cm long EasySpray column (ThermoFisher, Waltham, MA, USA, ES900) and eluted with a gradient from 0–28% buffer B over 180 min. Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 1.5 kV and an ion transfer tube temperature of 275 °C. MS scans were acquired at 120,000 resolution in the Orbitrap and top speed mode was used for SPS-MS3 analysis with a cycle time of 3 s. MS2 was performed with CID with a collision energy of 35%. The top 10 fragments were selected for MS3 fragmentation using HCD, with a collision energy of 55%. Dynamic exclusion was set for 25 s after an ion was selected for fragmentation.Raw MS data files were analyzed using Proteome Discoverer v2.4 (ThermoFisher, Waltham, MA, USA), with peptide identification performed using Sequest HT searching against the mouse protein database from UniProt. Fragment and precursor tolerances of 10 ppm and 0.6 Da were specified, and three missed cleavages were allowed. Carbamidomethylation of Cys and TMTpro labelling of N-termini and Lys sidechains were set as a fixed modification, with oxidation of Met set as a variable modification. The false-discovery rate (FDR) cutoff was 1% for all peptides.Analysis of TMT-MS resultsFor each TMT-MS run, at least one LysoIP sample from cells expressing IRES-mCherry only was included as a negative control. Only proteins whose raw abundance in every sample was >1.5x the abundance of mCherry were considered for further analysis. Raw abundance of mCherry control was subtracted from the raw abundance of each sample, and each sample was normalized to the raw abundance of Lamp1. Progranulin-deficient cells have an increased number of lysosomes23, thus normalizing to a non-lysosomal standard would artificially increase the level of lysosomal proteins in progranulin-deficient samples. Gene Ontology analysis was performed using ShinyGO 0.77 (http://bioinformatics.sdstate.edu/go/).Quantification and statistical analysisData analysis was conducted using ImageJ (NIH) and Odyssey Imaging Systems (Li-Cor). GraphPad Prism was used for all statistical analysis except PK analysis, which used Microsoft Excel. Data are displayed as means ± standard deviation (SD). Ordinary one-way ANOVA was used to compare multiple groups with common control(s). Repeated measured two-way ANOVA was used for weight analysis. For TMT-MS analysis, fold changes were compared by multiple t-tests followed by two-stage step-up Benjamini, Krieger, and Yekutieli correction. To show statistical significance, * or # represents a p < 0.05. Replicate number shown in figure graphs.Primer Sequences Mouse Grn Forward5′-GTCCTGGGAGCCAGTTTGAA-3′Mouse Grn Reverse5′-CATCCCCACGAACCATCAAC-3′Mouse Cyclophilin Forward5′-TGGAGAGCACCAAGACAGACA-3′Mouse Cyclophilin Reverse5′-TGCCGGAGTCGACAATGAT-3′Human GRN Forward5′-GAGATGTCCCCTGTGATAATGTCA-3′Human GRN Reverse5′-CCACTCCCCAGACGTGAGTT-3′Human TPP1 Forward5′-GGTGGCTTCAGCAATGTGTTCC-3′Human TPP1 Reverse5′-GAAGTAACTGGATGGTGGCAGG-3′Human DPP7 Forward5′-CACCATCCAGTTACTTCAATGC-3′Human DPP7 Reverse5′-CTGACCCTCCACTTCTTCATTC-3′Human h36B4 Forward5′-TGCATCAGTACCCCATTCTATCA-3′Compound synthesisHRMS data for A21, A39, A40, A41 found in supplemental materialsSynthesis of 2-substituted 1,3-benzoxazol-5-amine derivativesThis synthesis was partly performed as described in Chancellor et al., 2011139.4.3 g polyphosphoric acid (PPA) was heated in a 5 ml glass vial to 110 °C under stirring. 0.3 g of respective thiophenecarboxylic/ benzoic acid derivative and 1 mol equiv. of 2,4-diaminophenol were added simultaneously to the heated PPA. While stirring, the mixture was heated to 180 °C until no starting material was left. LC-MS was used for reaction control.After complete conversion, the mixture was cooled to room temperature and to obtain the final product, water was added into the reaction vial to precipitate the crude solid. In some cases, the solid was recrystallized with hot ethanol for further purification.Synthesis of N-(2-substituted 1,3-benzoxazol-5-yl)-amide derivativesFor amine coupling the respective benzoxazole derivative received in first synthesis step (various amounts) was dissolved in DMF and 1.1 mol equiv. respective carboxylic acid was added. While stirring, 1 mol equiv. HATU and 3 mol equiv. DIPEA were added, and the reaction was kept at room temperature until all starting material was converted into the product. The amide was precipitated by adding ice to the reaction. Purification was achieved by column chromatography and the pure product was eluted with constant 3:1 Hexane: Ethyl Acetate solvent mixture.A01 – N-(2-phenylbenzo[d]oxazol-5-yl)benzamideYield: 57.2 mg (91%); 1H-NMR (400 MHz, Methanol-d4) δ 8.25 – 8.20 (m, 2H), 8.19 (d, J = 2.1 Hz, 1H), 7.99 – 7.92 (m, 2H), 7.68 (dd, J = 8.8, 2.0 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.60 – 7.47 (m, 6H); 13C-NMR (151 MHz, Methanol-d4) δ 168.79, 165.18, 148.68, 142.77, 136.93, 135.87, 132.92, 132.74, 130.00, 129.43, 128.45, 128.42, 127.63, 120.57, 113.17, 111.37; ESI-MS m/z = 315.2 [M + H+]A02 – N-(2-phenylbenzo[d]oxazol-5-yl)propionamideYield: 45.7 mg (85%); 1H-NMR (400 MHz, Methanol-d4) δ 8.24 – 8.18 (m, 2H), 8.09 (d, J = 2.1 Hz, 1H), 7.62 – 7.52 (m, 4H), 7.50 (dd, J = 8.8, 2.1 Hz, 1H), 2.43 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H); 13C-NMR (151 MHz, Methanol-d4) δ175.31, 165.24, 148.41, 142.88, 137.29, 133.01, 130.12, 128.48, 127.79, 119.50, 112.03, 111.45, 30.95, 10.24; ESI-MS m/z = 267.2 [M + H+]A03 – N-(2-phenylbenzo[d]oxazol-5-yl)pentanamideYield: 52.8 mg (89%); 1H-NMR (400 MHz, Methanol-d4) δ 8.21 (dd, J = 7.9, 1.9 Hz, 2H), 8.09 (d, J = 2.0 Hz, 1H), 7.61 – 7.54 (m, 4H), 7.51 (dd, J = 8.8, 2.1 Hz, 1H), 2.41 (t, J = 7.6 Hz, 2H), 1.71 (tt, J = 7.7, 6.5 Hz, 2H), 1.43 (h, J = 7.4 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H); 13C-NMR (151 MHz, Methanol-d4) δ 173.37, 163.93, 147.13, 141.56, 135.90, 131.71, 128.82, 127.21, 126.47, 118.29, 110.83, 110.16, 36.44, 27.71, 22.10, 12.96; ESI-MS m/z = 295.2 [M + H+]A04 – N-(2-phenylbenzo[d]oxazol-5-yl)isonicotinamideYield: 60.8 mg (92%); 1H-NMR (400 MHz, Methanol-d4) δ 8.75 (t, J = 3.6 Hz, 2H), 8.23 (dd, J = 8.0, 2.9 Hz, 3H), 7.95 – 7.90 (m, 2H), 7.71 (dt, J = 8.8, 2.0 Hz, 1H), 7.68 – 7.64 (m, 1H), 7.58 (tdd, J = 10.3, 8.6, 5.4, 2.6 Hz, 3H); 13C-NMR (151 MHz, Methanol-d4) δ 166.06, 165.34, 150.76, 148.93, 144.14, 142.86, 136.43, 132.99, 130.03, 128.47, 127.59, 122.98, 120.39, 113.18, 113.15, 111.54; ESI-MS m/z = 316.1 [M + H+]A05 – N-(2-phenylbenzo[d]oxazol-5-yl)nicotinamideYield: 59.8 mg (91%); 1H-NMR (400 MHz, Methanol-d4) δ 9.12 (d, J = 2.2 Hz, 1H), 8.72 (dd, J = 4.9, 1.6 Hz, 1H), 8.37 (dt, J = 7.9, 2.0 Hz, 1H), 8.24 – 8.18 (m, 3H), 7.69 (dd, J = 8.8, 2.1 Hz, 1H), 7.64 (dd, J = 8.8, 1.8 Hz, 1H), 7.62 – 7.53 (m, 4H); 13C-NMR (151 MHz, Methanol-d4) δ 166.12, 165.28, 152.55, 149.21, 148.83, 142.82, 137.20, 136.58, 132.96, 132.32, 130.01, 128.45, 127.59, 124.94, 120.41, 113.14, 111.50; ESI-MS m/z = 316.2 [M + H+]A06 – N-(2-phenylbenzo[d]oxazol-5-yl)picolinamideYield: 54.8 mg (87%); 1H-NMR (400 MHz, Methanol-d4) δ 8.72 (d, J = 4.4 Hz, 1H), 8.37 (d, J = 2.0 Hz, 1H), 8.27 – 8.22 (m, 3H), 8.02 (td, J = 7.7, 1.7 Hz, 1H), 7.75 (dd, J = 8.8, 2.1 Hz, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.64 – 7.55 (m, 4H); 13C-NMR (151 MHz, Methanol-d4) δ 165.38, 164.40, 150.83, 149.54, 148.80, 143.01, 138.93, 136.36, 133.02, 130.09, 128.51, 127.89, 127.72, 123.34, 119.92, 112.40, 111.65; ESI-MS m/z = 316.2 [M + H+]A07 – 2-(dimethylamino)-N-(2-phenylbenzo[d]oxazol-5-yl)acetamideYield: 38.5 mg (65%); 1H-NMR (400 MHz, Methanol-d4) δ 8.20 (d, J = 6.5 Hz, 2H), 8.12 (d, J = 2.0 Hz, 1H), 7.62 – 7.51 (m, 5H), 3.16 (s, 2H), 2.40 (s, 6H); 13C-NMR (151 MHz, Methanol-d4) δ 170.88, 165.18, 148.51, 142.79, 136.27, 132.90, 129.97, 128.40, 127.60, 119.48, 112.09, 111.45, 63.96, 45.99; ESI-MS m/z = 296.3 [M + H+]A08 – N-(2-phenylbenzo[d]oxazol-5-yl)pyrimidine-5-carboxamideYield: 55.1 mg (88%); 1H-NMR (400 MHz, Methanol-d4) δ 9.32 (s, 1H), 9.31 (s, 2H), 8.24 (d, J = 1.5 Hz, 1H), 8.23 (d, J = 1.4 Hz, 2H), 7.73 – 7.65 (m, 2H), 7.61 – 7.56 (m, 3H); 13C-NMR (151 MHz, Methanol-d4) δ 165.44, 164.02, 160.96, 157.31, 149.01, 142.95, 136.46, 133.07, 130.19, 130.09, 128.52, 127.65, 120.31, 113.11, 111.64; ESI-MS m/z = 317.2 [M + H+]A09 – N-(2-phenylbenzo[d]oxazol-5-yl)pyrimidine-4-carboxamideYield: 56.3 mg (88%); 1H-NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H), 9.43 (d, J = 1.4 Hz, 1H), 9.13 (d, J = 5.0 Hz, 1H), 8.40 (dd, J = 4.3, 2.1 Hz, 1H), 8.23 – 8.19 (m, 2H), 8.17 (dd, J = 5.1, 1.4 Hz, 1H), 7.93 (ddd, J = 8.8, 3.7, 2.1 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.64 – 7.60 (m, 3H); 13C-NMR (151 MHz, DMSO-d6) δ 163.45, 161.65, 160.11, 158.11, 156.91, 147.43, 141.90, 135.30, 132.27, 129.56, 127.54, 126.63, 119.23, 119.20, 111.85, 110.96; ESI-MS m/z = 317.2 [M + H+]A10 – N-(2-phenylbenzo[d]oxazol-5-yl)pyrimidine-2-carboxamideYield: 46.3 mg (74%); 1H-NMR (400 MHz, Methanol-d4) δ 9.01 (d, J = 4.9 Hz, 2H), 8.38 (d, J = 2.1 Hz, 1H), 8.23 (dd, J = 7.7, 1.9 Hz, 2H), 7.78 (dd, J = 8.8, 2.1 Hz, 1H), 7.68 (s, 1H), 7.67 (d, J = 5.0 Hz, 1H), 7.62 – 7.52 (m, 3H); 13C-NMR (151 MHz, Methanol-d4) δ 164.16, 161.05, 157.68, 157.33, 147.72, 141.73, 134.93, 131.79, 128.84, 127.28, 126.44, 123.11, 118.78, 111.40, 110.42; ESI-MS m/z = 317.2 [M + H+]A11 – 2-methoxy-N-(2-phenylbenzo[d]oxazol-5-yl)acetamideYield: 47.5 mg (85%); 1H-NMR (400 MHz, Methanol-d4) δ 8.19 (ddd, J = 6.5, 4.8, 2.0 Hz, 2H), 8.10 (dt, J = 3.5, 2.0 Hz, 1H), 7.61 – 7.51 (m, 5H), 4.06 (s, 2H), 3.51 (s, 3H); 13C-NMR (151 MHz, Methanol-d4) δ 170.48, 165.24, 148.71, 142.79, 135.87, 132.95, 130.02, 128.46, 127.62, 120.02, 112.73, 111.46, 72.79, 59.70; ESI-MS m/z = 283.2 [M + H+]A12 – N-(2-phenylbenzo[d]oxazol-5-yl)cyclohexanecarboxamideYield: 55.2 mg (88%); 1H-NMR (400 MHz, Methanol-d4) δ 8.20 (dd, J = 6.6, 1.9 Hz, 2H), 8.08 (t, J = 2.5 Hz, 1H), 7.60 – 7.52 (m, 4H), 7.51 (dd, J = 8.8, 2.1 Hz, 1H), 2.38 (tdd, J = 11.2, 4.6, 2.2 Hz, 1H), 1.91 (dd, J = 13.7, 2.0 Hz, 2H), 1.84 (dt, J = 12.9, 3.4 Hz, 2H), 1.73 (dt, J = 13.6, 3.5 Hz, 1H), 1.55 (qd, J = 12.6, 3.4 Hz, 2H), 1.37 (q, J = 12.9 Hz, 2H), 1.29 (tt, J = 12.9, 3.4 Hz, 1H); 13C-NMR (151 MHz, Methanol-d4) δ 177.52, 165.07, 148.25, 142.68, 137.17, 132.85, 129.97, 128.36, 127.63, 119.52, 112.03, 111.28, 46.95, 30.47, 26.64, 26.58; ESI-MS m/z = 321.3 [M + H+]A16 – N-(2-phenylbenzo[d]oxazol-5-yl)pyrimidine-2-carboxamideYield: 42.8 mg (54%); 1H-NMR (400 MHz, CDCl3) δ 8.22 (dd, J = 8.8, 5.4 Hz, 2H), 7.93 – 7.86 (m,1H), 7.58 – 7.44 (m, 2H), 7.39 (s, 1H), 7.20 (t, J = 8.6 Hz, 2H), 2.40 (t, J = 7.6 Hz, 2H), 1.75 (p, J = 7.6 Hz, 2H), 1.43 (h, J = 7.4 Hz, 2H), 0.96 (t, J = 7.3 Hz, 3H); 13C-NMR (151 MHz, CDCl3) δ 171.64, 166.24, 163.15, 147.71, 142.59, 135.11, 130.04, 129.95, 123.50, 118.39, 116.45, 116.23, 111.67, 110.57, 37.66, 27.86, 22.57, 13.98; ESI-MS m/z = 313.1 [M + H+]A17 – N-(2-(4-chlorophenyl)benzo[d]oxazol-5-yl)pentanamideYield: 13.8 mg (21%); 1H-NMR (400 MHz, CDCl3) δ 8.15 (d, J = 8.4 Hz, 2H), 7.90 (s, 1H), 7.51 (dd, J = 17.7, 8.3 Hz, 4H), 7.39 (s, 1H), 2.40 (t, J = 7.6 Hz, 2H), 1.74 (p, J = 7.6 Hz, 2H), 1.66 (s, 2H), 1.43 (h, J = 7.3 Hz, 2H), 0.96 (t, J = 7.3 Hz, 3H); 13C-NMR (151 MHz, CDCl3) δ 171.66, 163.06, 147.69, 142.54, 137.99, 135.18, 129.42 (2 C), 129.00 (2 C), 125.65, 118.61, 111.73, 110.63, 37.65, 27.85, 22.57, 13.98; ESI-MS m/z = 329.1 [M + H+]A20 – N-(2-(3-fluorophenyl)benzo[d]oxazol-5-yl)pentanamideYield: 25.5 mg (37%); 1H-NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 8.18 (s, 1H), 8.03 (d, J = 7.7 Hz, 1H), 7.93 (d, J = 9.5 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.70 – 7.63 (m, 1H), 7.54 (d, J = 8.9 Hz, 1H), 7.52 – 7.46 (m, 1H), 2.34 (t, J = 7.4 Hz, 2H), 1.60 (p, J = 7.4 Hz, 2H), 1.34 (dq, J = 14.4, 7.3 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C-NMR (151 MHz, DMSO-d6) δ 171.44, 161.16, 146.24, 141.52, 136.86, 131.80, 131.72, 128.66, 123.53, 117.90, 114.00, 113.76, 110.91, 109.88, 36.21, 27.30, 21.92, 13.84; ESI-MS m/z = 313.1 [M + H+]A21 – N-(2-(2-fluorophenyl)benzo[d]oxazol-5-yl)pentanamideYield: 36.9 mg (54%); 1H-NMR (400 MHz, DMSO-d6): δ 10.09 (s, 1H), 8.25 – 8.17 (m, 2H), 7.74 (d, J = 8.8 Hz, 1H), 7.72 – 7.65 (m, 1H), 7.58 – 7.52 (m, 1H), 7.51 – 7.41 (m, 2H), 2.34 (t, J = 7.5 Hz, 2H), 1.60 (p, J = 7.5 Hz, 2H), 1.34 (h, J = 7.3 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C-NMR (101 MHz, DMSO-d6): δ 171.43, 146.02, 141.27, 136.80, 134.15 (2 C), 130.52, 125.30, 117.87, 117.42, 117.21, 114.61, 110.84, 109.85, 36.21, 27.31, 21.92, 13.84; ESI-MS m/z = 313.1 [M + H+]. HRMS m/z = 313.1358 (calc 313.1347).A22 – N-(2-(3-chlorophenyl)benzo[d]oxazol-5-yl)pentanamideYield: 34.2 mg (51%); 1H-NMR (400 MHz, DMSO-d6): δ 10.09 (s, 1H), 8.20 – 8.10 (m, 3H), 7.74 – 7.68 (m, 2H), 7.64 (t, J = 7.8 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 2.34 (t, J = 7.4 Hz, 2H), 1.60 (p, J = 7.3 Hz, 2H), 1.34 (h, J = 7.3 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C-NMR (101 MHz, DMSO-d6): δ 171.44, 161.51, 146.26, 141.50, 136.86, 134.06, 131.76, 131.45, 128.45, 126.72, 125.91, 117.92, 110.92, 109.88, 36.21, 27.30, 21.92, 13.83; ESI-MS m/z = 329.1 [M + H+]A24 – N-(2-(2-chlorophenyl)benzo[d]oxazol-5-yl)pentanamideYield: 61.3 mg (57%); 1H-NMR (400 MHz, DMSO-d6): δ 10.11 (s, 1H), 8.22 (s, 1H), 8.14 (d, J = 7.7 Hz, 1H), 7.77 – 7.69 (m, 2H), 7.64 (t, J = 7.6 Hz, 1H), 7.57 (t, J = 7.8 Hz, 2H), 2.35 (t, J = 7.4 Hz, 2H), 1.61 (p, J = 7.3 Hz, 2H), 1.35 (dq, J = 14.2, 7.1 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C-NMR (101 MHz, DMSO-d6): δ 171.45, 160.83, 146.06, 141.23, 136.77, 132.96, 132.13, 131.96, 131.35, 127.84, 125.53, 118.00, 110.89, 110.01, 36.22, 27.31, 21.92, 13.83; ESI-MS m/z = 329.1 [M + H+]A28 – N-(2-(pyridin-4-yl)benzo[d]oxazol-5-yl)pentanamideYield: 48.3 mg (43%); 1H-NMR (400 MHz, DMSO-d6): δ 10.13 (s, 1H), 8.86 – 8.80 (m, 2H), 8.23 (s, 1H), 8.11 – 8.05 (m, 2H), 7.77 (d, J = 8.8 Hz, 1H), 7.58 (dd, J = 8.9, 2.0 Hz, 1H), 2.34 (t, J = 7.5 Hz, 2H), 1.60 (p, J = 7.5 Hz, 2H), 1.34 (h, J = 7.4 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C-NMR (101 MHz, DMSO-d6): δ 171.48, 160.88, 150.94 (2 C), 146.33, 141.37, 137.05, 133.52, 120.81 (2 C), 118.60, 111.17, 110.03, 36.21, 27.28, 21.92, 13.83; ESI-MS m/z = 296.1 [M + H+]A29 – N-(2-(pyridin-3-yl)benzo[d]oxazol-5-yl)pentanamideYield: 57.8 mg (52%); 1H-NMR (400 MHz, DMSO-d6): δ 10.10 (s, 1H), 9.36 – 9.32 (m, 1H), 8.80 (q, J = 5.9 Hz, 1H), 8.52 (d, J = 8.1 Hz, 1H), 8.19 (s, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.65 (dd, J = 7.9, 4.9 Hz, 1H), 7.55 (d, J = 8.9 Hz, 1H), 2.34 (t, J = 7.4 Hz, 2H), 1.60 (p, J = 7.4 Hz, 2H), 1.39 – 1.30 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C-NMR (101 MHz, DMSO-d6): δ 171.47, 152.38, 148.02, 146.25, 141.44, 136.89, 134.80, 124.42, 122.90, 117.93, 110.95, 109.87, 36.22, 27.31, 21.93, 13.84; ESI-MS m/z = 296.1 [M + H+]A30 – N-(2-(pyridin-2-yl)benzo[d]oxazol-5-yl)pentanamideYield: 18.7 mg (17%); 1H-NMR (400 MHz, DMSO-d6): δ 10.11 (s, 1H), 8.79 (d, J = 4.5 Hz, 1H), 8.32 (d, J = 7.9 Hz, 1H), 8.22 (s, 1H), 8.05 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H), 7.65 – 7.60 (m, 1H), 7.56 (d, J = 8.8 Hz, 1H), 2.34 (t, J = 7.4 Hz, 2H), 1.60 (p, J = 7.4 Hz, 2H), 1.34 (dq, J = 14.5, 7.3 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); ESI-MS m/z = 296.1 [M + H+]Amount was not enough for a clear 13C-NMR spectrumA33 – N-(2-(thiophen-3-yl)benzo[d]oxazol-5-yl)pentanamideYield: quantitative; 1H-NMR (400 MHz, DMSO-d6): δ 10.06 (s, 1H), 8.47 (d, J = 4.1 Hz, 1H), 8.11 (d, J = 1.8 Hz, 1H), 7.81 (dd, J = 5.1, 2.9 Hz, 1H), 7.74 (dd, J = 5.1, 1.2 Hz, 1H), 7.67 (d, J = 8.8 Hz, 1H), 7.50 (dd, J = 8.8, 2.0 Hz, 1H), 2.33 (t, J = 7.5 Hz, 2H), 1.60 (dt, J = 15.1, 7.5 Hz, 2H), 1.37 – 1.30 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C-NMR (101 MHz, DMSO-d6): δ 171.36, 159.81, 145.77, 141.59, 136.64, 129.45, 128.76, 128.33, 126.31, 117.22, 110.53, 109.71, 36.19, 27.31, 21.92, 13.83; ESI-MS m/z = 301.1 [M + H+]A34 – N-(2-(thiophen-2-yl)benzo[d]oxazol-5-yl)pentanamideYield: quantitative; 1H-NMR (400 MHz, DMSO-d6): δ 10.07 (s, 1H), 8.11 – 8.06 (m, 1H), 8.01 – 7.92 (m, 2H), 7.68 (d, J = 8.8 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.31 (dd, J = 4.7, 4.0 Hz, 1H), 2.34 (t, J = 7.5 Hz, 2H), 1.60 (dt, J = 15.0, 7.5 Hz, 2H), 1.35 (dt, J = 14.8, 7.4 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C-NMR (101 MHz, DMSO-d6): δ 171.38, 158.99, 145.83, 141.60, 136.80, 131.99, 130.54, 128.99, 128.57, 117.31, 110.54, 109.51, 36.19, 27.30, 21.92, 13.83; ESI-MS m/z = 301.1 [M + H+]A39 – N-(2-(p-tolyl)benzo[d]oxazol-5-yl)pentanamideYield: 71 mg (57%); 1H-NMR (400 MHz, DMSO-d6): δ 10.06 (s, 1H), 8.12 (s, 1H), 8.07 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.8 Hz, 1H), 7.50 (d, J = 9.8 Hz, 1H), 7.42 (d, J = 8.1 Hz, 2H), 2.41 (s, 3H), 2.33 (t, J = 7.5 Hz, 2H), 1.60 (p, J = 7.4 Hz, 2H), 1.34 (dq, J = 14.5, 7.3 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C-NMR (101 MHz, DMSO-d6): δ 171.37, 163.09, 146.11, 142.19, 141.74, 136.63, 129.97 (2 C), 127.26 (2 C), 123.76, 117.23, 110.60, 109.70, 36.21, 27.32, 21.93, 21.23, 13.84; ESI-MS m/z: 309.2 [M + H+]. HRMS m/z = 309.1599 (calc. 309.1598).A40 – N-(2-(m-tolyl)benzo[d]oxazol-5-yl)pentanamideYield: quantitative; 1H-NMR (400 MHz, DMSO-d6): δ 10.15 (s, 1H), 8.22 (d, J = 1.8 Hz, 1H), 8.10 – 8.03 (m, 2H), 7.77 (d, J = 8.8 Hz, 1H), 7.61 – 7.49 (m, 3H), 2.60 – 2.56 (m, 1H), 2.42 (t, J = 7.5 Hz, 2H), 1.68 (p, J = 7.5 Hz, 2H), 1.42 (h, J = 7.3 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H); 13C-NMR (101 MHz, DMSO-d6): δ 171.39, 163.03, 146.17, 141.69, 138.83, 136.67, 132.68, 129.31, 127.67, 126.41, 124.47, 117.42, 110.68, 109.78, 36.21, 27.32, 21.93, 20.94, 13.84; ESI-MS m/z: 309.2 [M + H+]. HRMS m/z = 309.1605 (calc. 309.1598).A41 – N-(2-(o-tolyl)benzo[d]oxazol-5-yl)pentanamideYield: 68 mg (55%); 1H-NMR (400 MHz, DMSO-d6): δ 10.07 (s, 1H), 8.18 (s, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.54 – 7.47 (m, 2H), 7.43 (dd, J = 14.8, 7.3 Hz, 2H), 2.74 (s, 3H), 2.34 (t, J = 7.5 Hz, 2H), 1.60 (p, J = 7.4 Hz, 2H), 1.34 (dq, J = 14.3, 7.2 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C-NMR (101 MHz, DMSO-d6): δ 171.39, 163.17, 145.66, 141.67, 138.28, 136.53, 131.98, 131.36, 129.51, 126.50, 125.49, 117.46, 110.60, 109.91, 36.23, 27.34, 21.92 (2 C), 13.84; ESI-MS m/z: 309.1 [M + H+]. HRMS m/z = 309.1605 (calc. 309.1598).Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Benzoxazole-derivatives enhance progranulin expression and reverse the aberrant lysosomal proteome caused by GRN haploinsufficiency
