Benzoxazole-derivatives enhance progranulin expression and reverse the aberrant lysosomal proteome caused by GRN haploinsufficiency

Domain: earth_science
Published: 2024-07-20
Editorial outcome: major_revision

Abstract
Heterozygous loss-of-function mutations in the GRN gene are a major cause of hereditary frontotemporal dementia. The mechanisms linking frontotemporal dementia pathogenesis to progranulin deficiency are not well understood, and there is currently no treatment. Our strategy to prevent the onset and progression of frontotemporal dementia in patients with GRN mutations is to utilize small molecule positive regulators of GRN expression to boost progranulin levels from the remaining functional GRN allele, thus restoring progranulin levels back to normal within the brain. This work describes a series of blood-brain-barrier-penetrant small molecules which significantly increase progranulin protein levels in human cellular models, correct progranulin protein deficiency in Grn+/− mouse brains, and reverse lysosomal proteome aberrations, a phenotypic hallmark of frontotemporal dementia, more efficiently than the previously described small molecule suberoylanilide hydroxamic acid. These molecules will allow further elucidation of the cellular functions of progranulin and its role in frontotemporal dementia and will also serve as lead structures for further drug development.

Introduction
Frontotemporal dementia (FTD) is the second most common form of early-onset dementia and is currently incurable1–3. It is characterized by progressive, neuronal degeneration of the prefrontal and anterior temporal lobes and has a highly heterogeneous clinical representation with cognitive decline, language deficits and behavioral changes in personality and social conduct4–7. Currently the only treatment options for FTD are limited to pharmacological relief of disease symptoms8,9. No therapies are available to delay or stop neurodegeneration.

In 2006, heterozygous loss-of-function mutations within the progranulin (PGRN) encoding GRN gene were identified as a cause of FTD, accounting for up to 20% of all genetic FTD cases10–14. The majority of mutations result in nonsense-mediated decay and PGRN haploinsufficiency10,12,14–17. Lipofuscin accumulation, dysregulated lysosomal lipid homeostasis, impaired autophagy, neuropathological aggregation of the RNA-binding protein TDP-43, and inflammation are key pathogenic hallmarks of FTD18–25. Further, homozygous loss of functional PGRN causes neuronal ceroid lipofuscinosis CLN11, an early-onset, neurodegenerative, lysosomal storage disorder26–28. Unfortunately the mechanisms involved in neurodegeneration associated with PGRN deficiency remain elusive.

One conjecture is that a key driver of PGRN deficiency related neurodegeneration is lysosomal dysfunction29. Impairments of autophagic flux30, lysosomal cathepsin D deficiency31, aberration of the lysosomal proteome25,32 and lipidome23 due to PGRN deficiency point to an important role of PGRN in lysosomal homeostasis.

PGRN is a highly conserved, secreted 88kDa glycoprotein primarily expressed by microglia and neurons in the central nervous system (CNS)33,34. Full-length PGRN is credited with neuroprotective and anti-inflammatory properties35–41 and its transcription is regulated alongside lysosomal biogenesis, autophagy, and cellular energy homeostasis genes42–44.

When endocytosed, PGRN is transported to the lysosome where it is processed into seven granulin subunits35,45–50. The functions of individual granulins are a focus of contemporary research51–54, but a role in lysosomal acidification55 and regulation of lysosomal enzymatic activity31,56–62 has been proposed. This underscores PGRN’s pivotal role in maintaining lysosomal homeostasis with relevance for multiple CNS disorders24,26,30,63–67.

Earlier work has used various methods to boost PGRN levels in FTD models9,68–78. These methods include adeno-associated virus (AAV) and recombinant protein-based therapies79–82. However, beyond preclinical animal studies, the delivery of viruses and proteins to all regions of the human brain presents significant challenges compared to administration of small molecules. Also, since patients likely need to be targeted and treated before the onset of symptoms and for the remainder of their lives, protein-based therapies could be cost-prohibitive and inefficient83,84.

Previous work from our group demonstrated administration of the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat) increased the expression of progranulin protein levels in vitro85. However, due to the poor side effect profile of SAHA as well as the limited PGRN-boosting effects we observed in vivo, we moved forward to develop non-toxic and reliable enhancers of PGRN both in vitro and in vivo.

This study uses small molecules to take advantage of the remaining functional allele of the GRN gene in PGRN-dependent FTD patients for PGRN protein production to restore PGRN levels back to normal. To accomplish this objective, we conduct high-throughput screening (HTS) to identify small-molecule PGRN enhancers that do not alter protein maturation, transport etc., and develop analogs of selected hits through a structure-activity-relationship (SAR) approach. Both initial hits and analog compounds are tested for the ability to increase Grn mRNA and PGRN protein levels. Subsequently, promising compounds are evaluated in vitro and in vivo using pharmacokinetic and pharmacodynamic profiling. Here we describe small molecules with the ability to restore PGRN levels in PGRN haploinsufficient models in vitro and in vivo and the ability to correct aberrantly lysosomal proteomes in FTD patient-derived cells. These small molecules will serve as lead structures for further development, optimization, and translational experiments.

Results
PGRN enhancers identified through high-throughput screening
Neuronally derived mouse Neuro-2a (N2A) cells stably transfected with a luciferase reporter under the control of the human GRN promoter were created to assess the effects of small-molecule treatment on GRN activation85. Approximately 200,000 small molecules were screened to probe for upregulation of GRN at the University of Texas Southwestern Medical Center (UTSW) HTS Core. Any small molecules with activity 3 standard deviations above the robust mean of the test population were considered a hit in the screen. Using this low-stringency cut-off, 15,820 compounds qualified as primary hits. Of these primary hits, 2144 compounds had a score of >100 and were marked for further evaluation (Supplementary Fig. 1). To further reduce our hits, we eliminated 87 small molecules which were considered to have generalized toxicity in twelve different non-small cell lung cancer (NSCLC) cell lines (Supplementary Fig. 1, red)86. We then chose the remaining top 1,280 hits to conduct a 3-point dose response point activity confirmation experiment using the original GRN luciferase-based assay (Supplementary Fig. 2, blue).

Unique compounds induce PGRN protein production in vitro
We next tested the top 127 commercially available compounds from the HTS small molecule confirmation experiment for their ability to upregulate PGRN. Each of the 127 selected compounds were tested for GRN-linked luciferase activation (Supplementary Fig. 2a) and toxicity (Supplementary Fig. 2b) over a dose range from 10 nM to 30 µM. The ATP-based CellTiterGlo™ (Promega) was utilized to assess toxicity. All compound doses resulting in more than a 20% reduction in ATP levels were excluded from further testing. After toxicity exclusions we selected the four most active concentrations of each compound to test their ability to induce PGRN protein expression. The treatment concentrations ranged from 100 nM-30 µM. N2A cells were harvested after 48h of treatment and cell lysates were immunoblotted to evaluate compound activity. Nine compounds (C), C15, C19, C40, C41, C66, C105, C107, C116, and C127 (Fig. 1A) increased PGRN protein to approximately our target value of 2x compared to vehicle levels (Fig. 1B, C) and were selected for further evaluation.

Three compounds boost PGRN levels in an in vivo model of FTD
After validating our selected compounds in vitro, we utilized an in vivo mouse model of FTD, a CNS disorder, to evaluate these compounds’ activity in the brain. We used intracerebroventricular (ICV) cannulation with osmotic pumps to deliver the compounds directly to the brain to avoid complications due to differences in blood-brain-barrier (BBB) penetrance, distribution, and metabolic stability. All compounds were loaded into pumps at a concentration of 10 µM pre-delivery. Animals were infused at a delivery rate of 1 µL per hour and sacrificed after 7 days. Brains were collected for immunoblotting (Fig. 1D). Three compounds, C40, C41 and C127, produced a statistically significant increase of PGRN protein similar to WT (Grn+/+) levels (Fig. 1E).

Pharmacokinetic profiling
Having established that C40, C41, and C127 were active in vivo when delivered directly to the brain, we next evaluated their pharmacokinetic (PK) profiles when given peripherally. Several in vitro experiments were conducted to predict drug absorption, distribution, metabolism, and excretion (ADME) (Supplementary Fig. 3; Supplementary Table 1). First, compounds were incubated with murine microsome S9 fractions to simulate liver metabolism. C40 and C127 had satisfactory half-lives of 72 min and 76 min respectively, while C41 was eliminated from further PK testing due to its low half-life of 8 min (Supplementary Fig. 3a). Next, plasma stability of the compounds was assessed by incubation in murine plasma and saline. Both C40 and C127 exhibited acceptable half-lives of 533 min and 139 min, respectively (Supplementary Fig. 3b). Finally, in the intestinal permeability assay, both compounds exhibited no signs of active transport into the intestinal lumen when compared to controls, and both displayed adequate permeability (Supplementary Table 1). C40 and C127 PK profiles were further evaluated in vivo. Treatment concentrations of 10 mg/kg were used due to reduced solubility of both compounds at higher concentrations. C40 and C127 were found to be BBB-penetrant after intraperitoneal (i.p.) injection (Supplementary Fig. 3c, d). The concentration peaked at similar time points for both compounds, but the level of drug absorption differed significantly. C40 plasma and brain maximum observed concentrations (Cmax) of 1498 ng/mL and 6246 ng/g, respectively, were significantly higher than that for C127 Cmax of 522 ng/mL and 1112 ng/g. However, both compounds demonstrated similar concentrations at 180 min. The Cmax was higher in the brain than in the plasma for both compounds, and the terminal half-life (t½) of each was similar at 59 min for C127 and 41 min for C40. Next, mice were dosed with C40 using oral gavage to compare per os (PO) vs. i.p. compound PK (Supplementary Fig. 3c, d). Oral t½ was longer than i.p. t½ at 65 min, and plasma and brain Cmax achieved 657 ng/mL and 1456 ng/g, respectively.

To evaluate enhancement of brain PGRN production in vivo, we treated Grn+/− mice i.p. with either 10 mg/kg C40 or vehicle. Whole mouse brains, which were harvested 24h after injection and evaluated by western blotting, revealed that C40 significantly increases PGRN protein with respect to vehicle treated Grn+/− controls (Fig. 1F, G). We reasoned that the efficacy of C40 and C127 might be limited by their ability to dissolve in solution in vivo (maximal dose of 10 mg/kg) and in vitro (maximal dose of 30 µM); therefore, we elected to produce analog (A) compounds to improve their activity and physicochemical properties.

Structure-Activity-Relationship (SAR) approach to increase solubility and activity
SAR mapping can be a valuable tool to improve lead compounds that do not yet possess optimal pharmacological properties. Thus, we conducted an approach to optimize activity and improve solubility relative to C40 and C127. We subdivided our lead structures into four parts: the core (benzoxazole scaffold), carboxamide linker, and two variable groups R1 and R2 (Fig. 2A). We chose R1 and 2 for subsequent modifications to preserve the core nature of C40 and C12787.

First, we substituted R1 with various moieties (Fig. 2B) and assessed all analogs for their GRN luciferase reporter activation efficacy from 10 nM to 30 µM over 48h of treatment. To compare our analogs with each other, we chose the lowest concentration (3 µM) at which most analogs saturated the GRN luciferase reporter assay (Fig. 2C).

Through this SAR mapping, we identified analog A03 which achieved the highest GRN activation (indicated by our luciferase assay). A03 had the highest conformational freedom at R1 among all analogs, a key property at this position. Since A03 also presented reasonable solubility in vitro assays, we chose this R1-modification as our subsequent lead compound to continue our optimization process by substituting R2 (Fig. 2D). GRN activation was measured for all synthesized A03 analogs and compared after 24h. (Fig. 2E). Most R2-substituted analogs (except A24, A28, A29) presented a similar or slightly higher activity compared to A03 at 3 µM (Fig. 2E).

During our optimization process, we evaluated the physicochemical properties of all analogs with a combination of experimental and computational approaches. These parameters describe the drug- and lead-likeness of a small molecule based on Lipinski’s Rule of Five and predict important characteristics for efficacy, solubility, and permeability88–90. Typical parameters to address are logS (aqueous solubility) and logP (lipophilicity) values. Both parameters are critical for accessibility and distribution of the drug within the system91–93. Our effort to optimize our compounds in terms of activity led in most cases to molecules with a higher logP value while maintaining or increasing the logS value compared to C40 (Fig. 2F). However, high logP compounds can suffer from poor solubility and off-target effects. Identification of lower logP compounds that maintain high activity remains a desirable goal.

Along with logS and logP, we also examined topological polar surface area (TPSA), molecular weight (MW), fraction of sp3 hybridized carbon atoms, and number of rotatable bonds using SwissADME94. For a molecule to be able to cross the blood-brain barrier, lipophilicity, MW, flexibility and TPSA are crucial parameters95,96. We conducted principal component analysis (PCA) (Fig. 2G) and hierarchical clustering (Fig. 2H) with these predicted parameters (including predicted BBB penetrance ability, availability as a P-gp substrate (i.e., the likelihood of being pumped out of the cell by p-glycoprotein1), and the measured luciferase assay activity) by using ClustVis97 to identify physicochemical differences and similarities between our analogs. All analogs could be subdivided into two groups, either having high TPSA scores and logS values or high lipophilicity and flexibility. Since A03 presented increased solubility compared to C40 and C127, we decided to further assess analogs with similar characteristics.

Compound analogs exhibit increased activity in vitro
To validate the results of the SAR-approach, twenty-six optimized analogs were evaluated in vitro over a dose range from 10 nM to 30 µM to establish luciferase activation (Supplementary Fig. 4a). Next, N2A cells were treated at four concentrations ranging from 1–30 µM for induction of PGRN protein expression. Based on western blot data and in line with our SAR-approach results, the four compounds that demonstrated the most robust up-regulation of PGRN protein in N2A cells were A21, A39, A40, and A41 (Supplementary Fig. 4b, c). Encouragingly, all four compounds displayed a higher activity at identical doses when compared to the initial hits C40 and C127, with an average fold-induction of 2.5–3.3x. We could not detect alterations in PGRN protein of intermediate lengths in our N2A treated cells (Supplementary Fig. 4D), although there is a possibility that our antibody was unable to bind to non-full length PGRN. Because PGRN is both a secreted and intracellular protein, we measured the relative levels of PGRN in N2A cell conditioned media and cell lysate after incubation with our compounds. We found that our compounds were able to increase PGRN in cell lysate but did not significantly increase extracellular PGRN in our in vitro model (Supplementary Fig. 5).

To determine if our four analogs were active in human cells, we treated U-373 MG Uppsala glioblastoma cells with 30 µM and 100 µM of each analog for 24h (Fig. 3A, B) using 100 µM A03 as a positive control. We evaluated our analogs in U-373 cells at 30 and 100 μM to test whether our analogs were more active than our original compounds and if they had increased solubility in media which would allow substantial increased treatment concentrations. A21, A40, and A41 induced between 2 and 7-fold increases in PGRN protein between the two tested concentrations.

We next validated our analogs in human dermal fibroblast (HDF) cells from one GRN control (HDFX) and two patients with GRN haploinsufficiency (HDFY and 2). HDF cells were treated for 24h using A21 and A41 at two concentrations and then harvested for mRNA and protein analysis. We utilized an antibody that has been shown to react to full-length human PGRN, as well as granulin fragments 2 and 3 (Fig. 3C, D)48. Evaluation of treated cells revealed that A21 and A41 significantly increased GRN mRNA and full-length PGRN in both sets of GRN mutant cells and granulin fragments 2, 3 in at least one set of GRN mutant cells (Fig. 3E). Further, A21 increased full length PGRN protein and GRN mRNA in a dose-dependent manner, while A41 reached its maximal effective concentration at 50 µM.

Compound analogs are non-toxic, rescue Progranulin levels in vivo, and have slow decay in vitro
Following the selection of active analogs of interest and verification of enhanced activity in vitro, we evaluated selected compounds in vivo. Increased stability of our analogs in solution allowed us to inject A21, A39, A40, and A41 i.p. at 50 mg/kg into Grn+/− mice. Whole brain was harvested 24h after injection and blotted for PGRN (Fig. 4A, B). Impressively, in Grn+/− mice A21 and A41 fully restored PGRN protein to Grn+/+ levels which C40 had failed to do (Fig. 4B and 1F, G). Since we are unable to treat C40 at higher concentrations, we cannot verify if the analogs have an elevated activity level at higher concentrations compared to C40, but the ability of our analogs to fully restore PGRN levels could be due to increased activity, increased stability in solution and subsequent increased dosing, or both. Importantly, the small molecule SAHA which we had previously shown corrects PGRN levels in vitro had no effect on brain PGRN levels when injected IP. This demonstrates a major advantage of our compounds over the previously published small molecule SAHA and suggests that our compounds represent a more feasible chemical basis for GRN-FTD therapeutic development.

We next aimed to test the safety of prolonged exposure to our analogs. To accomplish this, we injected Grn+/+ mice with 50 mg/kg A41 IP for 14 days. We measured the weights of A41 injected and vehicle injected mice daily and performed a complete blood count (CBC) analysis of all mice on day 14. We saw no noticeable changes in weight, grooming, energy, or overall external health after the 14-day treatment. In addition, CBC analysis did not detect any significant changes between A41 or vehicle treated mice. (Supplementary Fig. 5).

We further performed time course experiments (see Methods) to measure the persistence of the effect of our compounds on PGRN levels and found Grn mRNA and PGRN protein remained elevated in vitro for 12 and 16h, respectively, after drug removal (Fig. 4C, Supplementary Fig. 7a) and that PGRN protein level remained elevated in vivo up to 24h after treatment (Fig. 4D, E, Supplementary Fig. 7b). Lastly, we performed PK analysis of A21 and A41 after i.p. injection with 25 mg/kg of either compound. Both analogs had decreased Cmax compared to C40, and unlike C40, they did not preferentially partition into the brain; however, both A21 and A41 had much longer t½ compared to C40 (119 and 98 min, respectively) (Supplementary Fig. 3d). Most importantly, in A21 and A41, we achieved our goals to successfully improve on the stability of C40 in solution and use these analogs to significantly increase PGRN levels in vivo when compared to C40.

Progranulin-deficient cells demonstrate aberrantly lysosomal proteomes
While our compounds demonstrated robust ability to enhance PGRN levels, it was imperative to test their ability to correct a disease-relevant cellular phenotype. However, this required assessment of the general effect of PGRN haploinsufficiency in our model systems. Most of the work performed on PGRN biology centers on PGRN homozygous models, and any heterozygous models (i.e. models that best genocopy actual FTD patients) have displayed modest, if any, pathological phenotypes98,99. However, previous transcriptomic and lipidomic studies of GRN+/− patient and mouse samples pointed towards altered lysosomes23.

To exclusively investigate the lysosome of our model system, we performed a technique called LysoIP to specifically isolate lysosomes for downstream proteomic analyses100–102. Briefly, PGRN-deficient mouse embryonic fibroblasts (MEFs) or human-derived fibroblasts (HDFs) were engineered to express a lysosomal transmembrane protein, TMEM192, with a cytosolic 3xHA tag (Fig. 5A). These cells were then homogenized, intact lysosomes were isolated with anti-HA Dynabeads, and lysosomal proteins were extracted and subjected to tandem-mass-tag mass spectroscopy (TMT-MS) (Fig. 5B). LysoIP samples were checked for quality of extraction by western blot and post hoc Gene Ontology analyses of proteins obtained by TMT-MS (Fig. 5C, D, E)103. When compared to Grn+/+ MEFs, Grn+/− and Grn−/− lysosomes demonstrated 40/765 (5.23%) and 42/94 (44.7%) differentially regulated proteins (Fig. 5F, Supplementary Fig. 8a, Supplementary Data 3, 4). Interestingly, when plotting the relative changes versus Grn+/+ lysosomes for proteins detected in both Grn+/− and Grn−/− LysoIP analyses, a strong correlation (p<0.0001) appears between the two genotypes with a slope of ~1.5, suggesting complete PGRN loss affects the same proteins to an ~1.5-times greater extent than the loss of one copy of PGRN (Supplementary Fig. 8b). This demonstrates a possible gene dosage dependent effect of PGRN on the lysosomal proteome.

When compared to GRN+/+ HDFs, lysosomes from GRN haploinsufficient patient-derived fibroblasts demonstrated 260/1218 (21.3%) differentially regulated proteins (Fig. 5g, Supplementary Data 5). The difference in number of proteins analyzed between LysoIP analyses is most likely due to a difference in total protein amounts used between TMT-MS runs. We further performed TMT-MS on HDF whole-cell lysates (Supplementary Data 6) and found a strong correlation in changes between GRN+/+ and GRN+/− whole-cell lysate and LysoIP (Fig. 5H).

When comparing PGRN-haploinsufficient cell types, 16 proteins were significantly dysregulated (Fig. 5I). These proteins ensemble a broad functional spectrum, ranging from proteases (CTSA, CTSD, DPP7, and TPP1)104–109, glycosidases (GAA, GLA, MAN2B1, MANBA)110–112, and nucleases (DNASE2)113 to regulators of lysosomal cholesterol/lipid levels (NPC1 and EPDR1)114,115 and protein trafficking (IGF2R and SCAMP3)116,117. The wide range of lysosomal proteins altered in both mouse and human PGRN-deficient cells points to the function of progranulin as a key regulator of lysosomal homeostasis across species.

Progranulin-enhancing small molecules rescue the aberrant lysosomal proteome in FTD patient cells
Having discovered a robust lysosomal proteomic phenotype in PGRN haploinsufficient cells, we further tested the ability of one of our lead compounds, A41, to correct this phenotype.

First, we treated HDF cells from two separate GRN-FTD patients (i.e., PGRN haploinsufficient cells) with 50 μM A41 for 72h and measured the levels of cleaved (i.e., lysosomal) TPP1, a protein significantly decreased in both LysoIP and whole-cell lysate TMT-MS datasets. Mutations in TPP1 are the cause of another type of neuronal ceroid lipofuscinosis, CLN2, underlining its importance for lysosomal homeostasis and function105,107,118. TPP1 was decreased by ~50% in untreated haploinsufficient HDFs compared to controls, and treatment with A41 led to a substantial increase in TPP1 protein levels (Fig. 6A, B). To determine how A41 was inducing increased lysosomal protein levels, we performed RT-qPCR to measure mRNA in untreated HDFs and HDFs treated for 24 or 72h with 50 μM A41. We observed a time-dependent increase in GRN and TPP1 transcript levels as well as in DPP7 (a second lysosomal protease decreased in PGRN haploinsufficient HDFs) (Fig. 6C). Previous work has demonstrated the lysosomal regulator transcription factor EB (TFEB) is a regulator of PGRN transcription42. Because A41 was affecting PGRN transcription as well as the transcription of other lysosomal proteins, we tested whether it was functioning by activating TFEB. To accomplish this, we measured the nuclear localization (i.e., activation) of TFEB-GFP in a stably expressing 293T cell line upon addition of various concentrations of A41. Compared to DMSO control, A41 did not significantly increase the nuclear localization of TFEB until 100 μM, and this effect disappeared at 200 μM, suggesting there is no dose-dependence between A41 and TFEB activation (Fig. 6D, E). Furthermore, these results imply that A41 does not increase PGRN through TFEB since A41 is able to increase PGRN at levels lower than 100 μM.

To investigate the ability of A41 to correct lysosomal homeostasis, we treated Grn+/− MEF cells with 100 μM A41 for 24h prior to LysoIP. This concentration is sufficient to increase PGRN back to Grn+/+ levels in vitro. TMT-MS analysis of vehicle and A41 treated lysosomes demonstrated several key lysosomal proteins that are significantly altered in Grn+/− lysosomes are corrected upon treatment with A41 (Fig. 6F, J, Supplementary Data 7). Several of these proteins are key lysosomal catabolizing proteins that are mutated in various lysosomal storage disorders such as Tpp1, Manba, and Naga107,119,120. Interestingly, when comparing the relative changes versus Grn+/+ for proteins detected in every MEF LysoIP-TMT-MS experiment (Grn+/− with DMSO and A41 and Grn−/−) and performing principal component analysis (PCA), Grn+/+, Grn+/−, and Grn−/− all cluster separately, and A41 treated Grn+/− lysosomes cluster with Grn+/+ lysosomes (Fig. 6G). This demonstrates A41 is able to substantially correct the PGRN-deficient aberrantly lysosomal proteome.

To further demonstrate the ability of A41 to correct the PGRN-deficient aberrant lysosomal proteome, we performed unbiased LysoIP TMT-MS analysis on GRN+/− HDFs treated for 72h with 50 μM A41 compared to GRN+/+ HDFs. We then compared A41-treated GRN+/− HDF quantitative spectral count changes (vs GRN+/+ HDFs) to those in untreated GRN+/− HDFs (vs GRN+/+ HDFs) from our original LysoIP dataset and found significant differences in 287/547 (52.5%) proteins (Fig. 6H, K, Supplementary Data 8, 9). Of these, 147 proteins returned to GRN+/+ levels, demonstrating substantial correction of the aberrant GRN+/− lysosomal proteome with our PGRN-enhancing compound. PCA demonstrates the distinct clustering of GRN+/+ and GRN+/− proteomes as well as the correction seen with A41 treatment (Fig. 6I), adding further evidence to the validity of our compounds as therapies for GRN-FTD.

Methods
Inclusion and ethics statement
All required permissions were obtained from institutional review boards.

Experimental model and subject details
Cell Lines. All cell culture reagents and plates were purchased from Corning (Corning, NY, USA). Cells were maintained at 8.8% CO2 at 37°C. HEK293T 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 husbandry
The 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/6J 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 12h 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).

Compounds
All 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 screening
The 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 human 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 4h after plating and incubated for 24h 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. Bright Glo™ (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)

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)

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 assay
All 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 treatment. Neuro2A, 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 24h 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 3kDa cutoff (EMD Millipore, UFC500308). Lysate and concentrated conditioned media were then analyzed by western blot.

ICV cannulation
All 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/6J 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. injections
66 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 datasets.

Immunoblotting
All 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 1h in 5% BSA in TBS with 0.1% Tween 20 for 1h. Membranes were incubated with proper primary antibody solutions on a rocking shaker overnight at 4°C. Nitrocellulose membranes were incubated in fluorescent secondary for 1h and visualized using the Odyssey Imaging System (Li-cor, Lincoln, NE, USA). PVDF membranes were incubated for 2h 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 3724S, Lot 10); Anti-mouse CD107a 1:1000 dilution (BD Biosciences 553792, Lot 0000055197); Anti-EEA1 1:1000 dilution (Cell Signaling 3288S, Lot 8); Anti-Rab7 1:1000 dilution (Cell Signaling 9367S, Lot 3); Anti-GM130 1:1000 dilution (Thermo Fisher MA5-35107, Lot XD3555932); Anti-COXIV 1:1000 dilution (Cell Signaling 4850S, Lot 10); Anti-Lamin A+C 1:1000 dilution (Abcam ab133256, Lot 7); Anti-Calnexin 1:12,000 dilution (Cell Signaling 2679S, 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 fractions
Compound 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 table top, 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 110916
Ion 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 stability. Compound (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 table top, 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 110916
Ion 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 assay
Caco2-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 HBSS 0.5% 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 4000 Trap.

Caco2 Controls Prop Nal Quin Cim 031916.dam for control compound
MS 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 113016 Ion 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 113016
Ion 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 count
Whole 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 PCR
Total cellular RNA was isolated from cells using Trizol (Invitrogen, Waltham, MA, USA). and diluted 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 (Thermo-Fisher, Waltham, MA, USA). Cyclophilin and h36B4 were used for normalization.

TFEB activation assay
293T cells stably expressing TFEB-GFP were plated on coverslips and treated for 24h 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 24h and media was replaced by Earle’s balanced salt solution for 4h. 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 production
For lentivirus production, 70% confluent 15 cm plates of HEK293T 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 12–16h. 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 24h.

LysoIP
MEFs 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 24h, and 48h 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 (Thermo Fisher, 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 (Thermo Fisher, Waltham, MA, USA, 3451).

Tandem mass tag mass spectrometry
Samples 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 (Thermo Fisher, 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 (Thermo Fisher, 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 (Thermo Fisher, 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 side chains 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 results
For 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 analysis
Data 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.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors first screened a 200K library for transcriptional activators of a progranulin\npromoter-luciferase in N2 cells. Through a series of secondary screens, they narrowed their\nlarge hit list and settled upon a promising candidate, C40. Through SAR, they optimized their\nlead candidate for a number of criteria, including solubility, lipophilicity, and efficacy. They\nfurther test the analogs A21 and A41 compounds in a Grn+/- mice after single and multiple\ntreatments, in a time course, for PK and lysosomal improvement. For the latter, this consists\nof proteomic normalization of isolated lysosomes after compound treatment.\nThere are many positive aspects to this study, including the impressive initial screening\neffort, the SAR to improve the lead candidates, the multiple follow-up screens and rigorous\nuse of different cell and mouse models. However, there are issues that limit the overall\nimpact and interpretability of this study.\n1) The screening effort, while impressive in scope, only will identify transcriptional activators\nof PGRN, rather than protein stabilizing molecules. This should be acknowledged in the\nmanuscript.\n2) The Western blots only show a single progranulin band. Often, there are multiple\nprogranulin bands and it is particularly important to know if cleavage fragments are seen\nwith the compounds. Full blots should be included. In addition, the quantification in Figure\n3D does not make it clear if the full-length or cleaved progranulin is being measured. Again,\nthe full Western blots should be included.\n3) Additional characterization of the progranulin produced, including what portion is\nsecreted and/or cleaved, would be useful.\n4) While the improvement in lysosomal protein profile after A41 treatment is impressive,\nit is not an actual functional improvement in lysosomal function. The ability of compounds to\nnormalize progranulin functional outcomes would further improve the manuscript.\n5) On page 11, the conclusion that A41 is “safe” is an overstatement of the experimental\nresults.", "reviewer_2": "Upon the heterozygous loss-of-function mutations in the GRN gene as a major cause of FTD\nand potential function of small molecules on regulation of GRN expression reported in\nliterature, the authors here described the finding of a few small molecules by both virtual\nscreening and practical preparation which were used to enhance PGRN levels from the\nremaining functional GRN allele within the brain, with an attempt to develop small molecule\ndrugs for the treatment of FTD. Compared with the earlier work to boost PGRN levels in FTD\nmodels, application of these small molecules could effectively restore PGRN levels back to\nnormal in vitro and in vivo, and protect against the neurodegeneration caused by PGRN loss.\nThey have carefully made the research and provided substantial chemical and biological\nevidences to support their conclusions. In my opinion, the work is of much significance and\nwell organized, which can be accepted for publication in this journal after addressing the\nfollowing points.\n1. As other researchers have also reported that increasing the expression of progranulin\nprotein levels will be therapeutic to FTD previously, it is unnecessary for the authors to\nexaggerate their hypothesis that “restoring PGRN levels back to normal will protect\nagainst the neurodegeneration caused by PGRN loss”.\n2. High resolution ESI-MS data should be provided to ensure the accuracy of all the new\ncompounds. Moreover, purity of representative compounds like compounds C40, C41, C127\nand analogs A21, A39, A40 and A41 should be >95% as detected by HPLC.\n3. In the first in vitro test, nine compounds were found to be capable of increasing PGRN\nprotein to approximately the target value compared to vehicle levels, however, only three\ncompounds (C40, C41, and C127) enhanced PGRN levels in an in vivo model of FTD with a\nstatistically significant increase. It would better to make SAR analysis on the difference of in\nvitro and in vivo tests among these nine compounds.\n4. In the analog compounds of C40, no alkyl substituents for R2 were used to take the place\nof a phenyl group, while alkyl substituents for R1 were tried to replace a furanyl moiety.\nPlease give the reason. Besides, did the authors consider the substitution oppositely?\n5. They attributed the limited efficacy of C40 and C127 by their ability to dissolve in solution\nin vivo and in vitro. But I do not think the analog compounds A21 and A41 containing an\nalkyl chain showing higher activity in contrast to their parent compounds can only be due to\nthe solubility. In fact, as they stated later that a molecule to be able to cross the blood-\nbrain-barrier (BBB), lipophilicity, MW, flexibility and TPSA are crucial parameters for\ndeveloping drugs to cure diseases in brain.\n6. Compound A03 should be used as positive control in the determination of activity of four\nanalogs A21, A39, A40 and A41 in PGRN protein of U-373 MG glioblastoma cells in Figure 3. I\nwonder why they treated U-373 MG cells at such a high concentration (100 μM) of each\nanalog? Besides, in the following HDF cell tests, SAHA should be used as reference.\n7. On page 11, the authors summarized that the analogs were more active in vivo compared\nto the original compounds, possibly due to increased stability and subsequent increased\ndosing. This is contradictory to what they claim on page 8. Please explain about it.\n8. In validating analogs in human HDF cells from FTD patients, HDF cells were treated using\nA21 and A41for 24 hours for mRNA and protein analysis. While in measuring the levels of\ncleaved TPP1, HDF cells from FTD patients were treated using A41 for 72 hours. Please state\nwhy big different incubation time was adopted.\n9. In the mechanistic study, molecular docking between GRN protein and C40 and its\nanalogs (A21 and A41) are highly recommended to undertake if the GRN in PDB is available.\nIn addition to TFEB activation, further pharmacological mechanism of compound A41\nreversing an aberrant lysosomal proteome phenotype in PGRN-deficient models should be\nexplored as PGRN-regulated signaling pathways and gene knockout analysis.\n10. The safety profile of A41 needs to be further investigated as histopathological evaluation\nof brain tissue and several neurotoxicity assays.\n11. In Figure S1, please explain why non-small cell lung cancer (NSCLC) cell lines were used\nto assess the toxicity of the compounds.\nBesides, there are some minor errors or shortcomings needing to be corrected.\n(i) In Figure 1B, the protein bands of β-actin were inconsistent in C107-treated N2A cells,\nwhich could account for some observed changes of experimental condition, please be\nconsistent. Also, the protein bands in Figures 1D and 1F were not clear compared to these in\nFigure 1B, which need to be explained and rectified.\n(ii) On page 10, “Figure S2B” was probably cited in the incorrect place. Page 11, line 3, it was\nA40, not C40.", "reviewer_3": "In this manuscript the authors provide a description of a relatively thorough screening\nparadigm to identify small molecule therapeutic candidates intended to boost PGRN\nproduction in patients with GRN hapoloinsuficiency. Using a N2A cell model with a PGRN\ndependent luciferase promotor, they screened about 200,000 compounds for candidates\nthat robustly upregulated PGRN, then screened candidate toxicity using NSCLC cell line and\nATP-based assay, and selectedcandidates that boosted brain parenchymal PGRN after\ninterventricular introduction in a mouse model (though the mouse line is not clearly stated).\nNext, two candidates (C40 and C127) where assessed using in vitro assessments of liver\nmetabolism, plasma stability, and intestinal permeability, followed by en vivo assessments\nof BBB permeability after intraperitoneal injections in mice. The brain Cmax C40 was the\nreassessed after oral gavage (confirming a lower Cmax and long tmax). Finally, the PD effect\nof intraperitoneal C40 and C127 was assessed in sacrificed Grn-/+ mice using western blot\nassessments of whole brain samples. Of note their inital two primary candidates had limited\nsolubility in solution, so a SAR-mapping approach was then used to optimize for solubility\nand BBB permeability. Two resulting candidates A21 and A41 upregulated PGRN in their\nN2A cell model, U-373 Upsala Glioblastoma cells, and finally in fibroblasts reportedly derive\nfrom patients with GRN haploinsufficiency. IP delivery of both new candidates reportedly\nnormalized PGRN in Grn-/+ mice and toxicity was not detected for A41. In contrast to C40\nthe candidates did not however partition preferentially to the brain and they had a small\nCmax (albeit a longer tmax). Finally, using an tandem mass-spec proteomic approach, the\nauthors identified a proteomic signature in GRN haploinsufficient fibroblasts and mouse cell\nlines (relative to respective in species controls) and verified the ability of A41 to ameliorate\nproteins withing this signature, particularly proteins relevant to lysosomal function.\nOf note, there appears only one compound that underwent testing of oral bioavailability,\nC40, and this compound was apparently unable to normalize PGRN levels in Grn-/+ mice.\nThe other promising compounds do not appear to have oral bioavailability data. This is a\nworthwhile point because oral bioavailability likely adversely impacted the efficacy of FRM-\n0334, an HDAC inhibitor that was selected for previous human trial in GRN\nhaploinsufficiency (Ljubenkov et al). Despite optimization, some of the analog compound\ncandidates, like A41, appear to less-readily cross the blood brain barrier, so there are still\nongoing questions about its viability in future development\nIn the body of the text is sometime ambiguous what patients the fibroblasts were derived\nfrom. The authors should always explicitly state if each patient is symptomatic or\nasymptomatic and explicitly states if they do or do not have haploinsufficiency.\nThere appear to be multiple editorial comments in the discussion of the history of earlier\ntrials and the about the relative merits of different progranulin-boosting strategies. For\ninstance the author’s include the statement “delivery of viruses and proteins to all regions\nof the human brain presents significant challenges compared to administration of small\nmolecules.” Of note, in-human trials of small molecules to date (see Sha et al regarding\nnimodipine and Ljubenkov et al regarding HDAC inhibitors) have so far failed to show\nevidence of a pharmacodynamic impact on CSF PGRN.\nThe authors do not address the necessary next steps before their candidate compounds can\nmove into trials. While 4 interesting candidates have been identified it seems premature to\nconclude these compounds will “form a basis for the development of a feasible and cost-\neffective approach to the treatment of FTD caused by GRN haploinsufficiency”. These drugs\nstill need better defined oral bioavailability and PD data in other mammalian models,\nincluding non-human primates, before moving into human trials.", "reviewer_4": "GRN mutations are believed to contribute to the development of frontotemporal dementia\nby causing progranulin haploinsufficiency. Consequently, enhancing progranulin production\nfrom the unaffected allele appears to be a plausible approach for treatment.\nThe researchers employed luciferase-reporter-assays to conduct high-throughput screens,\nwhich enabled them to identify several small compounds capable of restoring the\nexpression level of PGRN in both in vitro and in vivo settings. SAR methodologies were\nadditionally employed to enhance the efficacy and certain pharmacological characteristics\nof these molecules. Furthermore, an investigation was conducted to assess the\neffectiveness of increasing progranulin (PGRN) levels in rectifying the abnormal lysosomal\nproteome in cells from patients with GRN-FTD. This was achieved by employing the LysoIP\nmethod in conjunction with lysosomal proteomics.\nI support the publication and congratulate them on this work!"}, "author_response": "Response to reviewers\nReviewer 1:\n1) The screening effort, while impressive in scope, only will identify transcriptional\nactivators of PGRN, rather than protein stabilizing molecules. This should be\nacknowledged in the manuscript. We thank the reviewer for this comment and have\nreworded the manuscript accordingly to address this. “...moved forward to develop\nnovel non-toxic and reliable enhancers of PGRN both in vitro and in vivo. We also\naimed for transcriptional activators rather than compounds which stabilize PGRN\nprotein to ensure proper PGRN protein function. To accomplish this objective, we...”\nOur goal was to identify transcriptional activators to take advantage of the residual\nfunctional allele and thereby restore normal PGRN mRNA levels without altering protein\nmaturation, transport, etc.\n2) The Western blots only show a single progranulin band. Often, there are multiple\nprogranulin bands and it is particularly important to know if cleavage fragments are seen\nwith the compounds. Full blots should be included. In addition, the quantification in\nFigure 3D does not make it clear if the full-length or cleaved progranulin is being\nmeasured. Again, the full Western blots should be included.\nFull length blots of HDF and N2A treated cells were added to Figure 3 and Figure S4 to\nfurther clarify questions about changes in full-length vs. cleaved progranulin.\n3) Additional characterization of the progranulin produced, including what portion is\nsecreted and/or cleaved, would be useful.\nWe have included a comparison of secreted versus internalized progranulin with and\nwithout our compounds in N2A cells and found that while internalized progranulin is\nincreased, there is no consistent change of progranulin concentrations in the media\n(Supplemental Figure 5). However, our compounds increased both full length\nprogranulin and cleaved granulins presumed to reside in the lysosome where they are\nthought to mediate their biological functions. To this extent, there is also evidence that\nobligate lysosomal progranulin was able to recapitulate the neurotrophic effects of wild\ntype progranulin the absence of extracellular progranulin, suggesting internal, not\nexternal progranulin is the primary role of progranulin function.\n4) While the improvement in lysosomal protein profile after A41 treatment is impressive,\nit is not an actual functional improvement in lysosomal function. The ability of\ncompounds to normalize progranulin functional outcomes would further improve the\nmanuscript.\nThis is a very good point, but unfortunately typical differences are only seen in GRN-/-\nmodels where we cannot test our compounds for improvement since our compounds\nare PGRN transcriptional activators, and no functional allele is present. Heterozygous\nanimal model systems do not present with robust and consistent differences compared\nto WT with the exception of those used in this manuscript.\n5) On page 11, the conclusion that A41 is “safe” is an overstatement of the experimental\nresults.\nWe updated the manuscript language to avoid overstating the results of our toxicity\nassessments.\nReviewer 2:\n1. As other researchers have also reported that increasing the expression of progranulin\nprotein levels will be therapeutic to FTD previously, it is unnecessary for the authors to\nexaggerate their hypothesis that “restoring PGRN levels back to normal will protect\nagainst the neurodegeneration caused by PGRN loss”. We appreciate this reviewer’s\npoint and have corrected our manuscript to avoid exaggerating our hypothesis.\n2. High resolution ESI-MS data should be provided to ensure the accuracy of all the\nnew compounds. Moreover, purity of representative compounds like compounds C40,\nC41, C127 and analogs A21, A39, A40 and A41 should be >95% as detected by HPLC.\nWe have included these data in the revised manuscript.\n3. In the first in vitro test, nine compounds were found to be capable of increasing\nPGRN protein to approximately the target value compared to vehicle levels, however,\nonly three compounds (C40, C41, and C127) enhanced PGRN levels in an in vivo\nmodel of FTD with a statistically significant increase. It would better to make SAR\nanalysis on the difference of in vitro and in vivo tests among these nine compounds.\nThe reviewer is referring to our analysis of initial hits from our primary screen. The data\ndescribed in Figure 1 reflect our efforts to prioritize compounds from the HTS to select\nones with in vivo activity for further profiling. These 9 compounds represent 8 different\nchemical scaffolds (C40 and C127 are analogs). A structural analysis across 8 distinct\nchemical scaffolds would not be informative, Accordingly, the data suggested that C40\nand C127 were suitable for additional study, as described in the remainder of the text.\n4. In the analog compounds of C40, no alkyl substituents for R2 were used to take the\nplace of a phenyl group, while alkyl substituents for R1 were tried to replace a furanyl\nmoiety. Please give the reason. Besides, did the authors consider the substitution\noppositely?\nA high priority for the program was to replace the furanyl group because furans can be\nenzymatically oxidized to form reactive, and therefore potentially toxic, metabolites. For\nthis reason, we aggressively modified the amide substructure with aryl and alkyl groups.\nThe optimization process was iterative, and we identified suitable replacements for the\nfuran. Accordingly, we initially limited our changes to R2 to focus on aryl rings because\na) the chemistry to provide the benzoxazole is more reliable with R2 = aryl, and b) early\nanalogs showed a robust SAR with substituted (hetero)aryl rings. Once we found\ncompounds with promising properties, we started to evaluate those in preclinical\nmodels, while starting the iterative process again to synthesize more different analogs.\nOngoing research will continue to explore the R2 group to further improve potency and\nfacilitate target identification.\n5. They attributed the limited efficacy of C40 and C127 by their ability to dissolve in\nsolution in vivo and in vitro. But I do not think the analog compounds A21 and A41\ncontaining an alkyl chain showing higher activity in contrast to their parent compounds\ncan only be due to the solubility. In fact, as they stated later that a molecule to be able\nto cross the blood-brain-barrier (BBB), lipophilicity, MW, flexibility and TPSA are crucial\nparameters for developing drugs to cure diseases in brain.\nWe appreciate this comment and have addressed this language in the manuscript text.\nIn brief, the analog compounds A21 and A41 show higher activity at the same\nconcentrations in N2A cells (Figure S4B), so we can see that in this assay C40 and\nC127’s ability to dissolve in solution is not a factor in its increased activity. The same is\ntrue of the 30 µM treatments in the U-373 MG glioblastoma cells. We have edited the\ntext to reflect that since we are unable to treat C40 and C127 at higher concentrations\nwe cannot say for sure if our analogs’ increased PGRN induction in the in vivo and in\ncell culture experiments is due to increased activity, ability to dissolve in solution (and\nthus an increase in dosing), or a combination of both.\n6. Compound A03 should be used as positive control in the determination of activity of\nfour analogs A21, A39, A40 and A41 in PGRN protein of U-373 MG glioblastoma cells in\nFigure 3. I wonder why they treated U-373 MG cells at such a high concentration (100\nμM) of each analog? Besides, in the following HDF cell tests, SAHA should be used as\nreference.\nStudies were conducted using 4 successive doses from 100 nM, 300 nM, 1 µM, 3 µM, 10\nµM, and 30 µM, so 100 µM would be the next step in that dosing chain.\nSince 30µM treatments of C40 and C127 were unable to induce PGRN increases in any\nhuman cell lines tested (data not shown) and 100 µM treatments were not possible\nbecause the compound was not form stable solutions in media, evaluating the analogs in\nthe U-373 cells at 30 μM and 100 μM would allow us to verify whether our new analogs\nwere more active than our original compounds and if they had increased stability in media\nwhich would allow substantial increases treatment concentrations. This reasoning has\nbeen added to the manuscript.\nWe used progranulin enhancing HDAC inhibitors as positive controls throughout the\nscreening process. Our goal was to identify drugs that could restore WT progranulin\nexpression levels (i.e., to increase progranulin levels by approximately 2-fold in\nheterozygous cells). Therefore, we used changes from baseline expression without\nknown drugs as our reference point. Since SAHA is not a therapeutic candidate, we did\nnot use it as a reference point for further analysis. This study's purpose was not to identify\ncompounds more potent than SAHA but to identify alternative small molecules with\nsufficient in vitro and in vivo activity to restore normal progranulin levels. This point has\nbeen added to the manuscript.\n7. On page 11, the authors summarized that the analogs were more active in vivo\ncompared to the original compounds, possibly due to increased stability and\nsubsequent increased dosing. This is contradictory to what they claim on page 8.\nPlease explain about it.\nThe manuscript has been updated to better explain this: “Since we are unable to\nadminister C40 at higher concentrations, we cannot test if the analogs have an elevated\nactivity level at higher concentrations [in vivo] compared to C40, so the ability of our\nanalogs to fully restore PGRN levels could be due to increased activity, increased\nstability in solution and subsequent increased dosing, or both.”\n8. In validating analogs in human HDF cells from FTD patients, HDF cells were treated\nusing A21 and A41 for 24 hours for mRNA and protein analysis. While in measuring the\nlevels of cleaved TPP1, HDF cells from FTD patients were treated using A41 for 72\nhours. Please state why big different incubation time was adopted.\nTo measure downstream effects of progranulin amplification, we first monitored rising\nprogranulin levels over 24 hours, as seen in our time course studies (Figure 4C). After\nthis, our logic was to provide the cells with 48 hours additional dosing so they could\nreach a new steady state in response to this increase in progranulin levels before\ndetermining the new steady state proteomic signature (including TPP1 levels) in our\ncellular models.\n9. In the mechanistic study, molecular docking between GRN protein and C40 and its\nanalogs (A21 and A41) are highly recommended to undertake if the GRN in PDB is\navailable. In addition to TFEB activation, further pharmacological mechanism of\ncompound A41 reversing an aberrant lysosomal proteome phenotype in PGRN-deficient\nmodels should be explored as PGRN-regulated signaling pathways and gene knockout\nanalysis.\nCompound binding to GRN cannot be ruled out at this point, but we have no reason to\nbelieve that our compounds directly interact with GRN since we are looking for\ncompounds that increase GRN transcription rather than stabilizing the protein itself.\nAdditionally, we aim to increase PGRN from its remaining functional allele in\nheterozygous systems, thus studying knock-out systems should have no effects, but we\nagree that it would be a great control to pinpoint the effect of our compounds solely on\nPGRN increase rather than systemic effects independent of PGRN increase.\n10. The safety profile of A41 needs to be further investigated as histopathological\nevaluation of brain tissue and several neurotoxicity assays.\nWhile we performed a basic, prolonged in vivo toxicity assessment of overall external\nhealth, weight and CBC profiling, more complex histopathological and neurotoxicity\nevaluations will be pursued once the compound structure has been further refined and\nreached an advanced stage of development.\n11. In Figure S1, please explain why non-small cell lung cancer (NSCLC) cell lines were\nused to assess the toxicity of the compounds.\nThe toxicity screen in the manuscript was performed by the High Throughput Screening\nCore at UT Southwestern Medical Center. The original goal was to optimize the library\nscreening assays using NSCLC cell lines, so these cell lines were used for initial toxicity\nstudies. The screening core does not assess compound toxicity in each cell line used in\ntesting in order to conserve key resources.\nBesides, there are some minor errors or shortcomings needing to be corrected.\n(i) In Figure 1B, the protein bands of β-actin were inconsistent in C107-treated N2A\ncells, which could account for some observed changes of experimental condition,\nplease be consistent. Also, the protein bands in Figures 1D and 1F were not clear\ncompared to these in Figure 1B, which need to be explained and rectified.\nThis change in β-actin was C107 specific and consistent between blots. Since this\ncompound was not further pursued due to lack of in vivo activity, the reasons for the\ndose dependent decrease in cellular β-actin were not explored.\nThe bands in Figures 1D and 1F are consistent with each other because the samples\nare from mouse brains. In order to visualize bands from our mouse brain lysates we\nutilize a different blotting protocol from the N2A lysate protocol (seen in Figure 1B) (see\nmethods for detailed differences). Also, higher concentration of primary antibody are\nnecessary to visualize the target protein in brain lysate, which in turn increases the\nbackground of the blots. We therefore would be unable to harmonize the appearance of\nthe blots due to the inherent difference in experimental procedures.\n(ii) On page 10, “Figure S2B” was probably cited in the incorrect place.\nPage 11, line 3, it was A40, not C40. We thank this reviewer for pointing out this error. It\nhas been corrected in the manuscript.\nReviewer 3:\nOf note, there appears only one compound that underwent testing of oral bioavailability,\nC40, and this compound was apparently unable to normalize PGRN levels in Grn-/+\nmice.\nThe other promising compounds do not appear to have oral bioavailability data.\nThis is a worthwhile point because oral bioavailability likely adversely impacted the\nefficacy of FRM-0334, an HDAC inhibitor that was selected for previous human trial in\nGRN haploinsufficiency (Ljubenkov et al). Despite optimization, some of the analog\ncompound candidates, like A41, appear to less-readily cross the blood brain barrier, so\nthere are still ongoing questions about its viability in future development.\nOral availability, blood-brain barrier permeability, and other relevant pharmacokinetic\nproperties of this compound and its derivatives will certainly have to be continuously\nassessed and optimized at later stages of development.\nIn the body of the text is sometime ambiguous what patients the fibroblasts were\nderived from. The authors should always explicitly state if each patient is symptomatic\nor asymptomatic and explicitly states if they do or do not have haploinsufficiency.\nThe genotype of each patient has been clarified in the text and figures, but the de-\nidentified data from the UCSF Memory and Aging Center did not include information\nabout the clinical status or symptoms in control or haploinsufficient patients due to\nprivacy regulations.\nThere appear to be multiple editorial comments in the discussion of the history of earlier\ntrials and the about the relative merits of different progranulin-boosting strategies. For\ninstance the author’s include the statement “delivery of viruses and proteins to all regions\nof the human brain presents significant challenges compared to administration of small\nmolecules.” Of note, in-human trials of small molecules to date (see Sha et al regarding\nnimodipine and Ljubenkov et al regarding HDAC inhibitors) have so far failed to show\nevidence of a pharmacodynamic impact on CSF PGRN.\nIn terms of HDAC inhibitors, the findings from Ljubenkov et al are consistent with our\nfindings which showed no effects of SAHA on brain progranulin levels (Figure 1E). Of\ncourse, CSF progranulin will need to be measured in humans. Progranulin\nmeasurement in mouse CSF is experimentally challenging, therefore we analyzed\nprogranulin levels in whole brain lysate.\nThe authors do not address the necessary next steps before their candidate compounds can\nmove into trials. While 4 interesting candidates have been identified it seems premature to\nconclude these compounds will “form a basis for the development of a feasible and cost-\neffective approach to the treatment of FTD caused by GRN haploinsufficiency”. These drugs\nstill need better defined oral bioavailability and PD data in other mammalian models,\nincluding non-human primates, before moving into human trials. We appreciate this comment and have addressed this accordingly in the\nmanuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Overall, we feel that the authors have addressed our concerns and further improved the\nmanuscript in response to the other reviewers. One minor point: to avoid confusion\nbetween progranulin cleavage fragments and the gene GRN, an alternative to “GRN” should\nbe used when referr to to granulins, in particular in figures. Cleavage product, PGRN\nfragment or even gran would be more clear.", "reviewer_2": "In the revised manuscript, the authors have given some reasonable answers or explanations\nto my concerns, but I am still not satisfied with parts of their replies, because it seems that\nthey have ignored a few of my requirements to provide extra data or make further study to\nsupport their work. For instance, it is necessary to offer high resolution ESI-MS data of the\nnew compounds and the purity of representative compounds by HPLC, but I cannot find the\ndata in the manuscript. As for point 4, their explanations, though acceptable, should be\ngiven in the text. In point 6, I insist that A03 should be used as positive control in the\ndetermination of activity in PGRN protein of U-373 MG glioblastoma cells, because A21,\nA39, A40 and A41 are its analogues. Finally, in the mechanistic study, they do not want to\nstudy the interaction of the compounds with GRN “since we are looking for compounds that\nincrease GRN transcription rather than stabilizing the protein itself”. For the reason they\nclaim, I believe they should undertake molecular docking to investigate the possible\ninteractions between GRN protein and C40 and its analogs (A21 and A41) to see whether\ntheir claim is true or not.\nOverall, I do not think the manuscript can be accepted before the authors address my all\nconcerns."}, "author_response": "Response to Reviewers:\nReviewer 1:\nOverall, we feel that the authors have addressed our concerns and further improved the manuscript\nin response to the other reviewers. One minor point: to avoid confusion between progranulin cleavage\nfragments and the gene GRN, an alternative to “GRN” should be used when referr to to granulins,\nin particular in figures. Cleavage product, PGRN fragment or even gran would be more clear.\nResponse:\nThank you for raising this point. To remove this ambiguity, processed GRN peptides have been\nrelabeled GRN 2/3 in the figures and are referred to as GRN fragments 2 and 3 in the text. This\nnomenclature reflects the specificity of the antibody and more precisely indicates what part of the\nprocessed PGRN is being detected.\nReviewer 2:\nIn the revised manuscript, the authors have given some reasonable answers or explanations to my\nconcerns, but I am still not satisfied with parts of their replies, because it seems that they have ignored a\nfew of my requirements to provide extra data or make further study to support their work. For instance, it\nis necessary to offer high resolution ESI-MS data of the new compounds and the purity of representative\ncompounds by HPLC, but I cannot find the data in the manuscript. As for point 4, their explanations,\nthough acceptable, should be given in the text. In point 6, I insist that A03 should be used as positive control in\nthe determination of activity in PGRN protein of U-373 MG glioblastoma cells, because A21, A39, A40 and\nA41 are its analogues. Finally, in the mechanistic study, they do not want to study the interaction of the\ncompounds with GRN “since we are looking for compounds that increase GRN transcription rather than\nstabilizing the protein itself”. For the reason they claim, I believe they should undertake molecular docking\nto investigate the possible interactions between GRN protein and C40 and its analogs (A21 and A41) to\nsee whether their claim is true or not.\nResponse:\nWe apologize for inadvertently omitting the HPLC and high resolution mass spectrometry data in\nour previous submission. They are now included as supplementary data.\nUsing A03 as a positive control: We had to resynthesize A03, because this was an obsolete\ncompound that was not further pursued for various reasons. We are uncertain why the reviewer thought\nthat using this discarded compound as a positive control was essential, but we have nevertheless repeated\nthe relevant experiments according to these wishes. The essentially identical reproduced results are now\nshown together with A03 as the positive control. The results naturally do not change the conclusions.\nMolecular docking studies were performed as described below. However, we are also not certain of the\nsignificance of doing this, since our compounds were selected as transcriptional enhancers of GRN\nexpression. There is no basis to assume that these compounds should interact with either PGRN or one\nof the processed granulins. We have no reason to believe that this is the case. Moreover, no\nexperimentally validated full-length PGRN structure is available. We therefore used two of the available\nmolecular docking programs (Autodock 4 and Neurosnap) to computationally dock our preferred and most\nactive compound A41 to the Alphafold human PGRN structure. As expected, no specific interaction was\nidentified. For these reasons, these data were not included in the manuscript.\nWe have further modified the text according to the reviewer’s requests. All changes and additions\nare highlighted in the revised text.\nFigure: Docking of A41 (red) to PGRN (grey) AlphaFold Model (AF- P28799-F1) by using A AutoDock4 or\nB the online tool Neurosnap resulted in random binding of A41 to PGRN.\nAll identified compounds in this manuscript were found in a screen for transcriptional regulators of PGRN.\nBased on our luciferase based assay, where luciferase is coupled to the GRN promotor, we found our\ncompounds to transcriptionally increase progranulin levels. No part of the screen was designed to identify\nmolecules that directly interact with progranulin. We have no evidence that A41 binds directly to\nprogranulin. However, out of deference to the reviewer, we attempted docking to human progranulin.\nAutoDock4 (AutoDockTools-1.5.7) was used to dock A41 to AlphaFold model of full-length human\nprogranulin (AF-P28799-F1). A blind docking approach was used where the complete protein was included\nin the grid box without predefining a binding pocket. Grid-based energy evaluation and all available\nrotational degrees of freedom for the small molecule were included by AutoDock4 using empirical free\nenergy scoring function and Lamarckian Genetic Algorithm. Additionally, we used an online platform,\nNeurosnap (https://neurosnap.ai/), to cross-validate our findings from AutoDock4. Here, DiffDockL was\nimplemented and used to dock A41 to the AlphaFold model of progranulin.\nNo preferred binding pocket was found and only unspecific binding occurred all over the structure with\nboth programs, thus this experiment revealed no compelling docked poses for A41 on progranulin. In the\nabsence of experimental or computational data supporting a direct A41-progranulin interaction, we have\nnot revised the text to discuss this potential mechanism."}, {"round": 3, "reviewer_comments": {"reviewer_2": "In this version of manuscript, the authors have offered sufficient data and extra test results\nto support their claims, which dispel my doubts. The manuscript can be now accepted for\npublication as is."}, "author_response": ""}]

Decision letter