Identification of KHS-101 as a Transcription Factor EB Activator to Promote α-Synuclein Degradation
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript identifies KHS-101 as a novel and brain-penetrant activator of TFEB and provides a well-structured and convincing set of cellular data demonstrating enhanced lysosomal function, increased autophagic activity, and accelerated clearance of pathogenic A53T α-synuclein. The experimental design is logical, the methodologies are appropriate and carefully executed, and the conclusions are, overall, well supported by the data. The study will be of interest to researchers working on autophagy–lysosome biology and neurodegenerative diseases.
The manuscript is generally well written and technically sound. I have only a small number of minor comments that, if addressed, would further strengthen the clarity and rigor of the study.
Minor comments
- Assessment of autophagic flux (Figure 3)
While the authors convincingly demonstrate increased LC3-II accumulation and its further elevation upon chloroquine co-treatment, the assessment of autophagic flux would be strengthened by including p62/SQSTM1 protein levels. Given that p62 is both transcriptionally upregulated by TFEB and selectively degraded through autophagy, immunoblot analysis of p62 would help distinguish enhanced autophagic flux from altered synthesis. Including p62/SQSTM1 protein data in Figure 3 would further support the authors’ conclusion. - Clarification of the role of chloroquine
Although chloroquine is widely recognized as a late-stage autophagy inhibitor, explicitly stating in the text that chloroquine blocks autophagosome–lysosome fusion and/or lysosomal degradation would improve clarity and help avoid potential misinterpretation by non-specialist readers, particularly in the context of LC3-II accumulation and autophagic flux assessment.
Author Response
Response to Reviewer 1 Comments
1.Summary
Thank you very much for taking the time to review this manuscript. Please find below our detailed, point-by-point responses to all comments, along with the corresponding revisions and corrections. All changes are clearly highlighted in the re-submitted manuscript files.
- Point-by-point response to Comments
Comment 1: Assessment of autophagic flux (Figure 3)
While the authors convincingly demonstrate increased LC3-II accumulation and its further elevation upon chloroquine co-treatment, the assessment of autophagic flux would be strengthened by including p62/SQSTM1 protein levels. Given that p62 is both transcriptionally upregulated by TFEB and selectively degraded through autophagy, immunoblot analysis of p62 would help distinguish enhanced autophagic flux from altered synthesis. Including p62/SQSTM1 protein data in Figure 3 would further support the authors’ conclusion.
Response 1: Thank you for this excellent and constructive suggestion. We fully agree that assessing p62/SQSTM1 protein levels is crucial to distinguish between enhanced autophagic flux and altered synthesis. As recommended, we have now performed immunoblot analysis for p62 protein under the same experimental conditions. The new data (Supplementary Figure 3D) show that co-treatment with KHS-101 and CQ leading to a greater accumulation of p62 compared to either treatment alone. This result, together with the observed LC3-II accumulation pattern, provides complementary evidence supporting our conclusion that KHS-101 enhances autophagic flux.
Comment 2: Clarification of the role of chloroquine
Although chloroquine is widely recognized as a late-stage autophagy inhibitor, explicitly stating in the text that chloroquine blocks autophagosome–lysosome fusion and/or lysosomal degradation would improve clarity and help avoid potential misinterpretation by non-specialist readers, particularly in the context of LC3-II accumulation and autophagic flux assessment.
Response 2: Thank you for this helpful suggestion to improve the clarity of our manuscript. As recommended, we have revised the relevant sentence in the Results section (Section 2.3) to explicitly state the mechanism of action of chloroquine. The updated text now reads:
“……in the presence of chloroquine (CQ), a lysosomal inhibitor that blocks autophagosome–lysosome fusion and subsequent cargo degradation.”
Reviewer 2 Report
Comments and Suggestions for AuthorsGeneral Comments:
The authors present a study in which they identify KHS-101 as a novel blood-brain barrier-permeable activator of TFEB, capable of enhancing lysosomal function and autophagic flux, thereby promoting the degradation of pathogenic α-synuclein in a cellular model of Parkinson’s disease. The topic is timely and of high relevance to the field of neurodegenerative diseases. While the study is conceptually sound and the experimental design is generally appropriate, several issues need to be addressed to strengthen the mechanistic insight, experimental rigor, and translational relevance of the work. I recommend major revision before the manuscript can be considered for publication.
Specific Comments by Section:
- Abstract
1) The abstract should briefly mention the neuroprotective potential of KHS-101, not just protein degradation.
- Introduction
1) Inadequate citation regarding "impaired TFEB nuclear localization in PD," especially the mechanism of interaction with α-synuclein.
2) Does not explicitly justify why a "BBB-permeable compound library" was chosen for screening.
3) Analysis of the limitations of existing TFEB activators is insufficient (for example, clinical trial failures).
- Results
1) No information provided on the source, structural diversity, or concentration range of the compound library.
2) No justification for using HeLa cells instead of neuronal cells for primary screening.
3) Figure 1 does not show false-positive controls during the screening process.
4) Only excludes known pathways; fails to propose any alternative mechanistic hypothesis.
5) No detection of TFEB phosphorylation sites to confirm if the effect is direct.
6) No validation of whether TFEB transcriptional activity is genuinely enhanced (e.g., reporter gene assay).
7) Majority of experiments conducted in HeLa cells, weakening neuronal relevance.
8) No verification of whether KHS-101 affects lysosomal pH or membrane permeability.
9) No detection of lysosomal membrane protein expression, for example, LAMP1/2.
10) No assessment of changes in α-synuclein oligomeric or fibrillar forms.
11) No evaluation of whether KHS-101 affects intracellular localization or secretion of α-synuclein.
12) No validation of its effect on wild-type α-synuclein.
- Discussion
1) Discussion of KHS-101's "novel mechanism" is too vague and lacks scientific hypotheses.
2) No direct comparison with other TFEB activators (for example, Rapamycin, Trehalose).
3) No discussion of potential adverse effects or off-target effects of KHS-101.
4) No proposal for future research directions (e.g., target identification, animal studies).
- Materials and Methods
1) Incomplete antibody information (e.g., supplier, catalog number, dilution ratio).
2) No mention of cell line authentication or mycoplasma testing.
3) Statistical methods described too briefly; no mention of normality testing.
4) No statement regarding availability of raw data.
- Figures and Supplementary Materials
1) Calculation method for "Nuclear Fraction" in Figure 1 is not explained in the methods.
2) Some figures (e.g., flow cytometry plots) do not show representative replicates.
3) Supplementary materials lack raw data or statistical information for repeated experiments, especially the WB blots.
Author Response
Response to Reviewer 2 comments
1.Summary
Thank you very much for taking the time to review this manuscript. Please find below our detailed, point-by-point responses to all comments, along with the corresponding revisions and corrections. All changes are clearly highlighted in the re-submitted manuscript files.
- Point-by-point response to Comments
Comments 1: Abstract- The abstract should briefly mention the neuroprotective potential of KHS-101, not just protein degradation.
Response 1：Thank you for this constructive suggestion. We fully agree that highlighting the therapeutic implication of our findings strengthens the impact of the abstract. As recommended, we have revised the abstract to explicitly state the neuroprotective potential of KHS-101. The revised text now reads: “KHS-101 significantly accelerates the degradation of pathogenic α-synuclein in a cellular model of Parkinson’s disease, suggesting its potential to mitigate α-synuclein-mediated proteotoxicity and hold neuroprotective potential.”
Comments 2: Introduction-Inadequate citation regarding "impaired TFEB nuclear localization in PD," especially the mechanism of interaction with α-synuclein.
Response 2：Thank you for this constructive suggestion. We have added a detailed description of the molecular mechanism by which α-synuclein directly interacts with and sequesters TFEB in the cytoplasm, thereby inhibiting its nuclear translocation. These revisions are supported by the key reference (DOI: 10.1073/pnas.1305623110), which demonstrates that α-synuclein toxicity leads to TFEB cytoplasmic retention and that TFEB activation rescues midbrain dopamine neurons from α-synuclein toxicity.
Comments 3: Introduction-Does not explicitly justify why a "BBB-permeable compound library" was chosen for screening.
Response 3：Thank you for raising this important point. To clarify our screening rationale, we have revised the corresponding paragraph in the Introduction to explicitly state that the limited brain penetration of previous candidates has been a key translational obstacle. The revised text now reads: "to directly address the paramount challenge of CNS delivery, we specifically employed a BBB-permeable compound library for our primary screen. This strategy prioritizes the identification of hits with inherent brain access potential from the outset, thereby avoiding the selection of leads that are inactive in the central nervous system in vivo."
Comments 4: Introduction-Analysis of the limitations of existing TFEB activators is insufficient (for example, clinical trial failures).
Response 4：Thank you for the suggestion to deepen the critique of existing TFEB activators. In response, we have expanded the relevant section to include concrete examples of clinical limitations. The updated paragraph now cites specific challenges: “The translational development is constrained by several factors. First, many potent TFEB activators, such as mTOR inhibitors, have shown limited efficacy in neurological clinical trials, often due to inadequate central nervous system exposure, systemic toxicity, or off-target effects. For instance, the mTOR-inhibitory activity of such compounds carries a risk of immunosuppression, and notably, an open-label Phase I trial revealed that rapamycin was undetectable in the cerebrospinal fluid of Alzheimer’s patients, underscoring its poor CNS penetration. Second, other activators like PKC agonists exhibit unacceptable safety profiles , such as tumor-promoting activity, which precludes their therapeutic use. Furthermore, natural products with TFEB-activating properties, such as trehalose, often suffer from low potency, requiring impractically high concentrations for clinical application.”
Comments 5: Results-No information provided on the source, structural diversity, or concentration range of the compound library.
Response 5：Thank you for this comment. We have now provided a detailed description of the compound library in the revised Results section (Section 2.1, first paragraph). Specifically, we state: “...we performed a high-throughput screen using a commercially available library of 734 structurally diverse, bioactive compounds with confirmed blood-brain barrier permeability. The screen was conducted at 10 μM...”. Further details regarding the library's source and the screening concentration, have also been expanded in the Materials and Methods section (see Section 4.3. High-content chemical screening).
Comments 6: Results-No justification for using HeLa cells instead of neuronal cells for primary screening.
Response 6：Thank you for raising this critical point. Our choice of HeLa cells for the primary high-throughput screen was based on the following considerations: The primary goal of this initial screen was to efficiently and reliably identify compounds capable of inducing TFEB nuclear translocation. HeLa cells provide exceptionally high transfection efficiency, robust growth, and uniform morphology, which are essential for achieving a stable, high signal-to-noise ratio in automated imaging and quantitative analysis. These properties are crucial for minimizing technical variability and ensuring the reliability of primary hit identification in a high-throughput setting. HeLa cells stably expressing TFEB-GFP are a widely used and well-characterized model system for studying TFEB regulation and nuclear translocation, as established in prior key studies (e.g., DOI: 10.1002/alz.12776). This allowed us to perform a focused and efficient screen based on a well-defined molecular readout.
We fully acknowledge that neuronal models are ultimately essential for evaluating therapeutic relevance. Therefore, the core validation of KHS-101's potential to promote autophagy flux and α-synuclein clearance was conducted in neuronal cell models (Figure 3H, Figure 4). Furthermore, in this revision, we have expanded the validation in neuronal models to include its key upstream functions: promoting TFEB nuclear translocation and enhancing lysosomal activity(Supplementary: Figure 1 & 3）.
Comments 7: Results-Figure 1 does not show false-positive controls during the screening process.
Response 7：Thank you for this valid point regarding the rigor of the screening data presentation. We would like to clarify that vehicle (DMSO) controls were included on every screening plate to establish the baseline fluorescence ratio. These DMSO controls constituted the primary internal control for each experiment. While the initial screening scatter plot (Figure 1A) focuses on highlighting the most promising hits (like KHS-101) against the positive control (Torin1) for clarity, we acknowledge that explicitly displaying the distribution of all negative controls would further strengthen the figure. More importantly, our hit validation strategy was specifically designed to eliminate false positives. As detailed in the subsequent panels of Figure 1 (D-G), any initial hit was only considered confirmed after passing rigorous secondary assays, including:
- Dose-response validation (Figure 1D): Demonstrating a concentration-dependent effect.
- Time-course validation (Figure 1E): Showing a time-dependent effect.
- Validation with endogenous TFEB (Figure 1F, G): Confirming the effect is not an artifact of the GFP-tagged overexpression system.
Therefore, while the primary screening visualization emphasizes hit selection, the stringent multi-step validation protocol ensures that the reported effects of KHS-101 are genuine and reproducible, effectively addressing the concern regarding false positives.
Comments 8: Results-Only excludes known pathways; fails to propose any alternative mechanistic hypothesis.
Response 8: We sincerely thank you for highlighting this important mechanistic question. Our primary goal was to discover and characterize a novel TFEB activator, a clear unmet need. We achieved this by: (i) ruling out all major known regulatory pathways, (ii) demonstrating dose- and time-dependent TFEB activation, and (iii) confirming functional downstream benefits, including enhanced α-synuclein clearance.
By systematically excluding canonical pathways, we provide strong evidence that KHS-101 acts through a new mechanism, revealing a previously unrecognized regulatory node for TFEB. Identifying the precise target is a natural and exciting next step, and we have now explicitly framed this in the revised Discussion (the last paragraph) as a key future direction. Our study provides both the chemical tool (KHS-101) and the rationale for this important follow-up work.
Comments 9: Results-No detection of TFEB phosphorylation sites to confirm if the effect is direct.
Response 9: We thank you for raising this important mechanistic point. We agree that determining whether KHS-101 directly modulates TFEB (e.g., via altering its phosphorylation) or acts through an upstream, unidentified component is a crucial question. It is important to note that TFEB nuclear translocation can be regulated by mechanisms beyond phosphorylation, such as direct protein-protein interactions or other post-translational modifications. Our comprehensive exclusion of all major known upstream kinases (mTORC1, AKT, ERK, PKC, calcineurin) indicates that KHS-101 does not activate TFEB through these canonical pathways, thus pointing to a distinct mechanism of action. Elucidating this precise mechanism is the clear and exciting focus of our ongoing research. We have strengthened the Discussion to frame this as the key future direction (the last paragraph).
Comments 10: Results-No validation of whether TFEB transcriptional activity is genuinely enhanced (e.g., reporter gene assay).
Response 10: We thank you for this suggestion. In our study, we assessed TFEB transcriptional activation by quantifying the mRNA levels of multiple endogenous TFEB/CLEAR target genes via qRT‑PCR. The significant upregulation of these genes, together with the observed functional outcomes—enhanced lysosomal activity, increased autophagic flux, and accelerated α‑synuclein clearance—provides robust, physiologically relevant evidence for genuine TFEB activation. While reporter assays can offer complementary insights, our dataset already offers a comprehensive functional validation. We have noted in the revised Discussion that reporter assays represent a potential future direction.(the last paragraph).
Comments 11: Results-Majority of experiments conducted in HeLa cells, weakening neuronal relevance.
Response 11: Thank you for highlighting this important point. To strengthen neuronal relevance, we have now performed key experiments in the dopaminergic neuronal cell line SH‑SY5Y. New data demonstrate that in SH‑SY5Y cells, KHS‑101 effectively promotes TFEB nuclear translocation, enhances lysosomal activity (Supplementary: Figure 1&3). Combined with the original SH-SY5Y cell data -increasing autophagic flux and α‑synuclein degradation (Figure 3H,I & Figure 4), these results robustly confirm the neuronal translatability of our findings and significantly reinforce the therapeutic relevance of KHS‑101 for Parkinson's disease.
Comments 12: Results-No verification of whether KHS-101 affects lysosomal pH or membrane permeability.
Response 12: Thank you for this suggestion. In our study, we employed two established functional assays to assess lysosomal activity. First, LysoTracker Red staining showed a marked increase in acidic compartments upon KHS‑101 treatment, indicating an expansion of the acidic lysosomal pool. Second, direct measurement of cathepsin B and L activity demonstrated a dose-dependent increase in proteolysis, confirming that newly formed lysosomes are functionally active. While measurements of intraluminal pH or membrane permeability could provide additional biophysical detail, the combination of elevated acidic compartments and enhanced hydrolytic capacity provides robust functional evidence that KHS‑101 enhances lysosomal activity. We have added a note in the revised Discussion acknowledging that more detailed biophysical studies represent a potential future direction.(
Comments 13: Results-No detection of lysosomal membrane protein expression, for example, LAMP1/2.
Response 13: We thank the reviewer for this valuable suggestion. We agree that providing complementary protein-level data would strengthen the evidence for lysosomal biogenesis. In direct response to this point, we have now performed quantitative Western blot analysis to assess the expression of the key lysosomal membrane protein LAMP1. The new data (Supplementary: Figure 3B), Combined with the original immunofluorescence data (Figure 2D,E), confirm a significant increase in LAMP1 protein levels upon KHS-101 treatment.
Comments 14: No assessment of changes in α-synuclein oligomeric or fibrillar forms.
Response 14: Thank you for raising this interesting mechanistic point. The primary goal of this study was to determine whether enhancing the autophagic-lysosomal pathway via KHS-101 could alleviate the overall burden of pathogenic α-synuclein, a key therapeutic objective in PD. Our data using both Western blot and immunofluorescence (Figure 4) clearly demonstrate a significant reduction in total α-synuclein levels upon treatment.
We agree that determining whether KHS-101 preferentially clears soluble oligomers, insoluble fibrils, or both is a fascinating and important question for understanding its precise mechanism of action. Distinguishing between these species represents a logical and important next step to elucidate the precise mechanism of clearance. Importantly, our data demonstrate that the clearance is dependent on a functional autophagic-lysosomal system (blocked by chloroquine, Figure 4E, F), which is known to be capable of degrading various aggregated species.
We have revised the Discussion to explicitly highlight the investigation of α-synuclein species specificity as a key direction for future research, stating: “Future studies will also aim to determine whether KHS-101 preferentially facilitates the clearance of specific pathogenic forms of α-synuclein, such as soluble oligomers or insoluble fibrils. ”This work establishes the essential proof-of-concept and provides the chemical tool needed to pursue those detailed mechanistic questions.
Comments 15: No evaluation of whether KHS-101 affects intracellular localization or secretion of α-synuclein.
Response 15: Thank you for raising this point regarding α-synuclein trafficking. Our study focused on establishing that KHS-101 reduces overall cellular α-synuclein levels via lysosome-dependent clearance. Investigating effects on its subcellular localization or secretion represents a valuable next step to further delineate the clearance mechanism. We have noted this direction in the revised Discussion.
Comments 16: No validation of its effect on wild-type α-synuclein.
Response 16: We thank the reviewer for raising this important point. Our study focused on evaluating the efficacy of KHS-101 in a well‑established cellular model of familial PD using the pathogenic A53T mutant α‑synuclein. Determining whether KHS-101 similarly enhances the clearance of wild‑type α‑synuclein is a logical and important next step to assess its broader therapeutic potential for sporadic PD. We have now explicitly stated this as a key future direction in the revised Discussion.
Comments 17: Discussion-Discussion of KHS-101's "novel mechanism" is too vague and lacks scientific hypotheses.
Response 17: Thank you for highlighting the need for greater mechanistic clarity. In response, we have substantially revised the Discussion to provide a more specific scientific hypothesis regarding the novel mechanism of KHS-101. The updated text: “Our data show that KHS-101 activates TFEB independently of all these canonical regulatory nodes. This, combined with its rapid onset of action, strongly suggests that KHS-101 may operates through a novel, previously uncharacterized mechanism. We hypothesize that it may either directly interact with TFEB to influence its conformation or subcellular localization, or it may modulate a novel upstream component of the TFEB regulatory network. This distinct mechanism of action not only differentiates KHS-101 from existing tool compounds but also positions it as a unique chemical probe to uncover new biology within the lysosomal regulatory axis.”
Comments 18: No direct comparison with other TFEB activators (for example, Rapamycin, Trehalose).
Response 18: Thank you for your suggestion. As recommended, we have revised the Discussion to include a direct comparison with other TFEB activators. The updated text now states: “In contrast to existing agents, KHS-101 demonstrates a distinct profile: it acts through an mTOR‑independent mechanism, suggesting a potentially improved safety margin regarding immune modulation; it induces rapid TFEB nuclear translocation at low micromolar concentrations, showing significantly greater potency than trehalose; and it elicits a genuine functional enhancement of the autophagic‑lysosomal pathway, unlike lysosomal inhibitors such as chloroquine, which indirectly trigger TFEB translocation via stress signaling . Our high‑throughput screen of a blood‑brain barrier‑permeable compound library successfully identified KHS‑101 based on this unique combination of properties.”
Comments 19: No discussion of potential adverse effects or off-target effects of KHS-101.
Response 19: Thank you for your suggestion. We have now included a dedicated discussion on this topic in the revised manuscript. Specifically, we note that in our current in vitro study, KHS-101 demonstrated no detectable cytotoxicity at concentrations effective for TFEB activation and functional enhancement (up to 10 μM), indicating a favorable preliminary therapeutic window. However, we explicitly acknowledge that a comprehensive evaluation of its potential off-target effects, long-term safety, and systemic toxicity requires rigorous future investigation in appropriate in vivo models. This point has been integrated into the Discussion to provide a balanced perspective on the compound's therapeutic potential and necessary next steps in its development.
Comments 20: No proposal for future research directions (e.g., target identification, animal studies).
Response 20: Thank you for this constructive suggestion. In the revised Discussion, we have now explicitly outlined several key future research directions essential for advancing KHS-101. These include: (1) target identification efforts using chemical proteomics or functional genomics to elucidate its precise molecular mechanism; (2) rigorous evaluation in animal models of Parkinson’s disease to confirm in vivo efficacy, pharmacokinetics, and safety; and (3) investigation of its effects on other disease-relevant protein aggregates (e.g., tau, huntingtin) to assess broader therapeutic potential. This structured framework provides a clear roadmap for the next stages of research.
Comments 21: Incomplete antibody information (e.g., supplier, catalog number, dilution ratio).
Response 21: Thank you for highlighting the need for complete antibody information. In the revised Materials and Methods section (Section 4.7 Western Blotting), we have now provided the full details for all primary antibodies used in this study, including: supplier, catalog number and dilution ratio.
Comments 22: No mention of cell line authentication or mycoplasma testing
Response 22: Thank you for this important comment. In the revised Materials and Methods​ section (Section 4.1. Cell culture), we have now explicitly stated that: “Cell lines were authenticated by short tandem repeat (STR) profiling and routinely tested to be free of mycoplasma contamination using a PCR-based detection method.”
Comments 23: Statistical methods described too briefly; no mention of normality testing.
Response 23: Thank you for this suggestion to improve the rigor of our statistical reporting. In the revised Materials and Methods section (Section 4.11. Statistical analysis), we have now provided a detailed description:“ Data are presented as mean ± SEM from at least three independent experiments. Normality of data distribution was assessed using the Shapiro-Wilk test. . For comparisons between two groups, unpaired two-tailed Student’s t-test was used for normally distributed data; otherwise, the Mann-Whitney U test was applied. For multiple-group comparisons, one-way ANOVA with Tukey’s post hoc test (parametric) or Kruskal-Wallis test with Dunn’s post hoc test (non-parametric) was used. Two-way ANOVA was employed for comparisons involving two independent variables. All analyses were performed using GraphPad Prism 8.0.1, and a p-value < 0.05 was considered statistically significant.”
Comments 24: No statement regarding availability of raw data.
Response 24: Thank you for this suggestion. In the revised manuscript, we have now explicitly stated that:" Data Availability Statement: The original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors."
Comments 25: Calculation method for "Nuclear Fraction" in Figure 1 is not explained in the methods.
Response 25: Thank you for pointing out this omission. We have now clarified the calculation method in the Materials and Methods section (Section 4.3. High-content chemical screening) as follows: "The nuclear translocation of TFEB‑GFP was quantified by measuring the total fluorescence intensity within the nuclear and cytoplasmic compartments of individual cells. The Nuclear Fraction was calculated as the ratio of total nuclear intensity to total cellular (nuclear + cytoplasmic) intensity, and normalized to the mean value of the vehicle (DMSO) control group on each plate."
Comments 26: Some figures (e.g., flow cytometry plots) do not show representative replicates.
Response 26: Thank you for this comment. The representative replicates have been provided in the Source Data file submitted with this revision.
Comments 27: Supplementary materials lack raw data or statistical information for repeated experiments, especially the WB blots.
Response 27: Thank you for this comment. The data for repeated experiments have been provided in the Source Data file submitted with this revision.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI agree to publish.
