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Chemoproteomic development of SLC15A4 inhibitors with anti-inflammatory activity

Nature Chemical Biology (2024)Cite this article

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Abstract

SLC15A4 is an endolysosome-resident transporter linked with autoinflammation and autoimmunity. Specifically, SLC15A4 is critical for Toll-like receptors (TLRs) 7–9 as well as nucleotide-binding oligomerization domain-containing protein (NOD) signaling in several immune cell subsets. Notably, SLC15A4 is essential for the development of systemic lupus erythematosus in murine models and is associated with autoimmune conditions in humans. Despite its therapeutic potential, the availability of quality chemical probes targeting SLC15A4 functions is limited. In this study, we used an integrated chemical proteomics approach to develop a suite of chemical tools, including first-in-class functional inhibitors, for SLC15A4. We demonstrate that these inhibitors suppress SLC15A4-mediated endolysosomal TLR and NOD functions in a variety of human and mouse immune cells; we provide evidence of their ability to suppress inflammation in vivo and in clinical settings; and we provide insights into their mechanism of action. Our findings establish SLC15A4 as a druggable target for the treatment of autoimmune and autoinflammatory conditions.

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Fig. 1: Chemoproteomic discovery of SLC15A4 inhibitors.
Fig. 2: Functional valuation of SLC15A4 chemical probes and target engagement studies.
Fig. 3: Pharmacological inhibition of SLC15A4 suppresses multiple innate signaling pathways in human immune cells.
Fig. 4: AJ2-30 suppresses TLR7-9 and NOD activation of mouse immune cells in an Slc15a4-dependent manner.
Fig. 5: Mechanistic characterization of SLC15A4 inhibition by AJ2-30.
Fig. 6: Pharmacological inhibition of SLC15A4 suppresses inflammatory cytokine production in lupus patient-derived PBMCs.

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Data availability

The data that support the findings in this work are available in the paper and the Supplementary Information. Uncropped, full immunoblot images and gels are provided in Supplementary Fig. 11. Gating strategies are described in Supplementary Fig. 12. Processed proteomic data are provided in the Supplementary Data. The Homo sapiens proteome database (UniProt, 2018, 42,358 sequences) or the Mus musculus proteome database (UniProt, 2021, 25,314 sequences) was used in proteomic searches. All raw proteomics data files have been deposited to the PRIDE repository and are available under accession numbers PXD045715 (for enrichment datasets; http://www.ebi.ac.uk/pride/archive/projects/PXD045715) and PXD045712 (for unenriched datasets; http://www.ebi.ac.uk/pride/archive/projects/PXD045712). Source data are provided with this paper.

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Acknowledgements

We thank Belharra Therapeutics for assistance with in vivo and kinome profiling studies. We also thank B. F. Cravatt for invaluable discussions and insights. This work was supported by National Institute of Allergy and Infectious Diseases R01 AI156268 (C.G.P.) and T32 AI007244 (J.M.W.).

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Author notes

  1. These authors contributed equally: Tzu-Yuan Chiu, Daniel C. Lazar, Wesley W. Wang.

Authors and Affiliations

  1. Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA

    Tzu-Yuan Chiu, Wesley W. Wang, Jacob M. Wozniak, Appaso M. Jadhav, Weichao Li & Christopher G. Parker

  2. Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA

    Daniel C. Lazar, Nathalia Gazaniga, Argyrios N. Theofilopoulos & John R. Teijaro

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Contributions

J.R.T. and C.G.P. conceived of and supervised this study. D.C.L., W.W.W. and T.-Y.C. performed functional characterization of compounds in immune cells. T.-Y.C., W.W.W., D.C.L. and J.M.W. performed experiments to characterize the mechanism of action of compounds. T.-Y.C., W.L. and J.M.W. performed proteomic experiments. A.M.J. was responsible for synthesis and characterization of all new compounds. D.C.L. and N.G. performed in vivo analysis of compounds and generated data from the lupus patient cohort. N.G. assisted in flow cytometry analysis. A.N.T. provided reagents and assistance with experimental design. W.W.W., D.C.L., T.-Y.C., J.R.T. and C.G.P. contributed to the preparation of the original manuscript and the preparation of the figures. All authors assisted with the review and editing of the manuscript.

Corresponding authors

Correspondence to John R. Teijaro or Christopher G. Parker.

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Competing interests

C.G.P., J.R.T., D.C.L. and A.M.J. are inventors on a patent application (WO2021174023) submitted by The Scripps Research Institute that covers small-molecule inhibitors of SLC15A4. C.G.P. and J.R.T. are founders of and scientific advisors to Belharra Therapeutics, a biotechnology company interested in using chemical proteomic methods to develop small-molecule therapeutics. All other authors declare no competing interests.

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Nature Chemical Biology thanks Claire Steppan and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Fully functionalized fragment (FFF) probe library and cell models for SLC15A4 inhibitor characterization, Related to Fig. 1.

a, HA-SLC15A4 is localized to lysosomes in CAL-1 cells. Immunostaining of SLC15A4 in CAL-1 cells stably overexpressing EGFP or HA-tagged SLC15A4. Cells were co-stained with LAMP1 as a lysosome marker. Scale bar: 10 μm. Data are representative of two independent experiments. b, c, FFF-21 and 21c inhibit TLR7/8 mediated signaling. THP1-DUAL reporter cells were co-incubated with R848 (5 μg/mL) and escalating doses of FFF-21 or 21c. IRF inhibition was monitored by measuring activity of secreted luciferase. NF-kB inhibition was monitored by measuring activity of secreted alkaline phosphatase. Data is plotted as the mean ± s.d. (n = 3 biological independent replicates). d, Mutant human SLC15A4 (L14A, L15A, L318A, V319A) results in amplified MDP induced NOD activation relative to WT (24 h) as measured by luciferase activity in A549 cells expressing NOD2 and NF-κB luciferase reporter. Data is plotted as the mean ± s.d. (n = 3). e, Concentration dependent inhibition of SLC15A4-dependent MDP transport by FFF-21. A549 cells were engineered to express an NF-κB-luciferase reporter, NOD2, and either membrane-localized SLC15A4. SLC15A4 transport cells were co-treated with FFF-21 and MDP (500 ng/mL) for 24 h. Data is plotted as the mean ± s.d. (n = 3 biological independent replicates).

Extended Data Fig. 2 Characterization of SLC15A4 chemical probes and control compounds, Related to Fig. 2.

a, A549 cells engineered to express an NF-κB luciferase reporter, human NOD2, and membrane-localized human SLC15A4 or SLC15A3. Cells were treated with AJ2-18 or AJ2-30 (5 μM) along with increasing quantities of MDP (24 h). Data is plotted as the mean ± s.d. (n = 3). b, A549 cells transfected with membrane trafficked murine Slc15a4 as well as NOD2 were stimulated with MDP and treated with AJ2-18 or AJ2-30 (5 μM) for 24 h. Luciferase activity was measured in the cells at 24 h post-stimulation. Data is plotted as the mean ± s.d. (n = 3). P-values are shown. c, THP1-DUAL reporter cells were co-incubated with 5 μg/ml R848 and escalating doses of AJ2-30 or AJ2-32 for 18 h. IRF inhibition was monitored by measuring activity of secreted luciferase. Data is plotted as the mean ± s.d. (n = 3). d, THP1-DUAL reporter cells were stimulated with MDP and TriDAP in the presence of AJ2-30 or AJ2-18 and SEAP levels were assessed at 24 h. Data is plotted as the mean ± s.d. (n = 4 biological independent replicates). P-values are shown. e, f, CETSA for AJ2-30 and AJ2-18 at varying doses. CAL-1 cells expressing HA-SLC15A4 were treated with increasing dosage of AJ2-30 or AJ2-18. Soluble and insoluble fractions were isolated by centrifugation and analyzed by immunoblot. Immunoblots were quantified and plotted as the mean ± s.d. of three independent replicates in (f). Results are presented as mean ± s.d. of at least n = 3 independent experiments. Statistical analysis was performed using ANOVA analysis followed by multiple comparisons test. P-values are shown.

Source data

Extended Data Fig. 3 AJ2-30 inhibition of endo-lysosomal TLR signaling is selective and dependent on SLC15A4, Related to Fig. 4.

a, AJ2-30 does not inhibit TLR7/8 signaling in primary macrophages. Primary human derived macrophages were treated with AJ2-18 or AJ2-30 (5 μM), polarized with IFN-γ, and then stimulated with R848 (5 μg/mL) for 22 h. b, AJ2-30 does not inhibit production of IgG2c, IgM, or CD86 expression in wild-type mouse B cells after LPS treatment. c, AJ2-30 does not inhibit production of IgG2c, IgM in feeble mouse B cells after R837 and CpG-B treatment. d, AJ2-30 suppresses CD86 expression in wild-type but not feeble B cells. Results are presented as the mean ± s.d. of n = 3 (n = 4 for a) independent experiments. Statistical analysis was performed using ANOVA analysis followed by multiple comparisons test.

Extended Data Fig. 4 Mechanistic characterization of SLC15A4 inhibition by AJ2-30, Related to Fig. 5.

a, IFNB mRNA expression assessed by quantitative PCR in CAL-1 cells treated with compounds (5μM) or DMSO. Results were normalized to beta actin and values indicate mean ± s.d. (n = 2). b, FACS analysis of STAT1 phosphorylation in WT and feeble mouse B cells with co-treatment of 1 μM CpG-B and 5 μM AJ2-30 (n = 3 biological independent replicates; mean ± s.d.). c, Immunostaining of TLR9 in WT mouse B cells. Cells were co-treated with AJ2-30 (5 μM) orDMSO and stimuli 1 μM CpG-B for 15 min before fixation. Cells were co-stained with LAMP1 as a lysosome marker. Scale bar: 2 μm. d, Quantitation of the colocalization coefficient of TLR9 with LAMP1 (n = 35 for DMSO versus n = 38 for AJ2-30 treatment; mean ± s.d.; unpaired two tailed t test). Images are representative of two independent experiments. e, Acidification of lysosomes upon CpG-B stimulation in B cells. WT mouse B cells were treated with either DMSO, AJ2-18 (5 μM) AJ2-30 (5 μM), or Bafilomycin A (250 nM) and stimulated with 1 μM CpG-B for the indicated periods and labeled with LysoSensor DND-189. The fluorescence was analyzed by flow cytometry. Data is plotted as the mean ± s.d. (n = 3). f, Effect of AJ2-30 on ATPase activity of the LAMP1-enriched cellular fraction. Isolated lysosomal fraction was treated with DMSO, AJ2-18 (5 μM), AJ2-30 (5 μM), or bafilomycin A1 (1 μM). ATPase activity was measure using EnzChek™ Phosphate Assay Kit following manufacturer’s instructions. Statistical analysis was performed using ANOVA analysis followed by multiple comparisons test. Data is plotted as the mean ± s.d. (n = 3). P-values are shown.

Extended Data Fig. 5 Mechanistic characterization of SLC15A4 inhibition by AJ2-30, Related to Fig. 5.

a, FACS analysis of mTORc1 activation by measuring the phosphorylation of mTOR (S2448), 4E-BP1 (T37/46), and S6 (S235/236) in human pDCs isolated from PBMCs. Primary human pDCs were treated with DMSO, AJ2-18 (5 μM), or AJ2-30 (5 μM) and then stimulated with CpG-A (1 μM) for 4 h. Statistical analysis was performed using ANOVA analysis followed by multiple comparisons test. Data is plotted as the mean ± s.d. (n = 3). P-values are shown. b, Proximity labeling experiments of V5-APEX2-SLC15A4 in CAL-1 cells. Scatter plot displaying the fold change (FC) of proteins enriched in V5-APEX2 tagged SLC15A4 cells versus HA tagged SLC15A4 (negative control) cells. Proteins considered enriched if FC between V5-APEX2-SLC15A4 and control >30, p < 0.05 (analyzed by unpaired two tailed t test). Data are presented as mean of n = 3 biologically independent samples. Enriched mTORC1 related proteins depicted as red circles, lysosomal associated proteins as green circles, and all other proteins as gray circles. Associated data set provided in Supplementary Data. c, GO Enrichment Analysis of enriched proteins identified in proximity labeling enriched datasets include mTOR signaling among top 3 enriched pathways59. d, Quantitation of indicated immunoprecipitated protein levels upon AJ2-30 treatment. Data presented as mean ± s.d. of n = 3 three biologically independent treatment samples and normalized to DMSO. Statistics were performed using ANOVA analysis. P-values are shown.

Source data

Extended Data Fig. 6 Mechanistic characterization of SLC15A4 inhibition by AJ2-30, Related to Fig. 5.

a, Immunostaining of mTOR in human B cells isolated from PBMCs. Cells were co-treated with AJ2-30 (5 μM) or DMSO and stimuli 5 μg/ml R848 or 1 μM CpG-B for 4h before fixation. Cells were co-stained with LAMP1 as a lysosome marker. Scale bar: 2 μm. b, Quantitation of the colocalization coefficient of mTOR with LAMP1 (n = 64, 75, 47, 74, 76 cells from left to right). Images are representative of two independent experiments (two healthy donors). Data are presented as box plots. Centre line indicated the median, upper to lower bounds indicates 75th – 25th percentile with whiskers at maximum and minimum values. Statistics was performed using Student two-tail t-test analysis. c, IRF7 activation was assessed by measuring the phosphorylation of IRF7 (S477/479) by flow cytometry in human pDCs isolated from PBMCs. Primary human pDCs were treated with DMSO, AJ2-18 (5 μM), or AJ2-30 (5 μM) and then stimulated with CpG-A (1 μM) for 4 h. Data is plotted as the mean ± s.d. (n = 3). Statistics were performed using ANOVA analysis. P-values are shown. d, Immunostaining of IRF5 and IRF7 in primary human pDCs. Cells were co-treated with compounds (5 μM) or DMSO and stimuli 1 μM CpG-A for 4h before fixation. Cells were co-stained with DAPI as a nucleus marker. Scale bar: 5 μm. e, Quantitation of the colocalization coefficient of IRF5 or IRF7 with DAPI (n = 121, 120, 153, 110 cells from left to right). Images are representative of three independent experiments. Data are presented as box plots and defined as Extended Data Fig. 8b. Statistics were performed using ANOVA analysis. P-values are shown. f, AJ2-30 suppresses TLR7/8 or TLR9-induced IRF5 and IRF7 translation in human B cells. Cells were co-treated with compounds (5 μM) or DMSO and stimuli (1 μM CpG-B or 5 μg/mL R848) for 8 h. Data are representative of two independent experiments.

Source data

Extended Data Fig. 7 AJ2-30 down-regulates SLC15A4 in CAL-1 and mouse B cells.

a, b, AJ2-30 down-regulates HA-SLC15A4. CAL-1 cells stably expressing HA-SLC15A4 were treated with compounds (20 μM), or DMSO for indicated time (a) or escalating doses at 4h (b). HA-SLC15A4 abundance was determined by immunoblot. Data are representative of three independent experiments. c, Human B cells were treated with compounds (10 μM) or DMSO for 16 h. Abundance of SLC15A4 was analyzed by quantitative proteomics and normalized to DMSO treated samples. Data is plotted as the mean ± s.d. (n = 4). P-values are shown. Associated data set provided in Supplementary Data. d, e, (d) mouse B cells were treatment with AJ2-30 (20 μM), AJ2-18 (20 μM), or DMSO for 4 h. (e) CAL-1 cells were treatment with AJ2-30 (10 μM) or DMSO for 16 h. Abundance of SLC15A4 was analyzed by quantitative proteomics and normalized to DMSO treated samples (n = 2). Associated data set provided in Supplementary Data. f, Localization of SLC15A4 in CAL-1 cells. CAL-1 cells stably expressing HA-SLC15A4 were co-treated with AJ2-30 (10 μM) or DMSO for different timepoints. 0h indicated cells were fixed before treatment. Cells were co-stained with LAMP1 as a lysosome marker. Scale bar: 10 μm. g, Quantitation of the colocalization coefficient of HA-SLC15A4 with LAMP1 (n = 44, 23, 32 cells from left to right; mean ± s.d.). Statistics were performed using ANOVA analysis. Images are representative of two independent experiments. h, Bafilomycin A blocks AJ2-30 degradation in CAL-1 cells. CAL-1 cells stably expressing HA-SLC15A4 were pre-treated with BafA for 1h and co-incubated with AJ2-30 (10 μM) or DMSO for 4h. HA-SLC15A4 abundance was determined by immunoblot. Images are representative of two independent experiments.

Source data

Extended Data Fig. 8 AJ2-30 suppresses costimulatory molecule expression of lupus patient B cells.

a, b, Expression of the costimulatory molecules CD80, CD86, and MHC-II on B cells following R837 or CpG-B stimulation when treated with either DMSO or AJ2-30 (5 μM) for 24h. Data is plotted (n = 8). c, Expression of the costimulatory molecules CD80, CD86, and MHC-II on unstimulated B cells when treated with either DMSO or AJ2-30 (5 μM) for 24 h. Data is plotted (n = 5). Statistical analysis was performed using Wilcoxon matched-pairs signed rank two-tailed test. P-values are shown.

Supplementary information

Supplementary Information

Supplementary Figs. 1–12, Tables 1–3 and Chemical Synthesis and Characterization.

Reporting Summary

Supplementary Data

Proteomics data (Supplementary Data 1–10). Table titles and legends are within the file.

Source data

Source Data Figs. 1, 2 and 5 and Extended Data Figs. 2, 6 and 7

Uncropped immunoblot images.

Source Data Fig. 2

Quantification of immunoblot images.

Source Data Extended Data Fig. 2

Quantification of immunoblot images.

Source Data Extended Data Fig. 5

Quantification of immunoblot images.

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Chiu, TY., Lazar, D.C., Wang, W.W. et al. Chemoproteomic development of SLC15A4 inhibitors with anti-inflammatory activity. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-023-01527-8

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