Abstract
Chemically induced proximity (CIP) has remarkably advanced the development of molecular and cellular therapeutics. To maximize therapeutic potential, there is a pressing need to expand the repertoire of CIP systems of translational values, favoring chemical ligands that are cost-effective, structurally simple, biocompatible, reversible and have minimal side effects. Here, we present a salicylic acid (SA)-mediated binary association system (SAMBA), evolved from a tobacco SA receptor, that enables rapid protein–protein heterodimerization in response to SA or aspirin after hydrolysis. We demonstrate the broad applicability of SAMBA in various biological contexts, including SA-dependent reprogramming of a protein-based reaction–diffusion system, graded gating of calcium channels, inducible initiation of receptor tyrosine kinase-mediated signaling and gene expression, and tunable activation of chimeric antigen receptor T cells. Our work establishes SAMBA as a versatile chemogenetic platform that allows temporal control of biological processes and therapeutic cells both in vitro and in vivo.
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Data availability
The datasets generated and/or analyzed during this study are available as Supplementary Information. Additional data may be available upon request. The SAMBA constructs used in the study were also deposited in the nonprofit plasmid repository Addgene (no. 236390). Source data are provided with this paper.
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Acknowledgements
We thank K. Zhang (from University of Illinois at Urbana-Champaign) for providing the plasmid containing TrkA-ICD. This work was supported by the National Institutes of Health (grant nos. R01GM144986 to Y.Z., R21AI174606 to Y.Z., R21NS125167 to X.Z., P01CA265748 to Y.H., R01DK132286 to Y.H. and R35HL166557 to Y.H.), the Center Prevention and Research Institute of Texas (RP250468 to Y.Z.), the Cancer Therapeutics Training Program (RP210043), the Welch Foundation (grant no. BE-1913-20220331 to Y.Z.) and the Leukemia & Lymphoma Society (to Y.Z.).
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Y.Z., Y.H. and T.W. conceived the ideas and directed the work. T.W. and YZ designed the study. T.W., S.L., Y.K., S.A., T.H., T.-H.L. and R.W. performed the experiments. T.W., S.L., S.A., Z.L and Y.Z. analyzed the results. F.W., G.M. and M.X.Z. provided intellectual input and technical support. Y.Z., Y.H. and T.W. wrote the manuscript. All the authors contributed to the discussion and editing of the manuscript.
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Y.Z. has submitted a patent application to the United States Patent and Trademark Office pertaining to the design and biomedical application aspect(s) of this work (no. 63/684,543; with Y.Z., Y.H. and T.W. as inventors). The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Initial screening of split sites in AtNPR4 to enable SA-induced proximity.
a) The predicted 3D structure of the SA-binding core region of AtNPR4 (residues 373-574; based on PDB entry: 6WPG). Four potential split sites explored in this study are marked with pink spheres. b) Quantitative analysis of cytosolic mCherry fluorescence changes for four different split configurations of AtNPR4 after treatment with 1 mM salicylic acid. n = 8 cells from three independent biological replicates (mean ± s.e.m.).
Extended Data Fig. 2 Truncated variants of NtNPR1.
a) Schematic illustration of NtNPR1 containing seven mutations (NtNPR1-7M) and six additional truncation variants (7M-TR1-6) tested in the study. N and C denote the indicated N- and C-terminal parts of engineered NtNPR1. b) Quantification of the cytosolic mCherry clearance ratio induced by 0.1 mM SA for the indicated truncated variants.
Extended Data Fig. 3 A CRAC channel blocker (La3+) was used to inhibit the Ca2+ influx in cells co-transfected with N-ST1 and C-TD in the presence of SA.
a) Time-course of whole cell inward currents at -100 mV. Upon whole-cell break-in (t = 0 s), a substantial inward Ca2+ current was observed in an external solution containing 10 mM Ca2+ with SA. When the standard external Ringer’s solution was replaced with a 20 mM Ca2+ external solution, an even larger Ca2+ current was detected. These currents were effectively blocked by La3+ at 50 µM. b) Quantification of the current density at 20 mM Ca2+, and 50 µM La3+. n = 4 from three independent biological replicates (mean ± s.e.m.).
Extended Data Fig. 4 Quantification of NFAT-dependent luciferase expression under the indicated conditions.
Measurement of NFAT-Luc reporter activity was performed in Jurkat T cells with stable expression of the indicated constructs. Engineered Jurkat cells were co-cultured with human CD19 (hCD19)-positive Raji lymphoma cells (indicated by solid blue circle) or hCD19-negative K562 cells (open blue circle), in the absence (open red square) or presence (solid red square) of 500 µM SA. Anti-HER2 CAR and Anti-HER2 SAMBA-CAR were used as control groups. n = 3 independent biological replicates (mean ± s.e.m.).
Extended Data Fig. 5 Dose-dependent NFAT-Luc reporter activity (a) and IL2 production (b) in Jurkat T cells co-expressing the indicated constructs.
n = 3 independent biological replicates (mean ± s.e.m.).
Extended Data Fig. 6 Comparison of SAMBA-CAR with two other CID-based CARs.
a) Schematic representation of the FKBP-FRB CAR and NS3a-GNCR1 CAR constructs. The FKBP-FRB and NS3a-GNCR1 pairs were used to replace SAMBA-N/C components, respectively. The FKBP-FRB CAR configuration was based on a published study. b) Quantification of NFAT-dependent luciferase expression for three CAR constructs under the indicated conditions. NFAT-Luc reporter activity was measured in Jurkat T cells stably expressing the indicated constructs. These engineered Jurkat cells were co-cultured with human CD19 (hCD19)-positive Raji lymphoma cells in the absence (open squares) or presence (solid squares) of 500 µM SA (red), 1 µM rapamycin (yellow), or 10 µM grazoprevir (purple), respectively. Compared to SAMBA-CAR, FKBP-FRB CAR exhibited higher basal activity even without rapamycin, while NS3a-GNCR1 CAR showed a lower activation response, thereby positioning SAMBA-CAR as an ideal CAR design. n = 3 independent biological replicates (mean ± s.e.m.).
Extended Data Fig. 7 Comparison of the performance of SAMBA-CAR and NS3a-GNCR1 CAR in recruiting part 2 from the cytosol to part 1 on the PM.
a-b) Confocal imaging was used to visualize the recruitment of part 2 (red) from the cytosol to the PM-anchored part 1 (green) in HeLa cells. Recruitment was monitored after overnight incubation with SA for SAMBA-CAR (a) and grazoprevir for NS3a-GNCR1 CAR (b). Scale bar, 5 µm. c) Quantification of the mCherry signal ratio at the PM versus the cytosol (FPM/Fcytosol) before and after drug treatment for the indicated groups. Data represents n = 8 cells from three independent biological replicates (mean ± s.e.m.).
Extended Data Fig. 8 Characterization of SAMBA-CAR T cells.
a) Quantification of CD69 expression levels in untransduced T cells, SAMBA-CAR and WT CAR T cells after overnight co-culture with tumor cells expressing the CD19 antigen. b) Quantification of IL-2 production reversibility by washing out SA from SAMBA-CAR T cells, followed by an additional 8-hour incubation. c) ELISA measurements of IFNγ production by the indicated CAR T cells after overnight co-culture with Raji cells. d) The percentages of CD4+ to CD8+ cells after 5 days of stimulation of the indicated CAR T cells. e) FACS quantification of exhaustion markers (LAG-3 and PD-1) following 5-day stimulation of the indicated CAR T cells. f) Cytotoxic activity of the indicated CAR T cells against Raji cells at varying effector-to-target cell ratios (10:1, 5:1, or 2.5:1). WT and SAMBA-CAR T cells were co-cultured overnight with Raji cells in the absence or presence of SA, and Raji cell viability was evaluated by bioluminescence signal changes. Data represent three independent biological replicates (mean ± s.e.m.). PBMCs were collected from blood samples of three healthy blood donors.
Extended Data Fig. 9 H&E staining images of major organs to assess potential systemic toxicity of SA in vivo.
SCID-Beige mice were treated with daily oral doses of PBS or SA (100 mg/kg) for 60 days, after which their organs were harvested for analysis. n = 5 biologically independent mice. Scale bar, 300 µm.
Extended Data Fig. 10 Metabolic panel analysis in the blood to evaluate potential systemic toxicity of SA on major organs.
SCID-Beige mice were treated with daily oral doses of PBS or SA (100 mg/kg) for 60 days, followed by blood collection and sent to Metabolic Phenotyping Core of University of Texas Southwestern Medical Center (UTSW) for biomarker analysis. Minimal changes were observed across eight biomarkers, indicating that chronic exposure to SA caused negligible side effects in mice. n = 5 biologically independent mice. ns, not statistically significant. The P values were calculated using the two-sided unpaired Student’s t-test. Abbreviations: ALT, alanine aminotransferase (liver function); AST, aspartate aminotransferase (liver function); ALKP, alkaline phosphatase (liver and bone function); BUN, blood urea nitrogen (kidney function); CK, creatine kinase (kidney function); LDH, lactate dehydrogenase (indicator of tissue damage); Na, sodium; K, potassium.
Supplementary information
Supplementary Information
Supplementary Figs 1–11, Table 1 and Refs.
Supplementary Video 1
Time-lapse confocal imaging of HeLa cells expressing mCherry-SAMBA-N-P2A-SAMBA-C-CAAX in response to the addition of 1 mM SA (0 to 350 s), washout (350 to 799 s), readdition of 1 mM SA (799 to 1,135 s), a second washout (1,135 to 1,535 s) and readdition of 1 mM SA (1,535 to 1,919 s). The SA-induced cytosol-to-PM translocation was fully reversible. Scale bar, 5 µm.
Supplementary Video 2
Animation of SA-inducible slowing down of the MinDE circuit frequency using the SAMBA system. After SA addition, SAMBA-N fused with MinE (MinE-EGFP-SAMBA-N; green channel) was recruited to ER-anchored SAMBA-C (ER-C or SP-TM-SAMBA-C; no color). With the diminished level of MinE in the cytoplasm, the oscillation frequency of mCh-MinD (red channel) was reduced. The time point for SA addition was indicated in the video. Plasmids used in the assay are depicted in Fig. 2b. Scale bar, 5 µm.
Supplementary Video 3
Animation of SA-inducible activation of the MinDE oscillatory circuit via inserting the SAMBA components into MinE at the split site of Q55/I56 (MinE-55). On SA addition, the split MinE (green channel) was reassembled to restore its function, subsequently reactivating mCh-MinD oscillation (red channel). The time point for SA addition was indicated in the video. Plasmids used in the assay are depicted in Fig. 2g. Scale bar, 5 µm.
Supplementary Video 4
Animation of SA-inducible activation of the MinDE circuit frequency via inserting the SAMBA system into MinE at split site of G70/D71 (MinE-70). On SA addition, the split MinE (green channel) was reassembled to restore its function, subsequently reactivating mCh-MinD oscillation (red channel). The time point for SA addition was indicated in the video. Plasmids used in the assay were depicted in Fig. 2g. Scale bar, 5 µm.
Supplementary Video 5
Time-lapse confocal imaging of HeLa cells was used to monitor SA-induced recruitment of 2N (part 2 of split SAMBA-CAR shown in Fig. 5b) from the cytosol to PM-embedded 1C (part 1 of split SAMBA-CAR in Fig. 5b) in HeLa cells. Split SAMBA-CAR could be reversibly reassembled near the PM in a SA-dependent manner. The time points for SA addition or washout were indicated. Scale bar, 5 µm.
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Wang, T., Liu, S., Ke, Y. et al. Repurposing salicylic acid as a versatile inducer of proximity. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-01918-z
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DOI: https://doi.org/10.1038/s41589-025-01918-z
