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. 2024 Apr 12;15(5):653–658. doi: 10.1021/acsmedchemlett.4c00048

Structural Evaluations of a Selective Human STINGA230 Agonist and Its Use in Macrophage Immunotherapies

Zhichao Tang †, Junxing Zhao †, Ying Li ⊥, Shallu Tomer §, Manikandan Selvaraju †, Nicholas Tien §, Diyun Sun ⊥, David K Johnson ∥, Anjie Zhen §,*, Pingwei Li ⊥,*, Jingxin Wang †,*
PMCID: PMC11089548  PMID: 38746895

Abstract

graphic file with name ml4c00048_0008.jpg

Previously we identified a non-nucleotide agonist BDW568 that selectively activates the human STINGA230 allele. Here, we further characterized the mechanism of BDW568 and highlighted its potential use for selectively controlling the activation of engineered macrophages that constitutively express STINGA230 as a genetic adjuvant. We obtained the crystal structure of the C-terminal domain of STINGA230 complexed with BDW-OH (active metabolite) at 1.95 Å resolution. Structure–activity relationship studies revealed that all three heterocycles in BDW568 and the S-acetate side chain are critical for retaining activity. We demonstrated that BDW568 could robustly activate type I interferon signaling in purified human primary macrophages that were transduced with lentivirus expressing STINGA230. In contrast, BDW568 could not stimulate innate immune responses in human primary peripheral blood mononuclear cells in healthy donors in the absence of a STINGA230 allele. This high STING variant specificity suggested a promising application of STINGA230 agonists in macrophage-based therapeutic approaches.

Keywords: STINGA230, Agonist, Crystallography, Macrophage, Immunotherapy

The cyclic GMP-AMP synthase (cGAS)-STINGpathway is crucial for recognizing self or foreign double-stranded DNA and activating type I interferon (IFN-I) signaling via the interferon regulatory factor 3 (IRF3) axis.1,2 This pathway is indispensable for the innate immune responses in mammalian cells in events of bacteria or DNA virus infections.3,4 Pharmacological STING (STimulator of InterferoN Genes) activation has been demonstrated as a potential promising approach for the treatment of cancer and virus infections,5−10 as well as vaccine adjuvants.11−13 Human STING has genetic polymorphisms. Recent genetic analysis demonstrated that the most common STING allele (R71/G230/R232/R293) is present in 59.2% of the human population, followed by H71-A230-Q293 (HAQ) which occurs in 20.4%, H232 in 13.7%, A230-Q293 (AQ) in 5.2%, and Q293 in 1.5%.14 It was demonstrated that the STING alleles with H232 are defective in response to the stimulation of the natural ligand 2′,3′-cGAMP in vitro.3,15 In contrast, the biological impact of the STING-HAQ allele is controversial. Some studies found that the HAQ allele has impaired cGAS-dependent responses compared to the major STING allele,14,16−18 which was also partially supported by genome-wide association studies (GWAS) showing that STING-HAQ is linked to decreased IFN-I response to vaccinia virus in the European population.19 Another study claimed that the difference in innate response between STING-HAQ and wild-type alleles was insignificant.20 In addition to the four amino acid residues mentioned above, other rare STING variants exist. For example, STING-V155M variant is a constitutively active STING that causes a recessive form of STING-associated vasculopathy with onset in infancy.21

mRNA-encoding STING variants (e.g., V155M) have shown efficacy as genetic adjuvants in mouse tumor models in a CD8+ T cell-dependent manner.22 However, precise temporal control of the activity of these engineered immune cells is needed to avoid overactivation and other adverse effects associated with excessive inflammation. For this purpose, a STING variant-specific agonist is needed. To our knowledge, almost no reported human STING agonists can discriminate among STING variants except for BDW568 (Figure 1).5−7,23,24 BDW568 is a STING agonist newly identified by our laboratory which can selectively activate STINGA230 alleles,25 including HAQ and AQ in the human population. No activity of BDW568 against STINGG230 (major allele) was observed, suggesting a nearly complete selectivity in naturally occurring STING variants.25 BDW568 is a methyl ester and can be quickly hydrolyzed by cytosolic carboxylesterase 1 (CES1) after cellular uptake to yield the active metabolite BDW-OH (Figure 1).25 Here, we report the crystal structure of the STINGA230 C-terminal domain (CTD)-BDW-OH complex and structure–activity relationship (SAR) studies. We also validate the activity of BDW568 in primary human cells from healthy individuals with G230 or A230 alleles or purified primary macrophages with a transduced exogenous STINGA230 allele.

Figure 1.

Figure 1

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Structures of natural and synthetic STING agonists. The specificity of the compounds is indicated below each compound name (mSTING = mouse STING).

To acquire structural information on the STINGA230-BDW-OH complex, we expressed small ubiquitin-related modifier (SUMO) fusion of human STINGA230 CTD (residue 155–341) and cocrystallized the protein with purified BDW-OH or with the STING endogenous ligand, 2′,3′-cGAMP. The crystal structures of the STINGA230 CTD complexed with BDW-OH and 2′,3′-cGAMP were obtained at 1.95 and 2.01 Å resolution, respectively (Table S1, Protein Data Bank (PDB) IDs: 8T5K, 8T5L). Comparing these two structures, we found that the overall conformation of the STINGA230-BDW-OH complex is similar to that of the STINGA230-2′,3′-cGAMP complex (Figure 2). The STINGA230 CTD forms a butterfly-shaped dimer. Unlike the apo form of STING,26 in which the two “wings” from the two STING monomers are wide open, the STING–ligand complexes adopt a closed conformation (Figure 2A,B).

Figure 2.

Figure 2

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Overlay of STINGA230-BDW-OH complex (PDB: 8T5K) with (A) STING apo form (PDB: 4EMU) and (B) STINGA230-2′,3′-cGAMP complex (PDB: 8T5L). The distance between residues H185 from both monomers is shown to illustrate open (∼47 Å) and closed (34–37 Å) conformations. Interactions between STINGA230 CTD and (C) BDW-OH (PDB: 8T5K) or (D) 2′,3′-cGAMP (PDB: 8T5L). Superimposition of the bound structures of BDW-OH and (E) 2′,3′-cGAMPor (F) MSA-2 (PDB: 6UKM).

As expected, two molecules of BDW-OH occupy the 2′,3′-cGAMP binding pocket in the STINGA230 homodimer in a symmetric manner. The carboxylic acid group of BDW-OH engages R238/R232 of STING via hydrogen bonding, and the pyrimidine ring of BDW-OH also forms a hydrogen bond with T263 (Figure 2C). In addition, the triazole ring of BDW-OH forms π–π stacking with Y167 of the STING (Figure 2C). These interactions between the ligand and STING were also observed in structures of STING complexed with 2′,3′-cGAMP (Figure 2D) and MSA-2 (PDB: 6UKM),5 respectively. An overlay of BDW-OH and 2′,3′-cGAMP revealed that the tricyclic structures of BDW-OH are in the same plane as the two nucleobases of 2′,3′-cGAMP, maintaining the π–π stacking with Y167 (Figure 2E). Similarly, the tricyclic structure of BDW-OH also overlaps well with the benzothiophene ring of MSA-2. Importantly, both BDW-OH and MSA-2 use carboxylic acid to tackle R238/R232 in similar conformations (Figure 2F). Therefore, we concluded that the binding mode of STINGA230 and BDW-OH resembles the STING-MSA-2 complex. MSA-2 was previously shown to pre-dimerize in solution with a dissociation constant (KD) of 18 mM.5 In 1H NMR spectra of MSA-2, the aromatic protons exhibited significant chemical shift changes in a concentration-dependent manner due to the shielding effects arising from stacking of aromatic rings.5 As expected, similar proton chemical shift profiles were observed with various concentrations of BDW-OH in 1H NMR spectra, comparable with those observed with MSA-2 (Figure 3A). The protons close to the aromatic rings (H1, H2, and H3) showed significant chemical shift perturbations, but the methylene (H4) in the side chain did not (Figure 3A). The calculated KD values of the BDW-OH dimer are 9.4 and 17.5 mM using the chemical shift data for H1 and H3, respectively.5

Figure 3.

Figure 3

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(A) 1H NMR spectra of BDW-OH at various concentrations, demonstrating significant chemical shift changes of the aromatic proton (H1) and two aryl methyl groups (H2, H3). (B) Overlay of STINGA230-BDW-OH complex (PBD: 8T5K) and STINGI230-DMXAA complex (PDB: 4QXP). (C) Location of the amino acid residue 230 that is essential for maintaining the STING activation activity of BDW568.

Residue 230 of human STING is located in the lid region of a STING dimer (Figure 3B).27 The lid region is a four-stranded, antiparallel β-sheet that covers the binding pocket of STING. It was demonstrated that single-point mutation in the lid region can control the pharmacological activation of STING. For example, G230I mutation in human STING sensitized the mouse STING-specific DMXAA to bind.27 Similarly, the I229G or I229A mutation in mouse STING desensitized the DMXAA binding (residue 229 in mouse STING is homologous to residue 230 in humans).27 In human STING, the distance between residue 230 and BDW-OH in the crystal structure was measured at ∼7 Å (Figure 3C), which exceeds the range for typical direct ligand–receptor interactions. We further hypothesized that residue 230 functions as a gate for the exit tunnel of the ligand. As such, STING variants with bulkier amino acid residue 230 (e.g., alanine and isoleucine) can probably retain ligands in the binding pocket better than glycine.25

To further probe the interactions between BDW-OH and STINGA230, we performed SAR studies and used an interferon-sensitive response element (ISRE) reporter gene assay in THP-1 cells to evaluate the compounds’ activity.25 The ISRE response to IFN-I signaling is a key downstream event of STING activation and is routinely used to quantify potency for STING agonists.6 The genotype of STING in THP-1 cells is homozygous STINGHAQ.14 In this assay, the endogenous STING activator, 2′,3′-cGMAP, showed an activity (illustrated as half-maximal effective concentrations, or EC50 values) of 11.8 ± 2.8 μM. We previously demonstrated that STING activation in THP-1 cells using BDW568 is solely dependent on A230, but not H71 or Q293.25 The crystal structure of STINGA230-CTD-BDW-OH showed that there is a small unoccupied cavity on the dimethyl thiophene side in BDW568 (Figure 3A). To probe the size of the cavity, we linked the 4,5-dimethyl groups on the thiophene ring (A) with one carbon (1) or two carbons (2) (Figure 4). The activity of compound 1 was retained while compound 2 was inactive (Figure 4), implying the cavity in the binding pocket can only accommodate one additional carbon. An isomer of BDW568 having an ethyl group at C5 and hydrogen at C4 (3) is also active. In contrast, removal of the dimethyl groups (4) significantly weakened the activity (Figure 4). Substituting either of the two methyl groups with a methoxy group (5, 6) or replacing the 5-methyl group on the thiophene ring with a bromo group (7) significantly reduced the activity (Figure 4).

Figure 4.

Figure 4

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STINGA230 activation activities of BDW568 analogs with modifications on A/B/C rings in THP-1 cells (NA = not active).

In addition, we found that the skeleton of the tricyclic structure is essential for STING activation. For example, analogs replacing the thiophene ring (A) with pyrrole (8), furan (9), or dimethoxybenzene (10) are completely inactive (Figure 4). This is consistent with our previous report, in which we demonstrated that replacing the pyrimidine ring (B) with pyridine (11) or 2-methylpyrimidine (12),25 or triazole ring (C) with imidazole (13),25 all failed to retain the activity. Similarly, breaking the triazole ring (C) by using an N-carbamoylacetamide group (14) was also intolerable (Figure 4).

BDW568 is required to be hydrolyzed by CES1 to yield the carboxylic acid metabolite to interact with STINGA230.25 We tested other esters, such as ethyl (15) and isopropyl (16) esters,25 and observed a slightly weakened activity (Figure 5). Further increasing the bulkiness of the ester to tert-butyl ester (17)25 or changing the linear ester into a lactone (18) completely diminished the activity, probably because these esters can no longer be recognized by CES1. We synthesized a cell-permeable carboxylic isostere compound (19)25 and observed complete loss of activity. We concluded that a carboxylic acid group in BDW568’s active metabolite is required for STING binding. The side chain also needs to maintain a proper length to engage the R238 residue in STING, since addition (20) or reduction (21) of the chain length completely diminished the activity (Figure 5). Surprisingly, the thioether group in the side chain is also crucial for STING activation. We replaced the sulfur atom with oxygen (22) or a methylene group (23), or oxidized the thioether into sulfoxide (24), and found all these alterations rendered the compounds inactive (Figure 5). We speculate that multiple factors might influence the compound’s activity, including prodrug recognition by CES1,28 metabolism leading to inactivation, and binding affinity to the STING protein. These factors may all contribute to the unexpected inactivity observed with conservative modifications (e.g., 22 and 23).

Figure 5.

Figure 5

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STINGA230 activation activities of BDW568 analogs with modifications on the side chain in THP-1 cells (NA = not active).

Next, we explored the potential application of BDW568 in immunotherapy. In the first step, we confirmed the activity of BDW568 in THP-1 cells by real-time quantitative PCR (RT-qPCR) and found that THP-1 cells have elevated expression of interferon stimulated genes (ISGs), including interferon-induced GTP-binding protein (MX1) and 2′,5′-oligoadenylate synthetase 1 (OAS1), after BDW568 stimulation (Figure 6A). We selected the MX1 gene as a marker to quantify IFN-I activation in the following experiments. Then, to examine the activity of BDW568 in primary human cells, we collected peripheral blood mononuclear cells (PBMCs) from 15 healthy donors and measured MX1 expression in the presence of BDW568 and 2′,3′-cGAMP. The PBMCs from donor 9 responded to BDW568, while those from all donors responded to 2′,3′-cGAMP (Figure 6B). We genotyped donor 9 and confirmed that this individual carries homozygous STINGA230. The other 14 donors do not have a STINGA230 allele, indicating that the host immune cells did not respond to BDW568 without STINGA230.

Figure 6.

Figure 6

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BDW568 stimulates ISG expression in THP-1 cells and primary PBMCs that carry the STINGA230. (A) THP-1 cells were stimulated with either vehicle or BDW568 (50 μM) for 6 h. Afterward, cells were harvested and RT-qPCR was performed to measure expression levels of MX1 and OAS1. (B) Primary PBMCs were collected from 15 independent healthy donors, and cells were then stimulated with BDW568 (50 μM) and 2′,3′-cGAMP (1 μg/mL) for 6 h. Afterward, the cells were harvested and RT-qPCR was performed to measure the MX1 expression level. Donor 9 (red) showed significant elevation of MX1 after BDW568 stimulation.

Encouraged by the promising results in human primary cells, we envision that BDW568 can be a useful probe to selectively activate the STING pathway in genetically engineered immunogenic cells that carry the STINGA230 allele. To examine if we can genetically engineer cells with STINGA230 to respond to BDW568, we generated lentivirus that will allow constitutive expression of full-length STINGA230 and a marker protein, which is a truncated inactive EGF receptor (EGFRt) (Figure 7). Monocyte-derived macrophages were generated from 3 independent healthy donors with STINGG230 alleles and transduced with EGFRt-STINGA230 lentivirus. Three days after transduction, cells were stimulated with vehicle or BDW568. We measured the expression levels of two ISGs, MX1 and IRF7. As expected, no significant elevations of these genes were observed in mock transduced cells or untransduced cells after BDW568 stimulation. In contrast, macrophages expressing EGFRt-STINGA230 showed a 10-fold increase in MX1 and a 5-fold increase in IRF7 transcriptions (Figure 7). These results imply a robust selectivity for pharmacological activation of STINGA230 engineered macrophages without affecting the untransduced cells. As such, a STINGA230-specific agonist can potentially be used in STINGG230 patients to selectively activate engineered cells in cellular therapy, such as chimeric antigen receptor (CAR)-macrophages, which recently showed promising activity to treat solid tumors.29

Figure 7.

Figure 7

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BDW568 stimulation showed upregulated expression of ISGs MX1 and IRF7 in primary macrophages transduced with lentivirus STINGA230 as compared with untransduced macrophages. CD14+ monocytes were differentiated into macrophages in the presence of M-CSF. Cells were transduced with lentivirus FG12-EF 1p-EGFRt-p2A-STINGA230 or mock transduced and stimulated with DMSO (vehicle) or BDW568 for 6 h. The expression levels of MX1 and IRF7 were measured by RT-qPCR (n = 3, ***P < 0.0005, **P < 0.01).

In summary, we obtained the crystal structure of STINGA230 with a newly reported STING agonist, BDW568. In the STINGA230-BDW-OH (active metabolite of BDW568) complex the STING dimer adopts a “closed” conformation, which is almost identical to that of the STINGA230-2′,3′-cGAMP (endogenous ligand) complex. By comparing the structure of the STINGA230-BDW-OH complex with other reported structures, we concluded that the binding mode of BDW-OH is similar to that of a known synthetic STING ligand, MSA-2. SAR studies demonstrated that the skeleton in all three heterocycles is crucial for BDW568’s activity and probably cannot be extensively modified. We also found that the S-acetate side chain is essential to maintain the activity. We confirmed that BDW568 could activate PBMCs with the STINGA230 allele, while the compound could not activate PBMCs with the major STINGG230 allele. Importantly, BDW568 could selectively activate macrophages that were transduced with STINGA230. Therefore, the compound may be used to activate STINGA230 engineered macrophages for macrophage-based immunotherapies, including CAR-macrophage therapies.

Acknowledgments

The research described herein was funded by the National Institutes of Health, grant numbers P20GM103638 (J.W.), R35GM147498 (J.W.), R01AI145287 (P.L.), R01DA052841 (A.Z.), R01AI172727 (A.Z.), and R21AI155117 (A.Z.), the University of Kansas Research Grant Opportunity (J.W.), and Welch Foundation grant A-2107 (P.L.). The work at UCLA was also supported by the UCLA AIDS Institute, the James B. Pendleton Charitable Trust, and the McCarthy Family Foundation. We thank M. Farrell at the University of Kansas and W. Liu at Texas A&M University for discussions.

Glossary

Abbreviations

STING

stimulator of interferon genes

cGAS

cyclic GMP-AMP synthase

2′,3′-cGAMP

2′,3′-cyclic GMP-AMP

IFN-1

type I interferon

IRF3

interferon regulatory factor 3

GWAS

genome-wide association studies

CES1

cytosolic carboxylesterase 1

CTD

c-terminal domain

SUMO

small ubiquitin-related modifier

SAR

structure–activity relationship

ISRE

interferon-sensitive response element

ISG

interferon stimulated gene

OAS1

2′,5′-oligoadenylate synthetase 1

PBMC

peripheral blood mononuclear cell

CAR

chimeric antigen receptor

EGFRt

truncated EGF receptor

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.4c00048.

  • Experimental details; data collection and refinement statistics for STINGA230–ligand complexes; chemical synthesis; 1H and 13C NMR spectra; purity analysis of active compounds (PDF)

Author Present Address

# Biological Sciences Division, Department of Medicine, Section of Genetic Medicine, University of Chicago, Chicago, Illinois 60637, United States

Author Contributions

‡ Z.T., J.Z., and Y.L. contributed equally. Z.T., M.S., and J.W. performed the chemical synthesis. Y.L., D.S., and P.L. expressed the STINGA230 protein, performed crystallography, and analyzed the structural data. J.Z., Y.L., and D.K.J. visualized the structures. J.Z., S.T., N.T., and J.W. performed biological evaluation in THP-1 cells and analyzed the data. S.T., N.T., and A.Z. performed STING stimulation experiments in primary cells. J.W. wrote the manuscript with input from all the authors.

The authors declare no competing financial interest.

Supplementary Material

ml4c00048_si_001.pdf (3.6MB, pdf)

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