CXCR2; CXCR1
SX-682 targets CXCR1 and CXCR2 [1]
SX-682 acts as an allosteric inhibitor of CXCR1 and CXCR2 [1]

In vitro activity: SX-682 is a brand-new, highly effective, and selective CXCR2 chemokine receptor antagonist that may have anticancer effects. The proliferation and progression of tumor cells are facilitated by the upregulation of the CXC chemokine receptor CXCR2 in a range of distinct tumor cell types. Reduced tumorigenesis and metastasis were the results of CXCR2 inhibition. SX-682 is a CXCR2 antagonist that may be used to treat melanoma and prostate cancer that is resistant to castration. Syntrix Biosystems, Inc. has initiated a phase I clinical trial combining SX-682 and pembrolizumab for the treatment of metastatic melanoma.

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SX-682 (1 μM) had no direct effect on the viability of MOC1 and LLC tumor cells, as assessed by impedance analysis; it also did not induce apoptosis in these tumor cells when exposed for 24 hours [2]
- SX-682 (1 μM) did not alter the extracellular matrix invasion capacity of MOC1 and LLC cells when CXCL1 (50 ng/ml) was used as the chemoattractant [2]
- SX-682 (1 μM) did not affect the ability of activated OT-I T cells to kill SIINFEKL-expressing MOC1 or LLC tumor cells in vitro, as measured by impedance analysis [2]
- In in vitro assays, SX-682 did not directly impact the immunogenicity of MOC1 and LLC tumor cells [2]

In vivo, the whole tumor accumulation of CXCL1 in MOC1 and LLC tumors is notably higher than in normal lung and oral mucosa, respectively, and is not reduced by SX-682 treatment. In both models, tumor-bearing mice exhibit higher plasma accumulation of CXCL1 than naive mice, and this accumulation increases after SX-682 treatment. SX-682 treatment of mice with MOC1 or LLC tumors starting 10 or 20 days after the tumor initiation does not change the expression of CXCR1 or CXCR2 on tumor cells in vivo. [2] Tumor PMN-MDSC expression of the genes that produce H2O2, superoxide dismutase 1/2 or cell surface TGF-β, is not significantly changed after receiving in vivo SX-682 treatment. [3]

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In wild-type B6 mice bearing MOC1 or LLC tumors, treatment with SX-682 via chow (started on day 10 or day 20 after tumor implantation) significantly reduced the infiltration of CXCR2⁺ PMN-MDSCs into tumors, while having no significant effect on Ly6Gᵒ Ly6Cʰⁱ myeloid cell accumulation in tumors, spleens or bone marrow [2]
- SX-682 treatment enhanced the infiltration of endogenous CD8⁺ and CD4⁺ TILs into MOC1 and LLC tumors; it also upregulated PD-L1 expression on tumor cells, and this upregulation was dependent on CD8⁺ T cells [2]
- Combination of SX-682 with PD-1 mAb (200 μg i.p. twice weekly for 4 injections) significantly enhanced tumor growth control and improved survival in mice bearing established MOC1 or LLC tumors, compared with monotherapy of either agent; depletion of CD8⁺ cells abrogated this synergistic effect [2]
- In RAG1⁻/⁻ mice bearing SIINFEKL-expressing MOC1 or LLC tumors, SX-682 treatment (started on day 7) increased the tumor infiltration of adoptively transferred OT-I T cells (1 × 10⁶ cells on day 10) and enhanced tumor growth inhibition and survival benefit from adoptive T cell transfer [2]
- In wild-type C57BL/6 mice bearing MOC2 tumors, SX-682 treatment (started on day 7, for 7 days) combined with adoptively transferred KIL NK cells (5×10⁶ cells three times weekly for 2 weeks) significantly reduced tumor growth and improved survival, compared with monotherapy of SX-682 or KIL cells alone [3]
- SX-682 treatment inhibited the trafficking of fluorescently labeled splenic PMN-MDSCs and M-MDSCs into MOC2 tumors in adoptive transfer experiments (1×10⁷ MDSC/mouse), and enhanced the tumor infiltration, IFNγ production and granzyme B expression of adoptively transferred KIL NK cells [3]

Tumor cell viability and apoptosis assay [2]
: MOC1 and LLC tumor cells were plated in increasing doses of SX-682, and cell viability was evaluated by impedance analysis over time. For apoptosis assessment, the tumor cells were exposed to SX-682 (1 μM) for 24 hours, then stained with apoptosis-detecting reagents and analyzed by flow cytometry to determine the proportion of apoptotic cells.
- Cell invasion assay [2]
: MOC1 and LLC cells were seeded in the upper chamber of invasion chambers with extracellular matrix-coated membranes; the lower chamber contained 10% FBS (positive control) or CXCL1 (50 ng/ml) as the chemoattractant, with or without SX-682 (1 μM). After incubation, the number of cells that invaded through the membrane was counted to assess invasion capacity.
- T cell killing assay [2]
: SIINFEKL-expressing MOC1 or LLC cells were co-cultured with activated OT-I T cells in the presence or absence of SX-682 (1 μM). The killing effect of T cells on tumor cells was monitored in real-time by impedance analysis, and the percentage of tumor cell killing was quantified at 8 hours.
- NK cell function assay [3]
: Splenic NK cells or KIL cells were isolated from mice, and co-cultured with MOC2 tumor cells at different effector-to-target (E:T) ratios in the presence or absence of PMN-MDSCs and SX-682. The killing ability of NK cells was assessed by impedance analysis, and the quantification of tumor cell killing was performed at 12 hours.

wild-type C57BL/6 mice subcutaneously injected with MOC2 cells (1x105 cells in PBS)
500 mg/kg
Oral gavage

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MOC1/LLC tumor model in wild-type B6 mice [2]
: Wild-type B6 mice were subcutaneously implanted with MOC1 or LLC tumor cells to establish tumor models. SX-682 was administered via chow, starting on day 10 or day 20 after tumor implantation, and tumor growth was monitored regularly. For combination therapy with PD-1 mAb, PD-1 mAb (200 μg per mouse) was injected intraperitoneally twice weekly for a total of 4 injections, starting on day 10. CD8⁺ cell depletion was achieved by intraperitoneal injection of anti-CD8 antibody (clone YTS 169.4, 200 μg) twice weekly.
- SIINFEKL-expressing MOC1/LLC tumor model in RAG1⁻/⁻ mice [2]
: RAG1⁻/⁻ mice were implanted with SIINFEKL-expressing MOC1 or LLC tumor cells. SX-682 chow was given starting on day 7 after tumor implantation. On day 10, 1 × 10⁶ OT-I T cells were adoptively transferred via intravenous injection. Tumor growth and mouse survival were recorded, and tumor infiltration of OT-I T cells was analyzed 12 hours after transfer by flow cytometry.
- MOC2 tumor model in wild-type C57BL/6 mice [3]
: Wild-type C57BL/6 mice were implanted with MOC2 tumor cells. SX-682 treatment was initiated on day 7 after tumor implantation and continued for 7 days via chow. Adoptive transfer of KIL NK cells was performed at a dose of 5×10⁶ cells per mouse, three times weekly for 2 weeks, starting on day 7. Tumor growth was measured every few days, and survival was monitored for an extended period.
- MDSC adoptive transfer assay [3]
: Splenic PMN-MDSCs or M-MDSCs were isolated from mice bearing 14-day-old MOC2 tumors, fluorescently labeled, and adoptively transferred (1×10⁷ cells per mouse) into mice bearing 15-day-old MOC2 tumors treated with control or SX-682 chow (started on day 7). Tumors were harvested 18 hours after transfer, and the number of fluorescently labeled MDSCs in tumors was quantified by flow cytometry.
- NK cell adoptive transfer and functional analysis [3]
: KIL NK cells were fluorescently labeled and adoptively transferred into mice bearing day 10 MOC2 tumors treated with or without SX-682 (started on day 7). Tumor infiltration of KIL cells was analyzed by fluorescent imaging and flow cytometry 4 hours after injection; 24 hours after injection, tumor leukocytes were isolated, stimulated with PMA/Iono plus brefeldin, and the expression of IFNγ and granzyme B in KIL cells was detected by flow cytometry.

SX-682 is a small molecule inhibitor with high oral bioavailability.[2][3]

[1]. Nature . 2017 Mar 30;543(7647):728-732.

[2]. JCI Insight . 2019 Apr 4;4(7):e126853.

[3]. Clin Cancer Res . 2020 Mar 15;26(6):1420-1431.

SX-682, a CXCR1/2 inhibitor, is an orally bioavailable, selective, and reversible CXC motif chemokine receptor 1 (CXCR1) and 2 (CXCR2) antagonist with potential anti-inflammatory and antitumor activities. Upon administration, SX-682 selectively allosterically binds to CXCR1 and CXCR2, inhibiting the activation of these receptors by tumor-secreted chemokines. This inhibits CXCR1/2-mediated signaling, reduces the recruitment and migration of immunosuppressive myeloid-derived suppressor cells (MDSCs) and neutrophils in the tumor microenvironment (TME), suppresses inflammatory processes, and eliminates the immunosuppressive properties of the TME. This enables effector cells, such as natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), to kill and eliminate cancer cells. This can inhibit tumor cell migration, metastasis, angiogenesis, and proliferation. CXCR1 and CXCR2 are G protein-coupled receptors located on myeloid cells and certain tumor cells, playing a key role in the immunosuppressive properties of the tumor microenvironment, tumor metastasis, treatment resistance, and myeloid cell suppression. They play a key role in inflammation and are upregulated in a variety of inflammation-driven diseases. SX-682 is a small molecule allosteric inhibitor of CXCR1 and CXCR2 designed to block the migration of MDSCs to tumors [1][2][3] - The therapeutic effect of SX-682 is mainly achieved by inhibiting the recruitment of CXCR2⁺ PMN-MDSCs to the tumor microenvironment, thereby reducing MDSC-mediated immunosuppression and enhancing the efficacy of T-cell or NK-cell-based immunotherapies [2][3] - SX-682 has little direct antitumor effect on MOC1, LLC, and MOC2 tumor models, and its therapeutic benefits depend on the modulation of the tumor immune microenvironment [2][3]

C19H14BF4N3O4S

467.20

467.07

C, 48.85; H, 3.02; B, 2.31; F, 16.27; N, 8.99; O, 13.70; S, 6.86

1648843-04-2

90467234

Off-white to light yellow solid powder

1.53±0.1 g/cm3(Predicted)

3

11

7

32

606

0

S(C1N=CC(=CN=1)C(NC1C=CC(=CC=1)F)=O)CC1C=C(C=CC=1B(O)O)OC(F)(F)F

SDUDZBCEHIZMFZ-UHFFFAOYSA-N

InChI=1S/C19H14BF4N3O4S/c21-13-1-3-14(4-2-13)27-17(28)12-8-25-18(26-9-12)32-10-11-7-15(31-19(22,23)24)5-6-16(11)20(29)30/h1-9,29-30H,10H2,(H,27,28)

[2-[[5-[(4-fluorophenyl)carbamoyl]pyrimidin-2-yl]sulfanylmethyl]-4-(trifluoromethoxy)phenyl]boronic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 11.76 mg/mL (25.17 mM) in 15% Cremophor EL + 85% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication (<60°C).
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.45 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)