¹²⁵I-SDF-1α-CXCR4 ( Ki = 79.7 pM ); ¹²⁵I-SDF-1α-CXCR4 ( Ki = 49.5 pM )
LY-2510924 (LY2510924) is a novel, potent, and selective peptide antagonist of the C-X-C chemokine receptor type 4 (CXCR4). [2]

LY2510924 selectively inhibits SDF-1-induced GTP binding with a Kb value of 0.38 nM and blocks SDF-1 binding to CXCR4 with an IC50 value of 0.079 nM. LY2510924 inhibits SDF-1-induced cell migration and SDF-1/CXCR4-mediated intracellular signaling in human lymphoma U937 cells that express endogenous CXCR4. The IC50 value of this inhibitor is 0.26 nM. Tumor cells stimulated with SDF-1 show concentration-dependent inhibition of phospho-ERK and phospho-Akt. LY2510924 does not appear to have any agonist activity, according to biochemical and cellular analyses[1]. The main effects of LY2510924 are to reduce stromal cell defense against chemotherapy and to inhibit AML cell proliferation with minimal induction of cell death[2].

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LY-2510924 rapidly and durably blocked surface CXCR4 on human AML cell lines (OCI-AML3, U937, MOLM-13) in a concentration-dependent manner, as measured by flow cytometry using anti-CXCR4 antibody 12G5. It was more potent than the small-molecule CXCR4 antagonist AMD3100. Receptor occupancy started within 1 minute and lasted up to 72 hours at 10 nM [2].
LY-2510924 (1 nM) abolished stromal cell-derived factor-1α (SDF-1α)-induced chemotaxis of OCI-AML3 cells and primary AML cells in Transwell assays, whereas 1 µM AMD3100 did not [2].
LY-2510924 (1 µM) inhibited SDF-1α-induced phosphorylation of ERK and AKT in OCI-AML3 and U937 cells, as shown by western blot analysis [2].
LY-2510924 (1 µM) inhibited the proliferation of OCI-AML3 cells over an 8-day culture period, both in the presence and absence of SDF-1α. It did not induce significant apoptosis in monoculture [2].
In a co-culture system with MS-5 stromal cells, LY-2510924 (1 µM) significantly reversed the protective effect of stromal cells against cytarabine-induced apoptosis in OCI-AML3 cells [2]
.

LY2510924 selectively inhibits SDF-1-induced GTP binding with a Kb value of 0.38 nM and blocks SDF-1 binding to CXCR4 with an IC50 value of 0.079 nM. LY2510924 inhibits SDF-1-induced cell migration and SDF-1/CXCR4-mediated intracellular signaling in human lymphoma U937 cells that express endogenous CXCR4. The IC50 value of this inhibitor is 0.26 nM. Tumor cells stimulated with SDF-1 show concentration-dependent inhibition of phospho-ERK and phospho-Akt. LY2510924 does not appear to have any agonist activity, according to biochemical and cellular analyses[1]. The main effects of LY2510924 are to reduce stromal cell defense against chemotherapy and to inhibit AML cell proliferation with minimal induction of cell death[2].

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In NSG mice engrafted with OCI-AML3/Luc/mCherry cells, daily treatment with LY-2510924 (starting day 12 post-injection) significantly reduced bioluminescence imaging (BLI) signal (tumor burden) compared to controls. Histological analysis showed less leukemic infiltration in bone marrow (BM), spleen, and liver. This translated into a significant prolongation of median survival (40 days vs 26 days in controls) [2].
In NSG mice engrafted with primary AML cells (high CXCR4 expression), daily LY-2510924 treatment (starting day 25) initially mobilized leukemic cells into circulation (increased at 3 and 24 hours after first dose) and subsequently reduced leukemic burden in blood, BM, and spleen by day 45. Treatment also significantly prolonged median survival (56 days vs 44 days in controls) [2].
In the OCI-AML3/Luc/GFP xenograft model, LY-2510924 monotherapy prolonged survival compared to controls. Its combination with chemotherapy (cytarabine/doxorubicin) resulted in the lowest tumor burden (by BLI and histology) and the longest median survival (62 days), showing a synergistic effect [2].
Mechanistically, phospho-flow cytometry on leukemic cells from primary AML xenografts showed that LY-2510924 treatment reduced phosphorylation of AKT and ERK in vivo. Gene expression profiling (GEP) of leukemic cells from treated mice suggested loss of SDF-1α/CXCR4 signaling, reduced proliferation, and induction of myeloid differentiation, supported by increased CD11c+ cells in tissues [2].
Western blot analysis on sorted leukemic cells from treated mice showed that LY-2510924 attenuated phospho-AKT, led to GSK-3β dephosphorylation (activation), and decreased levels of activated β-catenin in vivo [2]
.

LY2510924 selectively inhibits SDF-1-induced GTP binding with a Kb value of 0.38 nM and blocks SDF-1 binding to CXCR4 with an IC50 value of 0.079 nM. LY2510924 inhibits SDF-1-induced cell migration and SDF-1/CXCR4-mediated intracellular signaling in human lymphoma U937 cells that express endogenous CXCR4. The IC50 value of this inhibitor is 0.26 nM. Tumor cells stimulated with SDF-1 show concentration-dependent inhibition of phospho-ERK and phospho-Akt. LY2510924 does not appear to have any agonist activity, according to biochemical and cellular analyses[1]. The main effects of LY2510924 are to reduce stromal cell defense against chemotherapy and to inhibit AML cell proliferation with minimal induction of cell death[2].

After harvesting U937 cells, they are gently rinsed once with chemotaxis assay buffer, which is made with 1× RPMI medium that contains 10 mmol/L HEPES, pH 7.5, and 0.3% BSA. At a density of 5 × 10⁶ cells/mL, the cells are then resuspended in assay buffer. A 96-well ChemoTx plate is used for the assay. Usually, the lower chamber was filled with 30 μL of SDF-1α (10 ng/mL) prepared in 1× chemotaxis buffer, while the upper chamber was plated with 50 μL of cell mixture, either with or without LY2510924. The plate is then incubated at 37°C for 2.5 hours. After the incubation period, the lower chamber is filled with 5 μL of CellTiter 96 AQ. The plate is then incubated at 37°C for 60 minutes. By measuring the absorbance at 492 nm, the migrated cells can be identified.

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CXCR4 Surface Expression and Occupancy Assay: AML cell lines or primary cells were treated with various concentrations of LY-2510924 or comparator for specified times. Cells were then harvested, stained with fluorescently labeled antibodies against different CXCR4 epitopes (e.g., 12G5-APC, 1D9-PE), and analyzed by flow cytometry. Antibody 12G5 binding is blocked when the receptor is occupied by the antagonist, serving as an inverse indicator of drug occupancy [2].
Chemotaxis (Migration) Assay: AML cells were placed in the upper chamber of Transwell inserts (5 µm pore size). The lower chamber contained medium with or without SDF-1α (e.g., 50 ng/mL) and with or without LY-2510924 or control. After incubation (e.g., 2.5-12 hours), cells that migrated to the lower chamber were counted. Results were expressed as the percentage of migrated cells relative to input [2].
Western Blot Analysis for Signaling Pathways: Cells were serum-starved overnight, pretreated with or without LY-2510924 (e.g., 1 µM for 1 hour), and then stimulated with SDF-1α (e.g., 100 ng/mL for 10 minutes). Cells were lysed, proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phosphorylated (e.g., p-AKT Ser473, p-ERK Thr202/Tyr204) and total forms of signaling proteins. Detection was performed using infrared secondary antibodies and an imaging system [2].
Co-culture Chemoprotection Assay: AML cells (e.g., OCI-AML3) were cultured alone or co-cultured with stromal cells (e.g., MS-5 or human mesenchymal stromal cells) for 72 hours in the presence of chemotherapy (e.g., cytarabine) with or without LY-2510924. Apoptosis in AML cells was quantified by flow cytometry using annexin V/DAPI staining [2]

Mice: MDA-MB-231 cells are injected intravenously into female SCID mice, and they receive subcutaneous injections of either vehicle (1×PBS) or LY2510924 (3 mg/kg) formulated in 1×PBS. The animals in Groups 1 and 2 are treated with either a vehicle or 3 mg/kg of LY2510924 twice a day for several days, starting one day prior to the injection of tumor cells. Treatment for Group 3 animals starts one day after tumor cell injection and lasts for 13 days, with each animal receiving 3 mg/kg of LY2510924 15 twice daily. Lung lobes are visible in histologic sections after lung tissues are preserved in 10% neutral-buffered formalin for at least 24 hours following treatment[1].

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Xenograft Models and Treatment: NOD/SCID/IL-2rγnull (NSG) mice were used. To establish leukemia, mice were intravenously injected with human AML cell lines (e.g., OCI-AML3 engineered with Luciferase and fluorescent markers) or primary AML cells from patients. Engraftment was monitored by flow cytometry of peripheral blood for human cells or by bioluminescence imaging (BLI). Once engraftment was confirmed, mice were divided into treatment groups. LY-2510924 was administered via daily subcutaneous injections. Treatment start days varied (e.g., day 8, 12, or 25 post-cell injection). In combination therapy studies, chemotherapy (cytarabine and/or doxorubicin) was administered intraperitoneally, typically 3 hours after LY-2510924 injection. Control groups received vehicle [2].
Assessment: Tumor burden was monitored longitudinally by BLI. At defined endpoints or when mice became moribund, mice were euthanized. Blood, bone marrow, spleen, and liver were collected. Leukemic infiltration was quantified by flow cytometry (using human-specific markers like CD45, CD34), histological examination with immunohistochemistry (e.g., for human CD45, CD11c), and multispectral image analysis. Survival was recorded as the primary endpoint [2]

The study mentioned that a previous Phase I study in patients with advanced cancer showed that LY-2510924 had good pharmacokinetic properties [2].

The study noted that, based on survival curves and the absence of additional deaths in the combination therapy group, the toxicity of LY-2510924 in combination with chemotherapy appeared to be similar to that of chemotherapy alone. The study also cited recent clinical studies of other CXCR4 antagonists that showed no additional myelosuppression when used in combination with chemotherapy. [2]

[1]. Identification of LY2510924, a novel cyclic peptide CXCR4 antagonist that exhibits antitumor activities in solid tumor and breast cancer metastatic models. Mol Cancer Ther. 2015 Feb;14(2):480-90.

[2]. Antileukemia activity of the novel peptidic CXCR4 antagonist LY2510924 as monotherapy and in combination with chemotherapy. Blood. 2015 Jul 9;126(2):222-32.

LY-2510924 is a cyclic peptide CXCR4 antagonist [2]. This study showed that LY-2510924 effectively blocks the SDF-1α/CXCR4 axis, acting not only as a chemosensitizer but also as a single drug in preclinical AML models, exhibiting intrinsic antileukemic activity [2]. Its mechanism of action includes: releasing leukemia cells from the protective microenvironment, inhibiting SDF-1α-induced pro-survival signaling pathways (PI3K/AKT, MAPK), inhibiting cell proliferation, weakening the Wnt/β-catenin signaling pathway, and inducing myeloid differentiation [2]. The results suggest that in future clinical applications of AML, more effective and longer-lasting CXCR4 inhibitors like LY-2510924 may be needed to achieve optimal antileukemic effects [2].

C62H88N14O10

1189.45

1188.68

C, 62.55; H, 7.37; N, 15.30; O, 14.78

1088715-84-7

25102787

White to off-white solid powder

1.3±0.1 g/cm3

1.641

0.58

14

13

25

86

2200

7

C([C@H]1C(NCC(N[C@H](CCC(N[C@H](C(N[C@H](C(N[C@H](C(N[C@@H](C(N1)=O)CCCNC(N)=N)=O)CCCCNC(C)C)=O)CC1C=CC(=CC=1)O)=O)CC1C=CC=CC=1)=O)C(=O)N[C@H](C(=O)N)CCCCNC(C)C)=O)=O)C1C=CC2C=CC=CC=2C=1

IJHWVENTEFSNBC-OFPUNPKKSA-N

InChI=1S/C62H88N14O10/c1-38(2)66-30-12-10-19-46(55(63)80)72-59(84)49-28-29-53(78)71-51(34-40-15-6-5-7-16-40)60(85)76-52(35-41-23-26-45(77)27-24-41)61(86)74-47(20-11-13-31-67-39(3)4)57(82)73-48(21-14-32-68-62(64)65)58(83)75-50(56(81)69-37-54(79)70-49)36-42-22-25-43-17-8-9-18-44(43)33-42/h5-9,15-18,22-27,33,38-39,46-52,66-67,77H,10-14,19-21,28-32,34-37H2,1-4H3,(H2,63,80)(H,69,81)(H,70,79)(H,71,78)(H,72,84)(H,73,82)(H,74,86)(H,75,83)(H,76,85)(H4,64,65,68)/t46-,47-,48+,49+,50-,51-,52+/m0/s1

(2S,5R,8S,11R,14S,20R)-N-[(2S)-1-amino-1-oxo-6-(propan-2-ylamino)hexan-2-yl]-2-benzyl-11-[3-(diaminomethylideneamino)propyl]-5-[(4-hydroxyphenyl)methyl]-14-(naphthalen-2-ylmethyl)-3,6,9,12,15,18,23-heptaoxo-8-[4-(propan-2-ylamino)butyl]-1,4,7,10,13,16,19-heptazacyclotricosane-20-carboxamide

LY2510924; LY-2510924; LY 2510924; Cyclo[Phe-Tyr-Lys(iPr)-D-Arg-2-Nal-Gly-D-Glu]-Lys(iPr)-NH2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO: ≥ 125 mg/mL (~105.1 mM)
Water: ~100 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (1.75 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
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 (1.75 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (1.75 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.)