CX3CR1 ( IC50 = 0.32 nM )

JMS-17-2 (10 mg/kg; intraperitoneal injection; twice daily for three days) significantly reduced the amount of tumors in the visceral and peripheral organs in SCID mice [1].

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Building on the target validation studies described above, we synthesized JMS-17-2, a small-molecule antagonist of CX3CR1, by exploiting important pharmacophoric features of non-specific chemokine antagonists and combining these with drug-like elements of G-protein coupled receptor ligands (Fig.3A and methods). JMS-17-2 potently antagonizes CX3CR1 signaling in a dose-dependent fashion, as measured by inhibition of ERK phosphorylation (Fig.3B and C). Interestingly, the two concentrations of JMS-17-2 most effective in blocking FKN-induced ERK phosphorylation also significantly reduced the migration of breast cancer cells in vitro [1].

JMS-17-2 (10 mg/kg; intraperitoneal injection; twice daily for three days) resulted in significant reductions in peripheral and visceral organ tumors in SCID mice [1]. Animal model: SCID mouse (~25g) MDA-231 xenograft [1] Dose: 10 mg/kg Administration: intraperitoneal injection; twice daily for three weeks Results: Significant tumor growth in bones and visceral organs reduce.

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Researchers then sought to ascertain the effects of JMS-17-2 on the conversion of breast CTCs into skeletal DTCs, in our relevant pre-clinical model of metastasis. Pharmacokinetic evaluation of JMS-17-2 administered to mice at a dose of 10mg/Kg (i.p.) produced drug levels of 89ng/ml (210 nM) in blood measured one hour after dosing, which corresponds to a 20-fold increase over the lowest fully effective dose of this compound in vitro (10nM, see fig. 3B,C). Thus, a first group of mice received MDA-231 cancer cells pre-incubated with 10nM JMS-17-2, whereas a second group of animals was dosed with JMS-17-2 (10mg/Kg; i.p.) twice, one hour prior and three hours after the IC inoculation of cancer cells, to maximize target engagement. Remarkably, both experimental groups showed a reduction in DTCs of approximately 60% as compared to control animals treated with vehicle (Fig. 3D and E)

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Reducing tumor seeding directly impairs colonization and growth [1]
The prognostic value of breast DTCs detected in the bone marrow of patients has been established, with higher values corresponding to a poorer clinical outcome. Since JMS-17-2 did not completely abolish the seeding of breast CTCs to the skeleton, we aimed to determine if and to which extent the reduction in skeletal DTCs would translate into a long-term inhibition of tumor growth. To this end, mice were grafted with MDA-231 cells engineered to express both fluorescent and bioluminescent markers and monitored by in vivo imaging during the two weeks following IC injection. Furthermore, the presence of skeletal tumors and DTCs was also assessed post-necropsy by examining bone tissue sections by multispectral fluorescence microscopy. These experiments showed that, in contrast to control animals presenting multiple tumors both in the skeleton and visceral sites, seven of the eight animals that received cancer cells pre-incubated with JMS-17-2 were found free of tumors (Fig. 4A and B). Notably, these animals were also found devoid of microscopic tumor foci and DTCs when inspected for fluorescent signals with multispectral microscopy-based imaging of frozen tissue sections (Fig. 4C).

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To determine whether the pharmacologic targeting of CX3CR1 with JMS-17-2 would produce similar effects, mice were grafted with MDA-231 cells and at the first week post-IC injection were randomized and administered with either vehicle or 10mg/Kg JMS-17-2 i.p. twice daily for three weeks, based on the results of our pharmacokinetics studies with this compound. Animals were imaged weekly before being euthanized and we observed that treatment with JMS-17-2 led to a reduction in the number of tumor foci and overall tumor burden, at least as effectively as observed using CRISPRi (Fig. 5D and E). Taken together, these results point towards a crucial role of CX3CR1 in dictating seeding, colonization and progression of disseminated breast cancer cells. We decided to ascertain whether interfering with CX3CR1 functioning could alter the expression of genes with an established role in tumorigenesis. Thus, tumor tissues collected by LCM (Fig. 5F) were interrogated using Nanostring technology for the expression of 730 genes including 606 genes regulating 13 canonical signaling pathways and 124 cancer driver genes (PanCancer Panel). A comparative analysis of CRISPRi and JMS-17-2 treatment showed that nine genes were similarly altered, with WNT5a being the only up-regulated gene (Fig. 5G and table 1). Notably, both pharmacologic and genomic targeting of CX3CR1 resulted in a strong down-regulation of NOTCH3 (table 1) and significant deregulation of the Notch signaling pathway (Supplementary Fig. S4).

Pharmacophore design and synthesis of JMS-17-2 [1]
A potent CCR1 antagonist, was previously found to bind the cytomegalovirus receptor US28. Since FKN also binds potently to this receptor, we speculated that this compound would also engage CX3CR1. Thus, we synthesized this CCR1 antagonist (named Compound-1) and found it to be also a functional antagonist of CX3CR1 with an IC50 = 268 nM, established by measuring the inhibition of FKN-stimulated ERK1/2 phosphorylation detected with a plate-based assay. Subsequently, we optimized Compound-1 by modifying the diphenyl acetonitrile moiety and combining it with the right-hand aryl piperidine motif, which led to the discovery of a lead series and the compound JMS-17-2 (IC50 = 0.32 nM). The favorable potency shown by JMS-17-2 was combined with significant selectivity for CX3CR1 over other chemokine receptors such CXCR2 and CXCR1, for which this compound showed lack of activity at concentrations as high as 1μM as well as CXCR4 that was tested by Western blot analysis (a patent covering this compound was recently published [US no. 8,435,993] and a manuscript reporting synthesis and pharmacologic validation of JMS-17-2 in major detail has been submitted elsewhere).

In vitro stimulation of CX3CR1 and analysis of downstream signaling [1]
SKBR3 human breast cancer cells were serum starved for four hours before being exposed to 50nM recombinant human FKN for 5 minutes, with or without previous incubation with either a CX3CR1 neutralizing antibody used at 15μg/ml or the JMS-17-2 antagonist (10nM) for 30 minutes at 37 °C.

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Chemotaxis assay [1]
MDA-231 cells (1×105) were starved overnight and plated in the top chamber of transwell inserts (filters with 8-μm pore diameter) 200μl of serum-free culture medium. The inserts were transferred into a 24-well plate where each well contained 700μl of serum-free medium with or without recombinant human FKN (50nM). Positive controls were obtained using 10% FBS. For experiments involving JMS-17-2 and the CX3CR1 neutralizing antibody, cells were plated on the upper side of the filters in serum-free medium containing JMS-17-2 (1nM, 10nM and 100nM) or the antibody (15μg/ml) and then transferred to wells containing JMS-17-2 or the neutralizing antibody plus FKN. Cells were allowed to migrate at 37°C for 6 hours and at the end of the assay the cells still on the top of the filter were removed by scrubbing twice with a tipped swab. Cells migrated to the bottom of the filter were fixed with 100% methanol for 10 minutes; filters were then washed with distilled water, removed from the insert and mounted on cover glasses using mounting medium containing DAPI for nuclear staining. Two replicates were conducted for each condition, and five random microscope fields were used for cell enumeration, conducted with an Olympus BX51 microscope connected to the Nuance multispectral imaging system using version 2.4 of the analysis software (CRI). Three independent experiments were performed and results were presented as a ratio of cells that migrated under each condition relative to cells that migrated in control conditions (serum-free culture medium).

SCID mice (~25g) with MDA-231 xenograft
10 mg/kg
Aministered i.p.; twice a day for three weeks

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Model of tumor seeding [1]
For the pre-incubation experiments, MDA-231 cells in suspension were exposed to either a CX3CR1 neutralizing antibody (15μg/ml) or the JMS-17-2 compound (10nM in 0.1% DMSO) for 30 minutes (10 minutes at room temperature plus 20 minutes on ice), before being delivered to mice in the same pre-incubation suspension to maximize target engagement. Species- and class-matched irrelevant immunoglobulins (Rabbit IgG, 15μg/ml) or DMSO were used for the control groups. For the experiments requiring administration of JMS-17-2, animals were then treated i.p. with the CX3CR1 antagonist dissolved in 4% DMSO, 4% Cremophor EL in sterile ddH20 or just vehicle twice, one-hour prior and three hours after being injected with cancer cells. The dosing regimen was selected based on results from pharmacokinetic analyses. Mice were killed 24 hours post-injection, except for the experiments described in Fig. 3 A-C, for which mice were killed at two weeks post-injection. Blue-fluorescent beads, 10μm-polystyrene in diameter were included in the injection medium and visualized by fluorescence microscopy to validate injection efficiency. Mice showing non-homogenous distribution of or lacking fluorescent beads in tissue sections of lungs and kidneys were removed from the study.

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Model of established metastases [1]
One week after IC cell injection, animals were randomly assigned to control and treated group and then imaged for tumors in the skeleton and soft-tissue organs. Vehicle or JMS-17-2 (10mg/Kg) was administered i.p. twice/day, respectively, for the entire duration of the study while animals were imaged weekly.

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Pharmacokinetic analyses [1]
Mice were administered with 10mg/Kg of JMS-17-2 in 10% dimethylacetamide (DMAC), 10% tetraethylene glycol and 10% Solutol HS15 in sterile ddH2O. Animals were then anesthetized as described above and 300μl of blood samples were collected by cardiac puncture at the designated time points and transferred in K2EDTA tubes. Blood samples were placed on ice and tested after dilution. The measurement of JMS-17-2 concentrations in blood and brain tissue was outsourced to Alliance Pharma (www.alliancepharmaco.com).

Pharmacokinetic evaluation of JMS-17-2 administered to mice at a dose of 10mg/Kg (i.p.) produced drug levels of 89ng/ml (210 nM) in blood measured one hour after dosing, which corresponds to a 20-fold increase over the lowest fully effective dose of this compound in vitro [1].

[1]. Novel Small-Molecule CX3CR1 Antagonist Impairs Metastatic Seeding and Colonization of Breast Cancer Cells. Mol Cancer Res. 2016 Jun;14(6):518-27.

Recent evidence indicates that cancer cells, even in the absence of a primary tumor, recirculate from established secondary lesions to further seed and colonize skeleton and soft tissues, thus expanding metastatic dissemination and precipitating the clinical progression to terminal disease. Recently, we reported that breast cancer cells utilize the chemokine receptor CX3CR1 to exit the blood circulation and lodge to the skeleton of experimental animals. Now, we show that CX3CR1 is overexpressed in human breast tumors and skeletal metastases. To assess the clinical potential of targeting CX3CR1 in breast cancer, a functional role of CX3CR1 in metastatic seeding and progression was first validated using a neutralizing antibody for this receptor and transcriptional suppression by CRISPR interference (CRISPRi). Successively, we synthesized and characterized JMS-17-2, a potent and selective small-molecule antagonist of CX3CR1, which was used in preclinical animal models of seeding and established metastasis. Importantly, counteracting CX3CR1 activation impairs the lodging of circulating tumor cells to the skeleton and soft-tissue organs and also negatively affects further growth of established metastases. Furthermore, nine genes were identified that were similarly altered by JMS-17-2 and CRISPRi and could sustain CX3CR1 prometastatic activity. In conclusion, these data support the drug development of CX3CR1 antagonists, and promoting their clinical use will provide novel and effective tools to prevent or contain the progression of metastatic disease in breast cancer patients.[1]

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A major clinical need is to identify effective treatment to delay disease progression in patients presenting with few metastatic lesions. It has been recently demonstrated that existing metastases function as active reservoirs of tumor cells, cross-seeding other metastases and generate additional lesions, which makes therapeutic treatments directed to effectively counteract cancer seeding urgently needed. The target validation achieved in vivo with CRISPRi silencing of CX3CR1 provided impetus to test the JMS-17-2 compound in animals reproducing early metastatic onset in patients. The results from these experiments are strongly indicative that impairing CX3CR1 can successfully limit metastatic cross-seeding. On the other hand, the unexpected observation that both JMS-17-2 treatment and CRISPRi drastically restricted the growth of single lesions and contained the overall tumor burden could not be justified by interfering with tumor seeding.
Therefore, to understand the mechanistic basis underpinning this role of CX3CR1 in regulating secondary tumor growth and identify the signaling pathway altered by targeting this receptor, we harvested tumor tissues from animals in the control, JMS-17-2 treated and CRISPRi experimental groups and conducted comparative transcriptome analyses using Nanostring technology. Using this uniquely informative approach, we found that nine genes were altered in a corresponding fashion by JMS-17-2 and CRISPRi-mediated gene silencing, thus revealing molecular mediators for the role of CX3CR1 in supporting survival and proliferation of disseminated breast cancer cells. Particularly relevant is the up-regulation of WNT5A, involved in non-canonical Wnt signaling and endowed with a suppressive activity on metastatic breast cancer. The down-regulation of SOST, highly implicated in bone-related disease nd PRLR, which promotes colonization of breast cancer cells in soft tissues, are equally compelling. Finally, the down-regulation of NOTCH3 and deregulation of Notch signaling pathway (Supplementary Fig. S4) are strongly indicative of a possible role of CX3CR1 antagonism mitigating the tumor-initiating properties regulated by this gene in breast cancer. Indeed, CX3CR1 transactivates the Epidermal Growth Factor signaling pathway in breast cancer cells, promoting cell proliferation in vitro and delaying mammary tumor onset in mouse models.
In conclusion, the work presented here introduces a conceptual shift in the treatment strategies for breast cancer patients. Furthermore, we have synthesized and functionally characterized the first lead compound in a novel class of potentially new drugs with novel mechanisms of action to be added to the arsenal of therapies to treat advanced breast adenocarcinoma.[1]

C25H26CLN3O

419.95

419.176

C, 71.50; H, 6.24; Cl, 8.44; N, 10.01; O, 3.81

1380392-05-1

JMS-17-2 hydrochloride; 2341841-07-2

57382073

White to off-white solid powder

1.3±0.1 g/cm3

603.8±55.0 °C at 760 mmHg

319.0±31.5 °C

0.0±1.7 mmHg at 25°C

1.661

4.8

0

2

5

30

585

0

ClC1C=CC(=CC=1)C1CCN(CCCN2C(C3=CC=CN3C3C=CC=CC2=3)=O)CC1

WOSMCMULWWHMIV-UHFFFAOYSA-N

InChI=1S/C25H26ClN3O/c26-21-10-8-19(9-11-21)20-12-17-27(18-13-20)14-4-16-29-23-6-2-1-5-22(23)28-15-3-7-24(28)25(29)30/h1-3,5-11,15,20H,4,12-14,16-18H2

5-[3-[4-(4-chlorophenyl)piperidin-1-yl]propyl]pyrrolo[1,2-a]quinoxalin-4-one

JMS-172; JMS-17-2; 1380392-05-1; 5-(3-(4-(4-chlorophenyl)piperidin-1-yl)propyl)pyrrolo[1,2-a]quinoxalin-4(5H)-one; 5-[3-[4-(4-chlorophenyl)piperidin-1-yl]propyl]pyrrolo[1,2-a]quinoxalin-4-one; JMS-17-21380392-05-1; 5-{3-[4-(4-chlorophenyl)piperidin-1-yl]propyl}-4h,5h-pyrrolo[1,2-a]quinoxalin-4-one; 5-{3-[4-(4-chlorophenyl)piperidin-1-yl]propyl}pyrrolo[1,2-a]quinoxalin-4-one; MFCD30489012; JMS172; JMS 17-2; JMS-17 2; JMS 17 2; JMS17-2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: 25~40 mg/mL (59.5~95.2 mM)
Ethanol: 10 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.95 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 (4.95 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.)