SHP2 (IC50 = 0.583 nM)
SHP2 phosphatase (PTPN11) [2]
IC50 = 0.58 nM (against full-length SHP2 enzyme in a biochemical assay) [2]

RMC-4550 exhibits a cellular IC50 of 39 nM in PC9 cells with a pERK readout, and it inhibits purified, activated full length human SHP2 with an IC50 of 1.55 nM. Up to 10 µM, RMC-4550 exhibits no discernible inhibitory activity against the catalytic domain of SHP2, a panel of 468 protein kinases, and a panel of 14 additional protein phosphatases. RMC-4550 has a high passive permeability (458 nm/s), an efflux ratio of 1, and low to moderate cross species in vitro intrinsic clearance (3.6-24 µL/min/million cells) in hepatocytes[1].

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RMC-4550 potently and selectively inhibits the activity of full-length SHP2 enzyme (IC50 = 0.58 nM) but shows no activity against the isolated SHP2 catalytic domain up to 10 μM, confirming its allosteric mechanism. It is highly selective across panels of 15 other phosphatases, 468 kinases, and 44 cellular targets. [2]
In a panel of 33 KRAS-mutant cancer cell lines screened using a 3D proliferation assay, cell lines bearing KRAS G12 missense mutations (especially G12C) were sensitive to RMC-4550 (IC50 < 2 μM). For example, in NCI-H358 (KRAS G12C) and MIA PaCa-2 (KRAS G12C) cells, RMC-4550 inhibited proliferation (IC50 ~0.1-0.3 μM), suppressed cellular RAS-GTP levels, and reduced phosphorylated ERK (pERK) levels (pERK IC50 ~46-53 nM). Treatment also induced caspase 3/7 activation in NCI-H358 spheroids, indicating pro-apoptotic effects. [2]
In the NF1-loss-of-function (NF1LOF) lung adenocarcinoma cell line H1838, RMC-4550 inhibited proliferation (IC50 ~46 nM), suppressed RAS-GTP levels, and reduced pERK levels (pERK IC50 ~4 nM). [2]
In cell lines with class 3 BRAF mutations (e.g., H1666 BRAF G466V), RMC-4550 suppressed RAS-GTP and pERK levels (pERK IC50 ~9 nM in H1666) and inhibited proliferation in H1666 cells (IC50 ~236 nM). In contrast, cell lines with class 1 (BRAF V600E, e.g., A-375) or class 2 (e.g., NCI-H1755) BRAF mutations were insensitive to RMC-4550 in proliferation and signaling assays up to 10 μM. [2]
Mechanistically, expression of a constitutively active SOS1 mutant (SOS-F) rendered HEK293 cells insensitive to RMC-4550-mediated inhibition of EGF-stimulated pERK, suggesting that SHP2 inhibition acts upstream of SOS1 to disrupt RAS-GTP loading. Genetic dependency data analysis (Project DRIVE) showed high correlation between PTPN11 (SHP2) knockdown and SOS1 or GRB2 knockdown, supporting their functional association in a core RAS-regulatory module. [2]

RMC-4550 has a half-life that is suitable for once-daily oral administration and a moderate to high bioavailability. RMC-4550 exhibits dose-dependent efficacy consistent with target modulation in the EGFR-driven KYSE-520 human esophageal cancer xenograft model, as measured by phospho-ERK inhibition in tumors. In this model, RMC-4550 demonstrates good tolerance at dosages that result in both maximum and sustained efficacy [1].

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In NCI-H358 (KRAS G12C) xenograft models in immunodeficient mice, daily oral administration of RMC-4550 at 3, 10, and 30 mg/kg produced dose-dependent tumor growth inhibition (TGI of 59%, 53%, and 96%, respectively). [2]
In MIA PaCa-2 (KRAS G12C) xenograft models, daily oral administration of RMC-4550 at 10, 30, and 60 mg/kg produced significant tumor growth inhibition (TGI of 74%, 83%, and 93%, respectively). [2]
A pharmacodynamics study in NCI-H358 xenografts showed that a single oral dose of 10 mg/kg RMC-4550 led to time-dependent suppression of tumor pERK levels, with maximal suppression observed at 2 and 8 hours post-dose, and partial recovery by 24 hours. [2]

A biochemical inhibition assay for full-length SHP2 was performed. The enzyme was activated using a bisphosphorylated peptide derived from GAB1. Catalytic activity was monitored using the fluorogenic substrate DiFMUP in 96-well plates. Reactions contained activated SHP2, DiFMUP, and serially diluted RMC-4550. Fluorescence increase (excitation 340 nm, emission 450 nm) was measured kinetically for 6 minutes to determine initial velocity. Concentration-response curves were fitted to calculate IC50 values. The assay for the isolated SHP2 catalytic domain was identical except the activating peptide was omitted. [2]
A phosphatase selectivity panel was assessed. Twelve protein tyrosine phosphatases and two serine/threonine phosphatases (PP1, PP2A) were expressed and purified. Inhibition assays for each phosphatase used conditions similar to the SHP2 assay, with enzyme-specific adjustments in concentration and reaction time. Fluorescence was read as an endpoint after incubation at room temperature. [2]

In Biotin-free RPMI supplemented with 0.1% fetal bovine serum, 0.02% bovine serum albumin, and 1% penicillin/streptomycin, 30,000 HEK-293 cells per well are plated in 96-well plates. The induction of SOS1 constructs is achieved by adding 0.1 μg/mL doxycycline and waiting a full day. For one hour, cells are treated with three-fold dilutions of RMC-4550 serially diluted in biotin-free medium supplemented with 1% penicillin/streptomycin and 0.02% bovine serum albumin (final DMSO concentration equal to 0.1%). After the last five minutes of medication administration, cells are lysed, stimulated with 50 ng/mL EGF, and their ERK1/2 phosphorylation is examined.

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A 3D cell proliferation assay was used for screening. Cells were seeded in 96-well plates suspended in 0.65% methylcellulose. The next day, initial cell viability was measured. Serial dilutions of RMC-4550 were added, and cells were incubated for seven days. Viability was measured using a luminescent cell viability assay. Data were normalized to day 0 and vehicle control to calculate IC50 values. [2]
ERK1/2 phosphorylation (pERK) analysis was performed using a bead-based immunoassay. Cells were plated in 96-well plates overnight, then serum-starved for 1 hour before treatment with RMC-4550 for 1 hour. Cells were lysed, and pERK levels were determined according to the assay kit protocol. [2]
Cellular RAS-GTP levels were measured using a plate-based ELISA kit. Cells were treated with RMC-4550 for 1 hour, then lysed. Lysates were incubated in wells coated with a Raf-1 RBD fusion protein to capture RAS-GTP, which was then detected with an anti-RAS antibody. [2]
Spheroid formation and proliferation assays were conducted. Cells were seeded in ultra-low attachment round-bottom 96-well plates to form spheroids over 72 hours. Formed spheroids were treated with RMC-4550 for five days, and viability was measured using a luminescent assay. [2]
Activated Caspase 3/7 assay in spheroids: NCI-H358 spheroids were treated with RMC-4550 or controls for 20 hours. Caspase 3/7 activity was measured using a luminescent substrate. [2]
Mechanistic rescue experiment: HEK293 cells with inducible expression of wild-type SOS1 (SOS-WT) or a constitutively active membrane-targeted mutant (SOS-F) were treated with RMC-4550 for 1 hour, stimulated with EGF for the final 5 minutes, and then lysed for pERK analysis. [2]

Female (6-8 week old) athymic nude mice implanted with NCI-H358 (Balb/c strain background) or MIA PaCa-2 (NCR nude strain background) tumor cells subcutaneously in the flank
3-60 mg/kg
PO

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For efficacy studies, female athymic nude mice (6-8 weeks old) were implanted subcutaneously with NCI-H358 or MIA PaCa-2 tumor cells. When tumors reached an average volume of ~200 mm³, mice were randomized into groups. RMC-4550 was dissolved in a vehicle composed of Captisol/50 mM acetate buffer pH 4.6 (10%/90%, w/v). The compound was administered daily via oral gavage at specified doses (3, 10, 30, 60 mg/kg). Tumor volumes were measured regularly. Animals were euthanized when the mean tumor volume in the control group reached ~1500 mm³ (day 25 for H358, day 22 for MIA PaCa-2). [2]
For pharmacodynamics studies, mice bearing NCI-H358 xenografts received a single oral dose of RMC-4550 (10 mg/kg) or vehicle. Groups of mice were euthanized at 2, 8, or 24 hours post-dose. Tumors were collected, snap-frozen, and processed for protein extraction and Western blot analysis of pERK and total ERK. [2]

[1]. Cancer Res (2018) 78 (13_Supplement): 4878.

[2]. bioRxiv 188730.

RMC-4550 is a novel, highly potent, and selective allosteric inhibitor of SHP2 (PTPN11). [2] It was discovered through biochemical and cellular screening combined with structure-guided medicinal chemistry optimization. [2] This compound is considered a targeted therapeutic strategy for treating cancers driven by nucleotide cycling oncogenic RAS (e.g., KRAS G12C), RAS-GTP-dependent oncogenic BRAF (type 3 mutants), or the absence of the tumor suppressor NF1. These alterations are present in a significant proportion of non-small cell lung cancer (NSCLC) and other tumors. [2] Mechanistically, RMC-4550 blocks the SHP2/SOS1/GRB2 module by inhibiting SHP2, thereby reducing GTP binding of RAS, inhibiting the activation of the MAPK pathway (pERK), and thus inhibiting oncogenic signaling. [2]

C21H26CL2N4O2

437.3627

436.14

C, 57.67; H, 5.99; Cl, 16.21; N, 12.81; O, 7.32

2172651-73-7

2172651-73-7

134183206

White to light yellow solid powder

2.5

2

6

3

29

563

2

C[C@H]1[C@H](C2(CCN(CC2)C3=NC(=C(N=C3CO)C4=C(C(=CC=C4)Cl)Cl)C)CO1)N

IKUYEYLZXGGCRD-ORAYPTAESA-N

InChI=1S/C21H26Cl2N4O2/c1-12-18(14-4-3-5-15(22)17(14)23)26-16(10-28)20(25-12)27-8-6-21(7-9-27)11-29-13(2)19(21)24/h3-5,13,19,28H,6-11,24H2,1-2H3/t13-,19+/m0/s1

[3-[(3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl]-6-(2,3-dichlorophenyl)-5-methylpyrazin-2-yl]methanol

RMC-4550; RMC 4550; RMC4550

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)

Ethanol: 22~100 mg/mL (50.3~228.6 mM)
DMSO: ~11 mg/mL (~25.2 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.72 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 25.0 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: ≥ 2.5 mg/mL (5.72 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 25.0 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: ≥ 2.5 mg/mL (5.72 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 25.0 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.)