Ras (inhibits SOS-mediated and EDTA-induced nucleotide exchange; binds to the effector binding groove between switch I and II, locking Ras in an open-like conformation) [1];
ARF6 (inhibits activation, blocks GPCR internalization) [1];
No IC50/Ki values reported for direct target binding; in cellular assays, inhibition of AT1R internalization IC50 = 10 μM, B2R internalization IC50 = 11 μM, β2AR internalization IC50 = 15 μM, AT1R-mediated ERK1/2 activation IC50 = 5 μM, EGFR-mediated ERK1/2 activation IC50 = 4 μM, EGFR-mediated Akt activation IC50 = 4-5 μM [1].

Rasarfin effectively inhibits coagulant-induced ERK1/2 signaling through GPCR, MAPK and Akt signaling through EGFR, and by inhibiting ischemia [1]. Rasarfin inhibits ARF6 and inhibits GPCR internalization [1]. Rasarfin causes MDA-MB- Rasarfin inhibits AP-2 recruitment to β-arrestin2 and receptor internalization, both processes requiring active ARF6 [1]. 231 cells decreased in a dose-dependent manner [1].

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Rasarfin (50 μM) inhibited AngII-induced AT1R internalization by >90% (BRET and confocal microscopy) with IC50 = 10 μM; inhibited BK-induced B2R internalization (IC50 = 11 μM) and isoproterenol-induced β2AR internalization (IC50 = 15 μM) [1].
Rasarfin did not block β-arrestin1/2 recruitment to AT1R or B2R at the plasma membrane, but prevented β-arrestin2 trafficking with receptors into endosomes, causing its accumulation at clathrin-coated pits (confocal microscopy) [1].
Rasarfin inhibited ARF6 activation (GST-GGA3-PBD pull-down) by >70% following AngII stimulation; no effect on ARF1. In a BRET-based ARF activation assay, Rasarfin (50 μM) blocked AngII-promoted signal by 40-60%. Overexpression of dominant-negative ARF6-T27N mimicked the effect of Rasarfin on AT1R internalization kinetics [1].
Rasarfin completely inhibited AngII-, BK-, isoproterenol-, and EGF-mediated ERK1/2 phosphorylation (Western blot) with IC50 for AT1R and EGFR of 5 μM and 4 μM, respectively. It also inhibited EGF-mediated Akt activation (IC50 4-5 μM) [1].
Kinase binding assay (KINOMEScan, 384 kinases) showed high selectivity (S(10)=0.008); Rasarfin did not bind EGFR, BRAF, Akt, PI3K, MEK, ERK, GRKs, Src, or AAK1 [1].
Rasarfin inhibited AngII-mediated Ras activation (GST-Raf1-RBD pull-down and BRET-based Ras biosensor) with significant reduction at 50 μM. In an in vitro mant-GTP exchange assay, Rasarfin dose-dependently inhibited SOS1-catalyzed nucleotide exchange on purified H-Ras (10-50 μM) and also inhibited EDTA-induced (Mg2+ chelation) exchange, indicating direct action on Ras independent of GEF. The compound also inhibited K-Ras WT, but not K-Ras-Y32A mutant (where Tyr32 is critical for binding) [1].
Rasarfin did not inhibit AT1R-mediated Gαq, Gαi3, Gα12/13, PKC, Rho, or Rac activation (BRET sensors) [1].
In MDA-MB-231 breast cancer cells, Rasarfin (10-50 μM) reduced cell proliferation (IncuCyte) and inhibited Ras and ARF6 activities (GST pull-down). In A549 lung cancer cells, Rasarfin also inhibited proliferation (5-10 μM). Cell viability (MTT) was modestly affected compared to doxorubicin, with no major cytotoxicity [1].
Structure-activity relationship (SAR) with analogs showed that the chloro substituent on the aryl ring is critical for Ras inhibition and MAPK blockade; compound 21.8 (n-propyl instead of isopropyl) retained MAPK inhibition but lost internalization inhibition, demonstrating functional selectivity [1].

GST pull-down assays: HEK293 cells expressing AT1R were stimulated with AngII (1 μM) or EGF (100 ng/ml) for 2 min, lysed, and incubated with GST-GGA3-PBD (for ARF6), GST-Raf1-RBD (for Ras), or GST-Rhotekin-RBD (for Rho) coupled to glutathione sepharose beads. Bound proteins were eluted and detected by Western blot using anti-HA, anti-Ras, anti-RhoA, or anti-ARF6 antibodies. Activation was quantified as pull-down/total protein ratio [1].
BRET-based small GTPase activation assays: HEK293 cells were transfected with biosensors consisting of a binding domain (e.g., GGA3-PBD for ARF, Raf1-RBD for Ras, Rhotekin-RBD for Rho, PAK-CRIB for Rac) fused to RlucII, together with rGFP-CAAX (PM anchor). After serum starvation and compound pretreatment, cells were stimulated with AngII (100 nM) and BRET (emission 410/80 nm for RlucII, 515/30 nm for rGFP) was measured using coelenterazine 400a. Kinetics and area under curve were analyzed [1].
In vitro mant-GTP exchange assay: Purified H-Ras or K-Ras (1.66 μg) was incubated in 2X exchange buffer with DMSO or Rasarfin (0.1-50 μM) in a 384-well plate. mant-GTP fluorescence (ex360/em440 nm) was recorded every 5 s for 30 s (baseline), then 40 mM EDTA or purified SOS1 (0.66 μg) was added, and fluorescence was measured every 30 s for 30 min. Delta RFU (post-addition minus pre-addition average) was calculated [1].
Kinase binding assay (KINOMEScan): Profiling of Rasarfin at 10 μM against 384 human kinases was performed by Eurofins/DiscoverX. Selectivity score S(10) = 0.008 (fraction of kinases with >90% inhibition) [1].
Molecular dynamics simulations: Rasarfin was docked into the Ras-SOS binding interface (PDB 1BKD) using MOE. The best-scoring pose was used for 3 × 4 μs MD simulations with CHARMM36m force field in ACEMD, followed by pharmacophore analysis in LigandScout. Spontaneous association simulations (3 × 2 μs) with five Rasarfin molecules randomly placed around Ras were also performed. Rasarfin stably bound in the groove between switch I and II, with key interactions: H-bond between amide carbonyl and Asp57/Ile55, hydrophobic contacts with Tyr40, Leu56, Thr20, and the chloro atom occupying a small cavity [1].

Cell proliferation assay (IncuCyte): MDA-MB-231 or A549 cells (1500 cells/well) were seeded in 96-well plates and treated with DMSO or Rasarfin (0.1-50 μM) for 96 h. Cell confluence was measured every 3 h using IncuCyte S3 software, and proliferation expressed as percentage confluence increase [1].
Cell viability assay (MTT): Cells were treated with Rasarfin or doxorubicin for 24-72 h, then 10 μL of 5 mg/mL MTT was added for 4 h, and absorbance at 590 nm was measured [1].
Western blot for ERK1/2 and Akt activation: HEK293 cells expressing AT1R, B2R, β2AR, or endogenous EGFR were serum-starved, pretreated with DMSO or Rasarfin (0.1-50 μM) for 30 min, stimulated with AngII (1 μM), BK (1 μM), isoproterenol (10 μM), or EGF (100 ng/ml) for indicated times, lysed in Laemmli buffer, resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-p-ERK1/2, anti-ERK1/2, anti-p-Akt, anti-Akt antibodies. Densitometry was performed using ImageLab [1].
Confocal microscopy for receptor and β-arrestin trafficking: HEK293 cells expressing AT1R-YFP or β-arrestin2-YFP with Flag-AT1R/HA-B2R were pretreated with DMSO or Rasarfin (50 μM) for 30 min, stimulated with AngII (100 nM) or BK (1 μM) for 15-30 min, and imaged using Zeiss LSM-510/710 with 405 nm excitation and 505-550 nm emission. Receptor internalization was assessed by loss of plasma membrane signal and accumulation in endosomes [1].
BRET-based receptor internalization assay: Cells expressing AT1R-RlucII, B2R-RlucII, or β2AR-RlucII together with rGFP-FYVE (endosomal anchor) were pretreated with compounds, stimulated with ligands, and BRET ratio (rGFP/RlucII) was measured. For AT1R, IC50 = 10 μM; for B2R, IC50 = 11 μM; for β2AR, IC50 = 15 μM [1].
BRET for β-arrestin/AP-2 interaction: Cells expressing β-arrestin2-RlucII and β2-adaptin-GFP10 were used; Rasarfin (50 μM) inhibited AngII-induced complex formation by only ~20%, whereas Barbadin (100 μM) inhibited >50% [1].
BRET for ARF activation: Cells expressing GGA3-PBD-RlucII and rGFP-CAAX were used; Rasarfin (50 μM) inhibited AngII-promoted signal by 40-60% [1].

Rasarfin at 50 μM did not cause abnormal cell morphological changes in HEK293 cells as assessed by brightfield microscopy, indicating low acute cytotoxicity [1].
In cell viability assays (MTT) on MDA-MB-231 and A549 cells, Rasarfin modestly reduced metabolic activity at 10-50 μM, but to a much lesser extent than doxorubicin, suggesting no potent cytotoxicity [1].
No systemic toxicity, LD50, or organ toxicity data are reported [1].

[1]. Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screen. Nat Commun. 2021 Aug 3;12(1):4688.

Rasarfin (originally compound 21) was identified from a high-throughput screen of ~115,000 small molecules using an endosomal BRET-based AT1R trafficking assay. It represents the first dual inhibitor of Ras and ARF6. Mechanism: binds to the SOS-binding groove on Ras (between switch I and II), locking the switches in an open conformation that prevents nucleotide exchange and effector interaction; also inhibits ARF6 activation, blocking clathrin-mediated GPCR internalization. Unlike catalytic Ras inhibitors, Rasarfin does not target the GTP-binding pocket but rather a protein-protein interaction interface [1].
SAR studies identified analogs with functional selectivity: compound 21.8 (n-propyl instead of isopropyl) retained MAPK inhibition but lost internalization inhibition, indicating that the ARF6 and Ras inhibitory activities can be dissociated [1].
Rasarfin is a tool compound to study the roles of Ras and ARF6 in GPCR signaling, endocytosis, and cancer cell proliferation. It is not FDA-approved or in clinical trials [1].
The chemical structure: not fully provided in the text but compound 21 is depicted in Fig. 1c; its analogs and CAS# 674359-73-0 correspond to the compound [1].

C23H24CLN3O3

425.907964706421

425.15

C, 64.86; H, 5.68; Cl, 8.32; N, 9.87; O, 11.27

674359-73-0

1396167

White to yellow solid powder

4.6

1

4

4

30

623

0

CC(C)C(=O)N1CCN(CC1)C2=C(C=C(C=C2)NC(=O)C3=CC4=CC=CC=C4O3)Cl

SRZCCUYFJZQLGE-UHFFFAOYSA-N

InChI=1S/C23H24ClN3O3/c1-15(2)23(29)27-11-9-26(10-12-27)19-8-7-17(14-18(19)24)25-22(28)21-13-16-5-3-4-6-20(16)30-21/h3-8,13-15H,9-12H2,1-2H3,(H,25,28)

N-[3-chloro-4-[4-(2-methylpropanoyl)piperazin-1-yl]phenyl]-1-benzofuran-2-carboxamide

Rasarfin

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)

DMSO: ~50 mg/mL (~117.4 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.87 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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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.88 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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 (Please use freshly prepared in vivo formulations for optimal results.)