KRas G12D

RMC-6236 (5 days) suppresses AsPC-1 (K-Ras G12D) cell viability with an IC50 of 1–10 μM[1].
RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. [2]

Oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985).[2]

RAS-RAF TR-FRET.[2]
Disruption of the interactions between wild-type KRAS or the mutant oncogenic RAS proteins and the RAS-binding domain of BRAF were assessed by time-resolved fluorescence energy transfer (TR-FRET) in reactions consisting of 12.5 nmol/L His6- KRAS [1–169], 50 nmol/L GST-BRAF [155–229], 10 nmol/L LANCE Eu-W1024 anti-6xHis antibody, 50 nmol/L Allophycocyanin-anti-GST antibody (PerkinElmer AD0059G), and 25 μmol/L CypA in reaction buffer (25 mmol/L HEPES-NaOH pH 7.3, 0.002% Tween20, 0.1% bovine serum albumin, 100 mmol/L NaCl, 5 mmol/L MgCl2). Compound or DMSO control (1% v/v) was added and incubated for 1.5 hours, and then TR-FRET was measured on a PerkinElmer Envision plate reader (excitation at 320 nm, 20 μs delay, 100 μs window, 2,000 μs time between flashes; emission at 665 nm and 615 nm in separate channels). The FRET ratio (665/615 nmol/L emission) was used to calculate % Inhibition as: [1 – (FRET ratio of sample – Average FRET ratio of positive controls)/(Average FRET ratio of DMSO control – Average FRET ratio of positive controls)] × 100%.

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CypA Binding Affinity (KD1). [2]
The binding affinity of compounds for CypA was assessed by surface plasmon resonance (SPR) on Biacore 8K instrument. AviTag-CypA was immobilized on a streptavidin sensor chip, and varying compound concentrations were flowed over the chip in assay buffer (10 mmol/L HEPES-NaOH pH 7.4, 150 mmol/L NaCl, 0.005% v/v Surfactant P20, 2% v/v DMSO). The SPR sensograms were fit using either a steady-state affinity model or a 1:1 binding (kinetic) model to assess the KD1 for CypA binding.

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RAS-Binding Affinity (KD2). [2]
The binding affinity of compound-bound CypA for the mutant oncogenic RAS proteins mentioned was assessed by SPR on Biacore 8K instrument. AviTag-RAS [1–169] was immobilized on a streptavidin sensor chip, and varying compound concentrations were flowed over the chip in assay buffer (10 mmol/L HEPES-NaOH pH 7.4, 150 mmol/L NaCl, 0.005% v/v Surfactant P20, 2% v/v DMSO, 25 μmol/L CypA). The SPR sensorgrams were fit using either a steady-state affinity model or a 1:1 binding (kinetic) model to assess the KD for RAS binding.

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AlphaLISA and MesoScale Discovery (MSD) Analysis of Cellular ERK Phosphorylation. [2]
NCI-H441, Capan-2, HPAC, or isogenic RAS-less MEF cells were seeded in tissue culture–treated 384- and 96-well plates and incubated overnight. The following day, cells were exposed to serial dilutions of compound or DMSO control (0.1% v/v) for specified time points using a Labcyte Echo 550 or Tecan D300e digital dispenser. Following incubation, cells were lysed, and the levels of ERK phosphorylation were determined using the AlphaLISA SureFire Ultra pERK1/2 (T202/Y204) Assay kit or MSD Multi-Array Assay Systems for Phospho/Total ERK1/2 Whole Cell Lysate Kit (K15107D), following the manufacturers’ protocols. Signal was detected using a PerkinElmer Envision with standard AlphaLISA settings, or a Meso QuickPlex SQ120 reader for MSD. For AlphaLISA, data were expressed as % of DMSO-treated control: 100–100 × (pERKDMSO – pERKtreated)/(pERKDMSO – pERKmedia). MSD signal from pERK1/2 was divided by MSD signal for total ERK1/2. The ratio was normalized to vehicle (% of pERK/total ERK = ((ratio pERKtreated/total ERKtreated)/(ratio pERKDMSO/total ERKDMSO)) × 100). For both assays, data were plotted as a function of log M [compound] with a sigmoidal concentration response (variable slope) model fitted to the data to estimate the inhibitor EC50 in Prism 9

PRISM Screening[2]
RMC-6236 was added to 384-well plates at 8-point concentration with 3-fold dilutions in triplicate. These assay-ready plates were then seeded with the thawed cell line pools. Adherent cell pools were plated at 1,250 cells per well, whereas suspension and mixed adherent/suspension pools were plated at 2,000 cells per well. Treated cells were incubated for 5 days, and then lysed. Lysate plates were collapsed together prior to barcode amplification and detection.

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2D Cell Proliferation Analysis. [2]
NCI-H441, Capan-2, and HPAC cells were seeded in tissue culture–treated 384- or 96-well plates and incubated overnight. Cells were exposed to serial dilutions of compound or DMSO control (0.1% v/v) using a Labcyte Echo 550 or Tecan D300e digital dispenser and incubated for 120 hours at 37°C. Doxycycline-inducible cell lines were retreated with doxycycline at the time of compound treatment. Cell viability was determined by CellTiter-Glo 2.0 reagent according to the manufacturers’ protocols. Luminescence was detected using a SpectraMax M5 Plate Reader of PerkinElmer Enspire. Luminescence signal was normalized to vehicle-treated wells [% vehicle = (lumtreated/mean(lumvehicle) × 100]. Data were plotted as a function of log molar [inhibitor], and a 4-parameter sigmoidal concentration response model was fitted to the data to calculate the EC50. Growth percentages were calculated by normalizing the treated cell counts to their respective untreated cell counts.

RMC-6236 Formulation. [2]
For in vitro studies, RMC-6236 was resuspended in dimethyl sulfoxide (DMSO) and used at 10 mmol/L stock concentration. For use in in vivo studies, RMC-6236 was prepared using formulation of 10/20/10/60 (%v/v/v/v) DMSO/PEG 400/Solutol HS15/water. The same vehicle formulation was used for all control groups.

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RMC-6236 Treatment. [2]
Tumor-bearing animals were randomized and assigned into groups (n = 1–10/group). The vehicle at 10 mL/kg or RMC-6236 at indicated doses was administered via oral gavage daily, and animals were treated for 28 days, or up to 90 days if PFS was being assessed. Animals were terminated early if the tumor burden reached a humane endpoint, or adverse effect was observed with body weight loss as a surrogate. For single-dose PKPD study, mice were randomized and assigned into groups (n = 3/dose/time point). A single dose of RMC-6236 was administered orally at either 3, 10, or 25 mg/kg. Blood and tissues, including the tumor, brain, colon, ear skin, and muscle, were harvested at indicated time points. Whole blood was collected in K2EDTA Microtainer tubes, incubated for 5 minutes, and snap-frozen in liquid nitrogen. The tissue was either fixed in 10% formalin or snap-frozen in liquid nitrogen for further analysis.[2]

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Mouse Blood and Tissue Sample Bioanalysis. [2]
The whole blood, tumor, brain, colon, and ear skin concentrations of RMC-6236 were determined using liquid chromatography–tandem mass spectrometry (LC/MS-MS) methods. Tissue samples were homogenized with a 10 × volume of homogenization buffer [methanol/15 mmol/L PBS (1:2; v:v) or 15 mmol/L PBS with 10% methanol]. An aliquot of whole blood or homogenized tissue (10, 20, or 40 μL) was transferred to 96-well plates (or tubes) and quenched with a 10 × volume of acetonitrile or 20 × volume of acetonitrile/methanol (1:1; v/v) with 0.1% formic acid containing a cocktail of internal standards (IS). After thorough mixing and centrifugation, the supernatant was diluted with water or directly analyzed on a Sciex 5500 or Sciex 6500+ triple quadrupole mass spectrometer equipped with an ACQUITY or Shimadzu UPLC system. A Halo 90Å AQ-C18 2.7 μm (2.1 × 50 mm) or an ACQUITY UPLC BEH C18 or C4 1.7 μm (2.1 × 50 mm) column was used with gradient elution for compound separation. RMC-6236 and IS (verapamil, celecoxib, glyburide, dexamethasone, or terfenadine) were detected by positive electrospray ionization using multiple reaction monitoring (RMC-6236: m/z 811/779; verapamil: m/z 455/165; celecoxib: m/z 382/362; glyburide: m/z 494/169; dexamethasone: m/z 393/373; terfenadine: m/z 472/436). The lower limit of quantification was 1 ng/mL or 2 ng/mL for blood, tumor, and other tissue.

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PK/PD Relationship. [2]
Concentrations of RMC-6236 in tumor or normal tissues and percentage of DUSP6 inhibition as compared with the vehicle control from individual animals were collected and analyzed post a single dose of RMC-6236 ranging from 0.3 to 100 mg/kg (Supplementary Table S6). A 3-parameter sigmoidal exposure–response model was fitted to the data in GraphPad Prism to derive EC50 and EC90 values.

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PK/Efficacy and PK/PD Modeling. [2]
For PK modeling, whole blood PK data from single or repeat dose administration of 25 or 40 mg/kg RMC-6236 to NCI-H441 xenograft tumor-bearing mice were used (Supplementary Table S9). RMC-6236 blood PK was best described using a one-compartment model with first-order absorption and elimination. Because intravenous data were not included in the modeling, the model was parameterized in terms of apparent clearance (CL/F) and volume of distribution (V/F), where F is the oral bioavailability.

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Specifically, to understand the responsiveness of tumors harboring diverse oncogenic Kras variants to RMC-6236 treatment, lung tumors were initiated in B6 mice using a barcoded lentivirus pool including vectors encoding oncogenic KRAS mutant (G12C, G12V, G12D, G12A, Q61H, or G13D) cDNAs (Lenti;KrasMUT;BC). Thirteen weeks post tumor initiation, mice were treated for 3 weeks with either: (i) vehicle (10% DMSO, 20% PEG400, 10% Solutol HS15, 60% water) po qd and 10 mg/kg isotype rat igg2a[2a3] ip biw; or (ii) RMC-6236 20 mg/kg po qd.

[1]. Use of sos1 inhibitors to treat malignancies with shp2 mutations. Patent WO2022060583A1.

[2]. Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers. Cancer Discov. 2024 Jun 3;14(6):994-1017.
[3]. Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy. Nature. 2024 May;629(8013):919-926.
[4]. Drugging RAS: Moving Beyond KRASG12C. Cancer Discov. 2023 Dec 12;13(12):OF7.
[5]. State-of-the-art and upcoming trends in RAS-directed therapies in gastrointestinal malignancies. Curr Opin Oncol. 2024 Jul 1;36(4):313-319.

RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). Significance: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.[1]

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RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).[2]

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Preliminary results from phase I trials respectively evaluating RMC-6236, a pan-RAS inhibitor, and HRS-4642, a KRASG12D inhibitor, indicate that both are safe and show promising signs of antitumor activity. These are just two of the candidate RAS therapies in a burgeoning development space as the field looks ahead to drugs that hit more than just KRASG12C.[3]

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Purpose of review: Overall, the review underscores the evolving landscape of KRAS-targeted therapy and the potential for these approaches to improve outcomes for patients with gastrointestinal malignancies. It highlights the importance of ongoing research and clinical trials in advancing precision medicine strategies for KRAS-driven cancers. This review provides a comprehensive overview of the RAS signaling pathway and its significance in gastrointestinal malignancies. Recent findings: The introduction of KRAS inhibitor represents a significant advancement in the treatment landscape for KRAS-mutant cancers. In this review, we discuss upcoming trends in KRAS-targeted therapy, including the development of mutant-specific direct KRAS inhibitors like MRTX1133 and pan-RAS inhibitors such as RMC-6236. It also explores indirect RAS inhibitors targeting upstream and downstream components of the RAS pathway. Additionally, the review examines other upcoming strategies like combination therapies, such as CDK4/6 and ERK MAPK inhibitors, as well as adoptive cell therapy and cancer vaccines targeting KRAS-mutant cancers. Summary: Targeting RAS has become an important strategy in treating gastrointestinal cancer. These findings in this review underscore the importance of a multidisciplinary approach, integrating advances in molecular profiling, targeted therapy, immunotherapy, and clinical research to optimize treatment strategies for patients with KRAS-mutant gastrointestinal malignancies.[4]

C44H58N8O5S

811.06

810.43

C, 65.16; H, 7.21; N, 13.82; O, 9.86; S, 3.95

2765081-21-6

2765081-21-6; 2765091-21-0 (racemate)

164726578

White to off-white solid

5.1

2

11

7

58

1470

5

FVICRBSEYSHKFY-UHFFFAOYSA-N

InChI=1S/C44H58N8O5S/c1-8-51-37-12-11-28-19-31(37)33(40(51)32-20-29(23-45-39(32)27(3)56-7)50-16-14-49(6)15-17-50)22-44(4,5)25-57-43(55)34-10-9-13-52(48-34)42(54)35(21-38-46-36(28)24-58-38)47-41(53)30-18-26(30)2/h11-12,19-20,23-24,26-27,30,34-35,48H,8-10,13-18,21-22,25H2,1-7H3,(H,47,53)

N-[21-ethyl-20-[2-(1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl]-17,17-dimethyl-8,14-dioxo-15-oxa-4-thia-9,21,27,28-tetrazapentacyclo[17.5.2.12,5.19,13.022,26]octacosa-1(25),2,5(28),19,22(26),23-hexaen-7-yl]-2-methylcyclopropane-1-carboxamide

RAS-IN-2; RAS In 2; RMC-6236; RMC 6236; RMC6236; EX-A6631; DA-77354; RMC-6236; Compound A122; RAS Inhibitor A122; (Z)-N-(11-ethyl-12-(2-(1-methoxyethyl)-5-(4-methylpiperazin-1-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-61,62,63,64,65,66-hexahydro-11H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-4-yl)-2-methylcyclopropane-1-carboxamide; RMC6236

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: ~250 mg/mL (~308.2 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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