KRas G12D (Kd = 0.2 pM)

MRTX1133 can reversibly bind to both activated and inactivated KRAS G12D mutants and inhibit their activity. It is a highly selective inhibitor of mutant KRAS. MRTX1133 is more specific to KRAS G12D than wild-type KRAS by a factor of more than 1000. MRTX1133 not only exhibits tumor regressions in the Panc 04.03 xenograft model, but also exhibits single-digit nM potency in a cellular proliferation assay.

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In addition to the KRAS G12C mutation, mutations such as KRAS G12D also play an important role in the occurrence and development of tumours. The KRAS G12D-specific inhibitor MRTX1133, developed by Mirati, can reversibly bind to the activated and inactivated KRAS G12D mutants and inhibit their activity. The specificity of MRTX1133 to KRAS G12D is more than 1000 times that of wild-type KRAS, and its half-life is more than 50 hours. In vitro experiments indicated that MRTX1133 has a dose-dependent inhibition of the KRAS signalling pathway activity and significantly reduced the size of tumours with KRAS G12D mutations in pancreatic and colorectal cancer models compared with the control group [1].

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Finally, the combination of the optimized three substituents on the 2-, 4-, and 7-positions of pyrido[4,3-d]pyrimidine led to the discovery of <MRTX1133, an exceptionally potent and selective KRASG12D inhibitor (Figure 10). MRTX1133 optimally fills the switch II pocket and extends three substituents to favorably interact with the protein (Figure 11), resulting in an estimated KD against KRASG12D of 0.2 pM. AlphaLISA data confirmed that binding of the inhibitor prevented SOS1-catalyzed nucleotide exchange and/or formation of the KRASG12D/GTP/RAF1 complex, thereby inhibiting mutant KRAS-dependent signal transduction. MRTX1133 inhibited ERK phosphorylation in the AGS cell line with an IC50 of 2 nM (see Table S2 for activity against a panel of KRASG12D cell lines). In a 2D viability assay, the IC50 of MRTX1133 was 6 nM against the same cell line, while demonstrating more than 500-fold selectivity against MKN1, a cell line which is dependent on KRAS for its growth and survival due to the amplification of wild-type KRAS.[2]

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Recent progress in targeting KRASG12C has provided both insight and inspiration for targeting alternative KRAS mutants. In this study, we evaluated the mechanism of action and anti-tumor efficacy of MRTX1133, a potent, selective and non-covalent KRASG12D inhibitor. MRTX1133 demonstrated a high-affinity interaction with GDP-loaded KRASG12D with KD and IC50 values of ~0.2 pM and <2 nM, respectively, and ~700-fold selectivity for binding to KRASG12D as compared to KRASWT. MRTX1133 also demonstrated potent inhibition of activated KRASG12D based on biochemical and co-crystal structural analyses. MRTX1133 inhibited ERK1/2 phosphorylation and cell viability in KRASG12D-mutant cell lines, with median IC50 values of ~5 nM, and demonstrated >1,000-fold selectivity compared to KRASWT cell lines [3].

MRTX1133 was discovered through an extensive structure-based activity improvement and shown to be efficacious in a KRASG12D mutant xenograft mouse tumor model. Intraperitoneal (IP) administration of MRTX1133 at 30 mg/kg in CD-1 mice resulted in sustained plasma exposure exceeding the free-fraction-adjusted pERK IC50 value in the KRASG12D mutant Panc 04.03 cell line for approximately 8 h. Encouraged by this result, we evaluated the ability to modulate KRAS-dependent ERK phosphorylation in the Panc 04.03 xenograft tumor model at 30 mg/kg BID (IP) and observed 62% and 74% inhibition of pERK signal at 1 and 12 h after the second dose, respectively. An antitumor efficacy study in this model resulted in MRTX1133 dose-dependent antitumor activity with 94% growth inhibition observed at 3 mg/kg BID (IP) and tumor regressions of −62% and −73% observed at 10 and 30 mg/kg BID (IP), respectively). In contrast, no significant antitumor activity was observed in the non-KRASG12D tumor model MKN1 (data not shown). [2].

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Intraperitoneal (IP) administration of MRTX1133 at 30 mg/kg in CD-1 mice (Figure 12) resulted in sustained plasma exposure exceeding the free-fraction-adjusted pERK IC50 value in the KRASG12D mutant Panc 04.03 cell line for approximately 8 h. Encouraged by this result, we evaluated the ability to modulate KRAS-dependent ERK phosphorylation in the Panc 04.03 xenograft tumor model at 30 mg/kg BID (IP) and observed 62% and 74% inhibition of pERK signal at 1 and 12 h after the second dose, respectively (Figure 13). An antitumor efficacy study in this model resulted in MRTX1133 dose-dependent antitumor activity with 94% growth inhibition observed at 3 mg/kg BID (IP) and tumor regressions of −62% and −73% observed at 10 and 30 mg/kg BID (IP), respectively (Figure 14). In contrast, no significant antitumor activity was observed in the non-KRASG12D tumor model MKN1 (data not shown).[2]

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MRTX1133 exhibited dose-dependent inhibition of KRAS-mediated signal transduction and marked tumor regression (≥30%) in a subset of KRASG12D-mutant cell-line-derived and patient-derived xenograft models, including eight of 11 (73%) pancreatic ductal adenocarcinoma (PDAC) models. Pharmacological and CRISPR-based screens demonstrated that co-targeting KRASG12D with putative feedback or bypass pathways, including EGFR or PI3Kα, led to enhanced anti-tumor activity. Together, these data indicate the feasibility of selectively targeting KRAS mutants with non-covalent, high-affinity small molecules and illustrate the therapeutic susceptibility and broad dependence of KRASG12D mutation-positive tumors on mutant KRAS for tumor cell growth and survival.[3]

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MRTX1133 demonstrates inhibition of KRAS-dependent signaling and tumor regression in xenograft models [3]
MRTX1133 was evaluated in immunocompromised mice bearing KRASG12D-mutant HPAC tumor xenografts for its effect on KRAS-mediated signaling and to characterize its anti-tumor activity over a range of dose levels and timepoints. MRTX1133 exhibited low intrinsic oral bioavailability in mice and was, therefore, administered at 3 mg kg−1, 10 mg kg−1 and 30 mg kg−1 dose levels via intraperitoneal (IP) injection to achieve sufficient systemic plasma exposure to develop an understanding of the relationship between the extent and duration of KRAS inhibition and anti-tumor activity in mice (Extended Data Fig. 3a). The IP route was selected owing to similar plasma disposition kinetics compared to intravenous (IV) administration as well as the feasibility for repeated dose administration to evaluate tumor response. MRTX1133 demonstrated complete pERK inhibition at both 1 hour and 6 hours after dose in cancer cells using immunohistochemistry supplemented with an image analysis algorithm designed to evaluate the fraction of biomarker-positive tumor cells (Fig. 2a and Supplementary Fig. 1). Dose-dependent reduction of the pERK-positive cell fraction was observed at the 12-hour and 24-hour post-dose timepoints, indicating partial recovery of this pathway at lower dose levels. The percentage of cancer cells expressing pS6 also exhibited a trend toward reduction in MRTX1133-treated tumors compared to vehicle-treated tumors, with near-complete inhibition observed at 1 hour and 6 hours after dose and a dose-dependent recovery by 12 hours and 24 hours at lower dose levels. Consistent with inhibition of KRAS-dependent signaling pathways, active RAS levels were determined in HPAC tumor lysates, and reduced RAS activity was observed at each post-administration timepoint up to 24 hours (Fig. 2b). MRTX1133 was well-tolerated at dose levels administered IP at up to 30 mg kg−1 twice daily (BID) in repeat-dose studies for up to 28 days, with no evidence of weight loss or overt signs of toxicity (Extended Data Fig. 3b). In a repeat-dose study, daily IP administration of MRTX1133 demonstrated dose-dependent anti-tumor efficacy, leading to near-complete responses (85% regression) in mice administered 30 mg kg−1 BID, 16% regression at the 10 mg kg−1 BID dose level and 81% tumor growth inhibition at 3 mg kg−1 BID (Fig. 2c). In the HPAC model, the percentage of cells positive for cleaved caspase-3 staining was increased with MRTX1133 treatment at all dose levels at 12 hours after treatment and at 30 mg kg−1 at both 12 hours and 24 hours after treatment (Fig. 2a and Supplementary Fig. 1). Thus, tumor regression observed was consistent with induction of apoptosis in the HPAC model.

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MRTX1133 was also evaluated for its impact on KRAS-dependent signaling and anti-tumor efficacy in additional KRASG12D-mutant xenograft models. In the Panc 04.03 model, active RAS levels in tumor lysates were significantly reduced 1 hour and 12 hours after administration of MRTX1133 at 3 mg kg−1, 10 mg kg−1 or 30 mg kg−1 administered IP for three consecutive doses at 12-hour intervals (Fig. 2d). Active RAS was also evaluated 24 hours after the final dose in the 30 mg kg−1 group, at which point active RAS remained inhibited. Administration of MRTX1133 also demonstrated dose-dependent inhibition of pERK in tumor lysates at 1 hour after the last dose in the BID × 7 days cohorts; however, pERK recovery was observed by the 12-hour timepoint at each dose level (Fig. 2e). Interestingly, pERK recovery at 12 hours after dose appeared to be incomplete after three consecutive doses compared to the complete pERK rebound observed after 7 days of BID administration, suggesting that pERK reactivation pathways may be increasingly engaged over longer administration schedules (Fig. 2e). MRTX1133 demonstrated dose-dependent anti-tumor efficacy over a repeat-dose schedule in the Panc 04.03 model, including marked regression at both the 10 mg kg−1 and 30 mg kg BID dose levels (60% and 74%, respectively) and approximate tumor stasis at 3 mg kg−1 BID (Fig. 2f). The similar degree of regression in the 10 mg kg−1 and 30 mg kg−1 BID dose groups suggests that 10 mg kg−1 BID is the maximally effective dose in the Panc 04.03 model. In the GP2D tumor model, a single dose of 30 mg kg−1 MRTX1133 was administered, and prominent inhibition of pERK by immunoblot at 1 hour and 6 hours after the dose with partial rebound by 12 hours was observed along with marked tumor regression (63%) over a BID repeat administration schedule (Extended Data Fig. 3c,d).

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MRTX1133 elicits distinct tumor response patterns in KRAS G12D mutant PDAC and CRC models [3]
To evaluate the breadth of anti-tumor activity across genetically and histologically heterogenous KRASG12D-mutant models, MRTX1133 was tested at a fixed dose of 30 mg kg−1 BID administered IP in a panel of human cell-line-derived and patient-derived xenografts. MRTX1133 induced 30% or greater tumor regression in 11 of 25 KRASG12D-mutant models (Fig. 3). Body weight loss did not exceed 15% for the duration of the study for each model represented. The extent of MRTX1133 anti-tumor activity was particularly notable in pancreatic cancer models where eight of 11 (73%) exhibited 30% or greater tumor regression (Extended Data Fig. 4a). By comparison, >30% regression was observed in two of eight (25%) CRC models. MRTX1133 did not demonstrate significant anti-tumor efficacy in all four non-KRASG12D-mutant models tested. Although treatment with MRTX1133 led to marked anti-tumor activity in most models tested, a subset of models was less sensitive to MRTX1133 and exhibited tumor growth inhibition or stable disease as a best response. Bioinformatic analyses were performed to identify molecular biomarkers that correlate with anti-tumor activity. Lower PTEN and CDKN2A RNA expression was associated with reduced anti-tumor activity; however, neither trend reached statistical significance (Extended Data Fig. 4b,c). The potential impact of reduced PTEN and CDKN2A RNA expression on anti-tumor activity was further interrogated using targeted CRISPR screens and combination studies with CDK4/6 or PI3Kα inhibitors, respectively, to assess the functional consequences of co-targeting these pathways in concert with KRAS inhibition in subsequent experiments. Overall, these data confirm that KRASG12D functions as an oncogenic driver across multiple cancer types and that inhibition of KRASG12D by MRTX1133 demonstrates KRASG12D-mediated and tumor-type-dependent efficacy, including marked cytoreductive activity in most PDAC models.

HTRF Binding Assay
A recombinant human KRAS 4B G12D protein (corresponding to amino acids 1-169, expressed in E. Coli with C-terminal Avi biotinylated tag MW=22 kDa) was incubated with the tested compound in buffer (50 mM HEPES pH 7.5, 5 mM MgCl2, 1 mM DTT, ~0.1% DMSO), 5 nM KRASG12D, 100 nM Tracer (compound 45) and 0.5 nM Tb-SA (Cisbio). After a 1-hour incubation at room temperature, the HTRF signal was measured with a Clariostar reader [(BMG) excitation filter (Ex Tr), dichroic filter (LP TP) and emission filters (F 665-10 and F 620-10)] according to the manufacturer’s instructions. The HTRF ratio was calculated using the formula: [emission 665/emission 620] * 10000. IC50’s were fit using Xlfit software (IDBS) with the Hill equation fixed to 1 (fit Background + Bmax/(1 + ((x/IC50)^Hill))).

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SPR [3]
The binding of MRTX1133 to human mutant KRASG12D and KRASWT enzyme was determined as follows. SPR was used to determine the kinetics and affinity of each compound versus KRASWT and KRASG12D. Experiments were performed using Series S SA sensor chips and a Biacore T200. Proteins were immobilized on the chip as follows. The sensor chip surface was conditioned using 1 M NaCl/50 mM NaOH (60 seconds at 10 μl min−1, 25 °C). Biotin (100 μM) was used to deactivate the reference cell (60 seconds at 10 μl min−1, 25 °C). Avi-KRASWT (corresponding to amino acids 1–169) and Avi-KRASG12D (corresponding to amino acids 1–169) (1 μg ml−1) were immobilized onto the experimental cells with a target of 700 response units (RU). Proteins were diluted in the assay running buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.5 mM TCEP, 0.03% Triton X-100, 5 μM GDP and 5% DMSO). Biotin (100 μM) was used to deactivate the remainder of the chip surface on the experimental cells (60 seconds at 10 μl min−1, 25 °C). The injection port was washed with a solution containing 50% isopropanol, 50 mM NaOH and 1 M NaCl between cycles.

Using more traditional experimental setups, MRTX1133 was determined to have a long dissociation half-life versus KRASG12D. Once the protein on the chip became saturated by compound, it was deactivated for the remainder of the experiment. To account for this, a new chip was used for each compound, and only a single injection was performed. Three blank running buffer injections were used to equilibrate the system and monitor baseline stability before each measurement. To capture a representative snapshot of each interaction, a single 3 nM injection of MRTX1133 was used. Empirically, it was determined that the optimal injection concentration for each compound was about equal to the IC50 value generated in a cell-based assay. A long contact time (1,050 seconds at 20 μl min−1, 25 °C) was used to slowly bring the protein to a saturated state, enabling a fit of the association kinetics. Compound dissociation was monitored for 12,600 seconds at 20 μl min−1, 25 °C. Solvent correction was run before and after each measurement containing compound using a 4.5–5.5% DMSO gradient. The experimental data were subtracted from the reference cell, and the third equilibration cycle was used to subtract baseline drift. Data were processed with T200 Bia Evaluation Software, and a binary fitting model was used. The ka, kd and KD values for each interaction are reported below (average ± s.d., n = 2). The KD and kd values generated for MRTX1133 versus KRASG12D are beyond what the instrument can accurately determine but are reported as an approximate value.

A similar experimental setup was used to measure the interaction of each compound versus KRASWT, but shorter dissociation half-lives allowed a single sensor chip to be used. The experimental and data-fitting protocol described above was used with the following modifications: two startup cycles were used to equilibrate the instrument after immobilization; a single 5 nM injection was selected; and the monitored dissociation time was reduced to 6,300 seconds.

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KRAS active and inactive assays [3]
MRTX1133 IC50 values were determined in inactive KRAS (GDP-loaded) biochemical binding assays and active KRAS (GMP-PNP-loaded) RBD binding assays. The ability of a compound to bind to inactive KRAS or KRASG12D was measured using a TR-FRET displacement assay. In a 10-µl assay, 5 µl of biotinylated KRAS or KRASG12D (corresponding to amino acids 1–169) was added to ECHO650 dispensed inhibitors in DMSO. Then, 5 ul of Cy5-labeled tracer described in ref. 10 and terbium streptavidin were added. The final assay conditions were 10 nM Cy5-labeled tracer, 0.5 nM terbium streptavidin and compound (1% DMSO final) in buffer (50 mM HEPES, pH 7.5, 5 mM MgCl2, 0.005% Tween 20 and 1 mM DTT). After a 60-minute incubation at room temperature, the reaction was measured using a BMG Labtech CLARIOstar Plus via TR-FRET. IC50 data were fit to a four-parameter IC50 equation and XLfit software (IDBS). One hundred percent of control (POC) was determined by using a DMSO control, and 0 POC was determined by using a concentration of control compound that completely inhibits binding of the tracer to KRAS.

The active KRAS (GMP-PNP loaded) was measured using TR-FRET in the same assay buffer. Biotinylated KRAS or KRASG12D was used in combination with GST-tagged Raf1-RBD and incubated with 2.5 nM anti-GST-d2 (Cisbio) and 0.5 nM terbium streptavidin in a 10-µl assay to pre-dispersed inhibitors in DMSO using an ECHO650. After a 60-minute incubation at room temperature, the reaction was measured using a BMG Labtech CLARIOstar Plus via TR-FRET. IC50 data were fit to a four-parameter IC50 equation using XLfit software (IDBS). One hundred POC was determined by using a DMSO control, and 0 POC was determined by using a concentration of control compound that completely inhibits binding of RAF-RBD to KRAS.

Protocol for AGS ICW Assay [2]
1. The KRASG12D mutant, AGS cells (ATCC CRL-1739), was grown in DMEM medium supplemented with 10% fetal bovine serum and Penicillin/Streptomycin. Cells were plated in black clear bottom tissue culture treated 96 well plates at a density of 20,000 cells/well and allowed to attach for 12-14 hours. The plated cells were treated with a 3-fold 9-point serial dilution of the compounds, with a top final concentration of 10 µM. The diluted compounds were added to plated cells at a final concentration of 0.5% DMSO. After 3 hours of drug treatment, the cells were fixed by incubating the plates in 50 µl of 4.0% formaldehyde at room temperature for 20 minutes. The formaldehyde was then dumped out, and 150µL of ice cold 100% methanol was added for 10 minutes to permeabilize the cells. Methanol was dumped out, and 100 µL of Licor blocking buffer (Li-Cor Biotechnology, Lincoln NE) was added for 1 hour at room temperature to inhibit non-specific antibody binding in the plates.

2. The amount of phospho-ERK was determined using an antibody specific for the phosphorylated form of ERK and compared to the amount of GAPDH. Primary antibodies used for the detection were added as follows: Phospho-ERK (Cell Signaling CS-9101) diluted 1:500 and GAPDH (Millipore MAB374) diluted 1:5000 in Odyssey blocking buffer + 0.05%Tween 20. The plates were incubated overnight at 4 °C. The plates were washed 3X with 150uL PBS + 0.1% Tween 20.
3. Secondary antibodies used to visualize primary antibodies were added as follows: Goat Anti-Rabbit-800 (LI-COR, 926-32211) and Goat Anti-Mouse680 (LI-COR, 926-68070) diluted 1:800 both in Odyssey blocking buffer +0.05% Tween20, and were incubated for 1 hour at room temperature. The plates were washed 3X with 150uL PBS +0.1% Tween20. Plates were imaged dry on a LiCOR Odyssey CLX plate reader. S192 4. The plates were analyzed by normalizing the phospho-ERK (Thr202/Tyr204) signal to the GAPDH signal for each well and percent of DMSO control values were calculated. IC50 values were generated using a 4-parameter fit of the dose response curve.

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Protocol for 2D Cell Proliferation Assay[2]
1. The KRASG12D mutant cell line, GP2d, was grown in DMEM medium supplemented with 10% fetal bovine serum and Penicillin/Streptomycin. The KRASWT cell line, MKN1 (JCRB0252), was grown in RPMI Cells supplemented with 10% fetal bovine serum, 10mM HEPES, 10mM Sodium Pyruvate, and Penicillin/Streptomycin. Cells were plated in white clear bottom tissue culture treated 96 well plates at a density of 2,000 cells/well and allowed to attach for 12-14 hours. Each cell line was also plated in 3 wells in a baseline plate at the same density to determine the luminescence RLU values of each cell line before the drug treatment. The baseline plate was read immediately before treating plated cells with drug by incubating each of the three plated wells per cell line with 30 µL of CTG reagents for 30 mins, covered from light and shaking vigorously. The luminescence RLU values were then read on the CLARIOstar microplate reader. The plated cells were treated a 3-fold 9-point serial dilution dose response of MRTX1133, with a top final concentration of 3µM. Diluted compounds were added in a final concentration of 0.5% DMSO. After 3 days of drug treatment, each plate was read on the CLARIOstar using the conditions for the baseline plate described above.

2. The data was analyzed by subtracting the baseline RLU values from the RLU values from the treatment plates after 3 days of MRTX1133 addition. Cell proliferation percent inhibition values were calculated by dividing luminescence unit values from each treated well by the average of the luminescence unit values in the vehicle-treated wells and multiplied by 100. Data were transposed and passed into GraphPad Prism to obtain IC50 values using a 4-parameter fit of the dose response curve.

Tumor Pharmacodynamic and Tumor Xenograft Studies [2]
Mice were maintained under pathogen-free conditions, and food and water were provided ad libitum. 6–8-weekold, female, athymic nude-Foxn1nu mice were injected subcutaneously with Panc 04.03 cells in 100 l of PBS and Matrigel matrix in the right hind flank with 5.0 x 106 cells 50:50 cells : Matrigel. Mouse health was monitored daily, and caliper measurements began when tumors were palpable. Tumor volume measurements were determined utilizing the formula 0.5 x L x W2 in which L refers to length and W refers to width of each tumor.  Tumor pharmacodynamic studies: When tumors reached an average tumor volume of ~400 mm3 , mice were randomized into treatment groups. Mice were treated by intraperitoneal injection with either vehicle consisting of 10% research grade Captisol in 50 mM citrate buffer pH 5.0 or MRTX1133 at 30mg/kg. Tumors and plasma were collected at 1 hour and 12 hours after a single dose to determine exposure levels. Tumor fragments were snap frozen in homogenization tubes with liquid nitrogen and homogenized with Lysis/Binding Buffer AM11 with protease and phosphatase inhibitors added fresh before use. Tumor lysates were then assayed for ERK1/2 phosphorylation. Xenograft studies: When tumors reached an average tumor volume of ~350 mm3 , mice were randomized into treatment groups. Mice were treated by intraperitoneal injection with either vehicle consisting of 10% research grade Captisol in 50 mM citrate buffer pH 5.0 or MRTX1133 in vehicle at 3, 10, or 30mg/kg BID.  Animals were monitored daily, tumors were measured 3 times per week, and body weights were measured 2 times per week. Data are expressed as mean +/- SEM. Statistical analysis of differences in mean tumor volume between vehicle and MRTX1133-treated cohorts was run using a two-tailed Student’s t-test with equal variance in Excel.

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The pharmacokinetic study was performed using oral solutions of MRTX1133 in 5% carboxymethyl-cellulose sodium (CMC-Na) at a concentration of 2.5 mg mL-1 (the dose was 25 mg kg-1 and 10 mL kg-1). The intravenous solution was prepared at a concentration of 5 mg mL-1 in polyethylene glycol 400 and dimethyl sulfoxide (8%). Twenty rats were randomly divided into two groups (ten rats each group), the rats in one group were given MRTX1133 by intravenous administration at 5 mg kg-1 and the other group by intragastric administration at 25 mg kg-1. Rats were slightly anesthetized with diethyl ether, blood samples were collected (approximately 150 µL) from the suborbital vein, and placed into heparinized tubes at 0.17 h, 0.33 h, 0.5h, 0.75 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, and 24 h after treatment. The rats were treated with saline at the same volume by gavage and used as control. Blood samples were centrifuged at 20,800 g for 10 min (at 4°C) and stored at - 80°C.

The distribution of MRTX1133 in the tissues was performed using 25 rats with a single intravenous administration of MRTX1133 at 5 mg kg-1. The blood samples were collected from abdominal aorta at 0.5 h, 1 h, 2 h, 6 h, and 24 h (five rats at each time point), then the organs such as the heart, liver, spleen, lung, kidney, intestine, and pancreas were immediately collected. The organs were washed with cold physiological saline (4°C), they were weighed and homogenized in cold saline solution (1:3, w/v). The homogenized samples were treated according to the method described in the sample preparation section. The excretion of MRTX1133 in the urine was also investigated by placing the rats in metabolic cages after intravenous administration of MRTX1133 at 5 mg kg-1 (one rat in each cage), the urine and feces were collected at 2 h, 6 h and 24 h after administration (five rats at each time point), and the content of MRTX1133 in urine and feces was detected by the LC-MS method.

The DAS 3.2 software package (edited by the Chinese Mathematical Pharmacology Society) was used for the pharmacokinetic data analysis and the non-compartmental model was applied. The oral bioavailability (F) of MRTX1133 was measured by comparing each area under the curve (AUC) 0-t value after intragastric (i.g.) and intravenous (i.v.) administration according to the equation: F = (AUC i. g./Dose i. g.)/(AUC i. v./Dose i. v.).[4]

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Animal/Disease Models: 6-8-week age, female/athymic nude-Foxn1nu mice (Panc 04.03 model)
Doses: 3, 10, 30 mg/kg
Route of Administration: Intraperitoneal injection/ip; twice a day for 28 days
Experimental Results: MRTX1133 dose-dependent suppressed tumor growth with a TGI of 94% observed at 3 mg/kg BID (IP) and TGIs of -62% and -73% observed at 10 and 30 mg/kg BID (IP), respectively.

Pharmacokinetics study [4]
MRTX1133 was detected in rat plasma samples after tail vein injection of 5 mg kg-1 and intragastric administration of 25 mg kg-1. The plasma concentration-time profile of MRTX1133 is shown in Figure 3, and the pharmacokinetic parameters are summarized in Table 5. The plasma concentration of MRTX1133 sharply increased after the oral administration, reaching the peak concentration at 45 min with the plasma Cmax of 129.90 ± 25.23 ng mL-1, suggesting that MRTX1133 was quickly absorbed. The t1/2 of MRTX1133 was 1.12 ± 0.46 h after oral administration and 2.88 ± 1.08 h after intravenous administration. MRTX1133 was bleow the limit of quantitation 6 h after oral administration and 8 h after intravenous administration. Wang et al. (2022) investigated the concentration of MRTX1133 in CD-1 mice after intraperitoneal administration of 30 mg kg-1. Their result showed that the C max of MRTX1133 was approximately 7,000 ng mL-1 at 1 h after intraperitoneal administration, almost 1,000 ng mL-1 at 4 h, 100 ng mL-1 at 8 h, and it was detected up to 24 h (approximately 50 ng mL-1), indicating that MRTX1133 has a long plasma half-life time, our results were quite different than those. The reasons of the inconsistent results might be different animals and administration routes. The AUC values for the oral and intravenous administration of MRTX1133 were 135.54 ± 46.51 and 927.88 ± 192.11 μg/L*h; thus, the F of MRTX1133 in rats was 2.92%. Suggesting that MRTX1133 had a very low bioavailability. The low bioavailability could be caused by different factors; therefore, it needed to be further studied in the process of formulation development.

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Tissue distribution and excretion study [4]
The tissue distribution of MRTX1133 was shown in Figure 4. MRTX1133 was widely distributed in the main organs, such as liver, kidney, lung, spleen, heart, pancreas, and intestine. The highest concentrations of MRTX1133 in the liver was 5,358.68 ± 1,062.23 ng g-1 at 1 h after administration, the highest concentrations in the kidney, lung and heart were 2,584.60 ± 609.56 ng g-1, 1,230.62 ± 125.94 ng g-1 and 879.29 ± 449.87 ng g-1, respectively, after 2 h. The highest concentrations in the spleen and pancreas were 1858.73 ± 224.31 ng g-1 and 155.74 ± 34.18 ng g-1, respectively, after 6 h. MRTX1133 was still detectable in the organs (except intestine) at 24 h after administration. The concentration of MRTX1133 in liver, spleen, kidney, and lung increased rapidly after administration, and they were higher than the concentration of the drug in plasma at 2 h after administration, which is indicated that MRTX1133 has high affinity to these organs. MRTX1133 was quickly transferred from serum to the liver, sleep, lung, and kidney, and it was widely distributed into the tissues. The testis was also assessed, but the results showed no MRTX1133.
The concentration of MRTX1133 in urine was 10.43% ± 2.89% (6.53%–13.54%) excreted through the kidney at 6 h after administration as the prototype drug, and 22.59% ± 3.22% (17.60%–25.92%) was excreted 24 h as the prototype drug. The result is shown in Figure 5. However, no MRTX1133 was found in the feces. The results of tissue distribution and excretion study revealed that MRTX 1133 might not be excreted through the biliary route. In addition, the excretion ratio through the kidney of the prototype drugs was very low because most drug might be metabolized into other components by the liver.
This study investigated the pharmacokinetic, distribution and excretion of MRTX1133 in rats, but some limitations are present. We only collected the feces 24 h after the administration of the drug, and the feces in other time was not collected, so we could not determine whether MRTX1133 was excreted with feces. Although MRTX1133 was not detected in the feces, bile was not collected; thus, it was not possible to accurately evaluate whether MRTX1133 was excreted through the bile. In addition, the metabolic process and main products of MRTX 1133 were not investigated in vivo.

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Results: The calibration curve for MRTX1133 in plasma and other homogenates was linear, with r 2 > 0.99. The intra- and inter-day accuracies were ranged from 85% to 115% and precision were within ± 10%. The matrix effect and recovery were within ± 15 %. The Cmax of MRTX1133 was 129.90 ± 25.23 ng/mL at 45 min after oral administration. The plasma half-life (t1/2) of MRTX1133 was 1.12 ± 0.46 h after oral administration and 2.88 ± 1.08 after intravenous administration. Its bioavailability was 2.92%. Furthermore, MRTX1133 was widely distributed in all the main organs, including liver, kidney, lung, spleen, heart, pancreas, and intestine. MRTX1133 was still detectable in liver, kidney, lung, spleen, heart, and pancreas after 24 h. The excretion ratio of prototype MRTX1133 through kidney was 22.59% ± 3.22% after 24 h. Conclusions: MRTX1133 was quickly absorbed, and widely distributed in the main organs. This study provided a reference for the quantitative determination of MTRX1133 in preclinical or clinical trials.[4]

[1]. KRAS-Mutant Non-Small Cell Lung Cancer: An Emerging Promisingly Treatable Subgroup. Front Oncol. 2021 May 3:11:672612.

[2]. Identification of MRTX1133, a Noncovalent, Potent, and Selective KRASG12D Inhibitor. J Med Chem. 2022 Feb 24;65(4):3123-3133.

[3]. Anti-tumor efficacy of a potent and selective non-covalent KRASG12D inhibitor. Nat Med. 2022 Oct;28(10):2171-2182.

[4]. Pharmacokinetics, bioavailability, and tissue distribution of MRTX1133 in rats using UHPLC-MS/MS. Front Pharmacol. 2024 Dec 19:15:1509319.

KRAS G12D Inhibitor MRTX1133 is an orally bioavailable reversible inhibitor of the oncogenic KRAS substitution mutation G12D, with potential antineoplastic activity. Upon oral administration, KRAS G12D inhibitor MRTX1133 specifically targets and noncovalently binds to KRAS G12D. This prevents KRAS G12D-mediated signaling and activation of downstream survival pathways. This leads to an inhibition of the growth of tumor cells that overexpress KRAS G12D. KRAS, a member of the RAS family of oncogenes, serves an important role in cell signaling, division and differentiation. Mutations of KRAS may induce constitutive signal transduction leading to tumor cell proliferation, invasion, and metastasis.

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Lung cancer, the leading cause of cancer-related deaths worldwide, can be classified into small cell lung cancer and non-small cell lung cancer (NSCLC). NSCLC is the most common histological type, accounting for 85% of all lung cancers. Kirsten rat sarcoma viral oncogene (KRAS) mutations, common in NSCLC, are associated with poor prognosis, likely due to poor responses to most systemic therapies and lack of targeted drugs. The latest published clinical trial data on new small-molecule KRAS G12C inhibitors, AMG510 and MRTX849, indicate that these molecules may potentially help treat KRAS-mutant NSCLC. Simultaneously, within the immuno-therapeutic process, immune efficacy has been observed in those patients who have KRAS mutations. In this article, the pathogenesis, treatment status, progress of immunotherapy, and targeted therapy of KRAS-mutant NSCLC are reviewed. [1]

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Through extensive structure-based drug design, MRTX1133 was identified as a noncovalent, potent, and selective inhibitor of KRASG12D. MRTX1133 suppresses KRASG12D signaling in cells and in vivo, and its antitumor benefit was demonstrated in a murine animal model. To the best of our knowledge, this is the first report in the literature of a small molecule inhibitor of KRASG12D that exhibits robust in vivo efficacy. These data support the potential for the advancement of an effective therapeutic against this “undruggable” target. The optimization process was facilitated by high-resolution X-ray crystal structures. In-depth binding mode analysis derived from cocrystal structures allowed the optimization of lipophilic contact of the inhibitor in the binding pocket and the identification of nonclassical hydrogen bonding and ion pair interactions, ultimately increasing selective binding affinity for KRASG12D by more than 1,000,000-fold relative to the initial hit 5B. MRTX1133 binds to the switch II pocket and inhibits the protein–protein interactions necessary for activation of the KRAS pathway. MRTX1133 not only possesses single-digit nM potency in a cellular proliferation assay, but also demonstrates tumor regressions in the Panc 04.03 xenograft model. A more comprehensive in vitro and in vivo pharmacological characterization of MRTX1133 will be disclosed in due course.[2]

KRASG12D, the most common oncogenic KRAS mutation, is a promising target for the treatment of solid tumors. However, when compared to KRASG12C, selective inhibition of KRASG12D presents a significant challenge due to the requirement of inhibitors to bind KRASG12D with high enough affinity to obviate the need for covalent interactions with the mutant KRAS protein. Here, we report the discovery and characterization of the first noncovalent, potent, and selective KRASG12D inhibitor, MRTX1133, which was discovered through an extensive structure-based activity improvement and shown to be efficacious in a KRASG12D mutant xenograft mouse tumor model. [2]

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These data indicate that discovery and pre-clinical development of high-affinity, mutation-selective, non-covalent inhibitors of KRASG12D and perhaps other KRAS mutant variants is feasible. As KRASG12D is the most prevalent of KRAS mutant alleles, the translation of these findings to a reality for patients with cancer harboring KRASG12D mutations would be highly impactful. Additionally, the anticipated therapeutic index for allele-specific inhibitors may provide an advantage in both facilitating maximal target inhibition and a favorable combinatorial therapy profile. These studies also provide insight toward the function of this mutation as an oncogenic driver in different tumor types and in the context of co-occurring genetic alterations. The ability to also characterize the effect of MRTX1133 on KRAS-dependent signaling and feedback pathways using molecular profiling approaches and functional genomics helps increase understanding of unique aspects of KRASG12D signaling. In turn, the understanding of KRASG12D signaling dynamics provides rational perspective on co-targeting of collateral dependencies. Collectively, the present studies provide renewed perspective on direct targeting strategies for KRAS and provide defining strategies to identify patients likely to benefit from single-agent or rationally directed combinations.[3]

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This study was the first to evaluate the pharmacokinetics, bioavailability, distribution, and excretion of MRTX1133 in rats. A sensitive, rapid, and reliable UHPLC-MS/MS method was developed to measure MTRX1133 in rat plasma, tissue homogenate and urine. The established method was successfully applied to the pharmacokinetic study of MTRX1133 in rats after administration by different routes. MRTX1133 is quickly absorbed after oral administration and widely distributed in the body. But the bioavailability is very low and only 24% of the drug were excreted through the kidneys by the original form. This study might provide a sufficient reference for the quantitative determination of MTRX1133, in preclinical or clinical studies/trials.

C33H31F3N6O2

600.6335

600.246

C, 65.99; H, 5.20; F, 9.49; N, 13.99; O, 5.33

2621928-55-8

2621928-55-8;

156124857

Yellow to brown solid powder

5.2

2

11

6

44

1100

2

C#CC1=C(C=CC2=CC(=CC(=C21)C3=NC=C4C(=C3F)N=C(N=C4N5CC6CCC(C5)N6)OC[C@@]78CCCN7C[C@@H](C8)F)O)F

SCLLZBIBSFTLIN-IFMUVJFISA-N

InChI=1S/C33H31F3N6O2/c1-2-23-26(35)7-4-18-10-22(43)11-24(27(18)23)29-28(36)30-25(13-37-29)31(41-15-20-5-6-21(16-41)38-20)40-32(39-30)44-17-33-8-3-9-42(33)14-19(34)12-33/h1,4,7,10-11,13,19-21,38,43H,3,5-6,8-9,12,14-17H2/t19-,20?,21?,33+/m1/s1

4-[4-(3,8-diazabicyclo[3.2.1]octan-3-yl)-8-fluoro-2-[[(2R,8S)-2-fluoro-1,2,3,5,6,7-hexahydropyrrolizin-8-yl]methoxy]pyrido[4,3-d]pyrimidin-7-yl]-5-ethynyl-6-fluoronaphthalen-2-ol

MRTX1133; MRTX 1133; MRTX1,133; 2621928-55-8; 4-[4-(3,8-Diazabicyclo[3.2.1]oct-3-yl)-8-fluoro-2-[[(2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl]methoxy]pyrido[4,3-d]pyrimidin-7-yl]-5-ethynyl-6-fluoro-2-naphthalenol; CHEMBL4858364; 4-(4-(3,8-Diazabicyclo[3.2.1]octan-3-yl)-8-fluoro-2-(((2R,7aS)-2-fluorohexahydro-1H-pyrrolizin-7a-yl)methoxy)pyrido[4,3-d]pyrimidin-7-yl)-5-ethynyl-6-fluoronaphthalen-2-ol; 4-[4-(3,8-diazabicyclo[3.2.1]octan-3-yl)-8-fluoro-2-[[(2R,8S)-2-fluoro-1,2,3,5,6,7-hexahydropyrrolizin-8-yl]methoxy]pyrido[4,3-d]pyrimidin-7-yl]-5-ethynyl-6-fluoronaphthalen-2-ol; MFCD34567005; MRTX-1133

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:50~100 mg/mL (83.3~166.5 mM)

Solubility in Formulation 1: 10 mg/mL (16.65 mM) in 10% SBE-β-CD/50 mM citrate pH 5.0 (add these co-solvents sequentially from left to right, and one by one), clear solution.

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Solubility in Formulation 2: 3.5 mg/mL (5.83 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 35.0 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 (4.16 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 4: 5%DMSO+40%PEG300+5%Tween80+50%ddH2O: 25mg/ml

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