wild-type ROS1; ROS1 G2032R; ROS1 fusions and resistance mutants

Zidesamtinib (72 h), with average IC50s of 0.4 nM, inhibits the growth of seven cell lines expressing wild-type ROS1 fusions[1].
Zidesamtinib (72 h), with average IC50s of 1.6 nM, inhibits the growth of six cell lines harboring ROS1 fusions with the G2032R mutation[1].
Zidesamtinib (72 h) potently inhibits the non-G2032R ROS1 mutants, with IC50s ≤ 1.5 nM[1].
Zidesamtinib (10-1000 nM; 4 weeks) inhibits the formation of colonies in NIH3T3 cells that express both ROS1 fusions with G2032R and wild-type ROS1 fusions[1].

Zidesamtinib (0.04-15 mg/kg; p.o. twice daily for 28 d) incites tumor regression in wild-type ROS1 xenograft models at all doses ≥0.2 mg/kg[1].

Biochemical Kinase Activity Assay[1]
Activity of purified kinases was measured using the PhosphoSens assay. Test compounds were dissolved in DMSO to 100-fold over the desired concentration and dispensed at 250 nL into a 384-well plate in a 3-fold dilution series. A 12.5 μL solution containing 2 mmol/L ATP with 26 μmol/L fluorogenic peptide substrate AQT0101 r AQT0104 in buffer (50 mmol/L HEPES pH 7.5, 0.01% Brij-35, 0.5 mmol/L EGTA, 10 mmol/L MgCl2) was added to the plate. The reaction was triggered by the addition of a 12.5 μL solution containing 0.5 nmol/L ROS1, 2 nmol/L ROS1 G2032R, 1 nmol/L TRKA, 3 nmol/L TRKB, or 0.5 nmol/L TRKC kinase domains in buffer (50 mmol/L HEPES pH 7.5, 0.01% Brij-35, 2% glycerol, 0.4 mg/mL BSA, 0.5 mmol/L EGTA, 10 mmol/L MgCl2). The final concentrations were 1 mmol/L ATP, 13 μmol/L peptide substrate (AQT0101 for ROS1 and ROS1 G2032R or AQT0104 for TRKA, TRKB, and TRKC), 0.25 to 1.5 nmol/L kinase (0.25 nmol/L ROS1; 1 nmol/L ROS1 G2032R; 0.5 nmol/L TRKA; 1.5 nmol/L TRKB; or 0.5 nmol/L TRKC), 50 mmol/L HEPES pH 7.5, 0.01% Brij-35, 1% glycerol, 0.2 mg/mL BSA, 0.5 mmol/L EGTA, and 10 mmol/L MgCl2. The plate was sealed, and fluorescence signal was recorded by a plate reader at λemission = 485 nm every 2 minutes for 120 minutes at 30°C. The change in fluorescence intensity over time during the initial, linear phase of the reaction is initial velocity (v). IC50 was calculated from the plot of initial velocity versus log (inhibitor concentration) regressed to the four-parameter logistic equation.[1]

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Kinase Panel Screening[1]
Inhibition of kinase activity was measured using radiolabeled [γ-33P]-ATP. A solution containing [γ-33P]-ATP was mixed with NVL-520, each of the 335 kinases, and the corresponding kinase substrate. NVL-520 was assayed at two concentrations: 100 nmol/L and 1 μmol/L. The concentration of ATP in this assay was close to the Michaelis–Menten constant (KM) for each kinase. The reaction was stopped after 1 hour, and the incorporation of 33P was quantified using a scintillation counter. Inhibition was measured by the residual activity of 33P: Lower residual activity indicated higher inhibitor potency for the specific kinase. Based on the 335-kinase screen, 24 kinases showing >50% inhibition were selected for focused IC50 determination using the same assay at 10 concentrations of NVL-520 in semilogarithmic steps (3 μmol/L, 900 nmol/L, 300 nmol/L, 90 nmol/L, …, 0.09 nmol/L). IC50 was determined from the plot of residual activity against log (inhibitor concentration) regressed to a four-parameter logistic equation. Kinome map illustration reproduced courtesy of Cell Signaling Technology, Inc.

Cell Viability Assay[1]
Protocols varied slightly with the testing site. For testing site A, A549 or stable Ba/F3 cells were seeded into 384-well plates, and test compounds were added in a 3-fold dilution series in complete culture medium containing 10% FBS. After a 72-hour incubation with the inhibitor, cell viability was measured using the CellTiter-Glo reagent. Untreated wells served as negative controls (no inhibition of proliferation), whereas wells treated with high concentrations of the nonspecific kinase inhibitor staurosporine served as positive controls (full inhibition of proliferation). IC50 was calculated from percent inhibition and log (inhibitor concentration) using four-parameter logistic regression.[1]
For testing site B, all inhibitors were prepared as 1 mmol/L stocks in DMSO. Plates were preseeded with 25 μL per well of complete medium using a Multidrop Combi Reagent Dispenser. Inhibitors were distributed onto 384-well plates at 2-fold of the indicated concentrations into 25 μL per well of complete medium using a D300 Digital Dispenser. Ba/F3 cell lines expressing wild-type or mutant ROS1 fusions were seeded at 1,000 cells per well in a volume of 25 μL using a Multidrop Combi Reagent Dispenser. Plates were incubated for 72 hours. Viability was measured using a WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]–based assay and read on a Biotek Synergy 2 plate reader. Each condition was assayed in triplicate. Data were normalized using Microsoft Excel, and IC50 values were calculated using a nonlinear regression analysis in GraphPad Prism.[1]
For testing site C, MGH193-1 and MGH9018-1 cells were plated at 4,000 cells/well in triplicate into 96-well plates. Cells were incubated with CellTiter-Glo after 5-day drug treatment, and luminescence was measured with a SpectraMax M5 Multimode Microplate Reader. GraphPad Prism (GraphPad Software) was used to graphically display data and determine IC50 values by a nonlinear regression model utilizing a four-parameter analytic method.[1]
In some human cancer cell lines, we observed that NVL-520 showed biphasic dose–response behavior. We attributed the first dose response to on-target growth inhibition caused by ROS1 inhibition and the second dose response to off-target cytotoxicity caused by pathways beyond ROS1 inhibition. In such cases, only the first IC50 values are reported and indicate on-target ROS1 inhibition.[1]

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Cellular Phosphorylation Assay[1]
For the Ba/F3 TRKB cell phosphorylation assay, cells were seeded into 384-well plates, and test compounds were added in a 3-fold dilution series in full culture medium + 10% FBS. Cells were stimulated with 100 ng/mL BDNF for 20 minutes. TRK phosphorylation was measured using the phospho-TRKA (Tyr674/675)/phospho-TRKB (Tyr706/Tyr707) AlphaLISA reagent. Untreated wells served as negative controls (no inhibition), whereas wells treated with high concentrations of the nonspecific kinase inhibitor staurosporine served as positive controls (full inhibition). IC50 was calculated from percent inhibition and inhibitor concentration using four-parameter logistic regression.[1]
NIH3T3 cells expressing EZR–ROS1 wild-type or mutant fusions were treated with the indicated concentrations of inhibitor for 3 hours prior to harvest. Cells were washed with phosphate-buffered saline (PBS) and harvested with cell lysis buffer supplemented with 0.25% deoxycholate, 0.05% SDS, and protease and phosphatase inhibitors. Protein concentrations were determined using the Pierce BCA Protein Assay. Lysates were extracted using Laemelli sample buffer supplemented with beta-mercapto­ethanol for 10 minutes at 75°C, and lysates were run on 4% to 20% precast gradient Bis-tris gels. [1]
MGH193-1 and MGH9018-1 were treated for 6 hours. Total protein lysates were analyzed by Western blotting with the following antibodies: phospho-ROS1 Y2274 (3078), ROS1 (3287), phospho-AKT S473 (4060), AKT (4691), phospho-ERK1/2 T202/Y204 (9101), ERK1/2 (9102), phospho-S6 S240/244 (5364), S6 (2217), and β-Actin (4970).[1]

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Colony Formation Assay[1]
Plates were preseeded with 0.8% agarose in complete medium with either DMSO or inhibitor (crizotinib, entrectinib, lorlatinib, or NVL-520 at 10, 100, or 1,000 nmol/L). Each inhibitor was paired with its own DMSO condition to serve as an accurate control. NIH3T3 cells expressing CD74–ROS1 or EZR–ROS1 wild-type or mutant fusions were plated in 0.4% agarose in complete medium at a density of 2,000 cells per 0.5 mL of agarose with DMSO or inhibitor at an identical concentration to the bottom layer. Plates were incubated for 4 weeks, and each well was fed 3× per week with 75 μL of complete medium with or without inhibitor to match each plated condition to prevent drying of the agarose. Plates were read after 3 and 4 weeks using GelCount. Colony counts were averaged by condition and normalized to the colony counts from paired DMSO conditions. Data analysis and visualization were performed using Microsoft Excel and GraphPad Prism.

Female athymic Nude-Foxn1^nu mice were implanted subcutaneously with tumor fragments from model CTG-0848[1]
0.04, 0.2, 1, 5, 15 mg/kg
Oral gavage twice daily for 21 days

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Subcutaneous Xenograft Studies.[1]
CTG-0848 PDX. [1]
The experimental procedures were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Champions Oncology, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Female athymic Nude-Foxn1nu mice were implanted subcutaneously into the left flank with tumor fragments from the CTG-0848 model. In the efficacy study, after tumors grew to 150 to 300 mm3, mice (n = 3–5/group) were randomized and administered vehicle or NVL-520 by oral gavage b.i.d. (12-hour intervals). In a separate pharmacokinetic (PK) and pharmacodynamic (PD) study, after tumors grew to 350 to 500 mm3, mice received a single dose or b.i.d. × 5 days of vehicle or NVL-520, and tumor and blood were collected at 1 hour and 12 hours (treatment only) after dose. The model was verified by vendor-provided NGS to contain a heterozygous CD74–ROS1 fusion.[1]

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Lu01-0414 PDX.[1]
All the procedures related to animal handling, care, and treatment in study were performed according to the guidelines approved by the IACUC of WuXi AppTec following the guidance of the AAALAC. Female Balb/c nude mice were implanted subcutaneously into the right flank with tumor slices (∼30 mm3) from the Lu01-0414 model. In the efficacy study, after tumors grew to an average tumor volume of 160 mm3, mice (n = 3–5/group) were randomized and administered by oral gavage b.i.d. (12-hour intervals) vehicle or NVL-520. In a separate PK and PD study, after tumors grew to an average tumor volume of 492 mm3, mice received a single dose or b.i.d. × 5 days of vehicle or NVL-520, and tumor and blood were collected at 1 hour and 12 hours (treatment only) after dose. The model was verified by vendor-provided NGS to contain a heterozygous SDC4–ROS1 fusion with a linkage between the SDC4 L66 residue and the ROS1 A1750 residue.[1]

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MGH9018-1 PDX. [1]
The study was conducted in accordance with the guidelines as published in the Guide for the Care and Use of Laboratory Animals and was approved by the IACUC of Massachusetts General Hospital. Xenografts were implanted subcutaneously into the flanks of female athymic nude (Nu/Nu) mice ages 6 to 8 weeks. Mice were maintained in laminar flow units in sterile filter-top cages with Alpha-Dri bedding. Mice were randomized into groups once the tumors had attained a volume of 150 mm3. The treatment groups were treated with drug solution dissolved in acid water once a day (crizotinib) or drug solution dissolved in 20% hydroxypropyl-b-cyclodextrin (HP-β-CD) twice a day at 9/15-hour intervals (NVL-520) by oral gavage. Tumor volumes were measured twice weekly and calculated using the formula: mm3 = 0.52 × L × W2. For protein assays, tumor-bearing mice were administered drugs for 3 days according to the above dosing schedule, and tumors were harvested 3 hours after the last treatment for Western blotting.[1]

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CTG-2532 PDX. [1]
The experimental procedures were performed according to the guidelines approved by the IACUC of Champions Oncology, accredited by AAALAC. Female athymic Nude-Foxn1nu mice were implanted subcutaneously into the left flank with tumor fragments from model CTG-2532. In the efficacy study, after tumors grew to 150 to 300 mm3, mice (n = 5/group) were administered vehicle, NVL-520, or repotrectinib by oral gavage b.i.d. (12-hour intervals) for up to 21 days (b.i.d. × 21 days) if tolerated. Repotrectinib (DC Chemicals) was dosed as a suspension solution containing 0.5% carboxymethylcellulose and 1% Tween-80. The dosing suspension was stored 2–8°C in the dark for up to 7 days with continuous stirring. In a separate PK and PD study, CTG-2532 tumors were subcutaneously implanted in mice and allowed to grow to 350 to 550 mm3 before a single dose of the vehicle or NVL-520 was administered. Tumor and blood were collected at 1 hour and 12 hours (treatment only) after dose for PK and PD analysis. The model was verified by vendor-provided NGS to harbor a CD74–ROS1 G2032R. Ba/F3 CD74–ROS1 G2032R CDX. All the procedures related to animal handling, care, and treatment in this study were performed according to guidelines approved by the IACUC of Pharmaron following the guidance of the AAALAC. Six- to 8-week-old female Balb/c nude mice were inoculated subcutaneously on the right flank with 1 × 106 Ba/F3 CD74–ROS1 G2032R cells. After the tumors reached a mean tumor volume of approximately 128 mm3, treatment was initiated, and tumors and body weight were measured at regular intervals.[1]

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Plasma drug concentrations were determined by LC/MS. Free drug concentrations were calculated by multiplying the total concentration values determined from PK experiments with the corresponding fraction unbound in mouse plasma (= 7.7% for NVL-520 and = 7.6% for crizotinib).[1]

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Kp,uu and Intracranial Studies[1]
All the procedures related to animal handling, care, and treatment in these studies were performed according to guidelines approved by the IACUC of Pharmaron following the guidance of AAALAC.[1]
NVL-520 was formulated as 1 mg/mL suspension in 20% HP-β-CD in deionized water. Lorlatinib was formulated as a 1 mg/mL solution in two equivalents of HCl + 20% HP-β-CD in deionized water. Compounds were administered orally to male Wistar Han rats (n = 3 each). After 1 hour, brain samples and plasma were collected, and brain samples were homogenized in PBS. Brain and plasma samples were precipitated by acetonitrile and centrifugation (4,700 rpm, 15 minutes). Drug concentrations in the supernatants were quantified by LC/MS-MS. Unbound fractions were determined using rapid equilibrium dialysis. Kp,uu was calculated as the ratio between unbound drug concentration in the brain and unbound drug concentration in the plasma.[1]
Ba/F3 CD74–ROS1 G2032R cells were transduced with viral particles containing the firefly luciferase gene and a neomycin resistance marker. Infected cells were selected on neomycin, and monoclonal cultures were established through limiting dilution. Successful transformants were confirmed by Sanger sequencing and bioluminescence. For the in vivo study, 1 × 105 Ba/F3 CD74–ROS1 G2032R luciferase cells were stereotactically implanted into the right forebrains of 6- to 8-week-old female Balb/c nude mice. After 5 days, mice were randomized based on mean bioluminescence signal into three groups of n = 7–10 mice each and received vehicle or NVL-520, 2 mg/kg, orally, b.i.d. Bioluminescence and body weight were measured at regular intervals until the end of the study (61 days after treatment start) or until animals met the criteria for euthanasia.

[1]. NVL-520 is a selective, TRK-sparing, and brain-penetrant inhibitor of ROS1 fusions and secondary resistance mutations. Cancer Discov. 2022 Dec 13;CD-22-0968.

Zidesamtinib is an orally available selective inhibitor of the receptor tyrosine kinase c-ros oncogene 1 (ROS1), with potential antineoplastic activity. Upon oral administration, zidesamtinib targets, binds to and inhibits wild-type, point mutants and fusion proteins of ROS1. This inhibits proliferation of ROS-1-driven tumor cells, including in tumor cells harboring certain ROS1 resistance mutations, such as the solvent front mutation G2032R and the S1986Y/F, L2026M, and D2033N resistance mutations. Inhibition of ROS1 leads to the disruption of downstream signaling pathways and the inhibition of cell growth of tumors in which ROS1 overexpressed, rearranged or mutated. ROS1, overexpressed in certain cancer cells, plays a key role in cell growth and survival of cancer cells. NVL-520 is able to penetrate the blood-brain barrier (BBB).

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The combined preclinical features of NVL-520 that include potent targeting of ROS1 and diverse ROS1 resistance mutations, high selectivity for ROS1 G2032R over TRK, and brain penetration mark the development of a distinct ROS1 TKI with the potential to surpass the limitations of earlier-generation TKIs for ROS1 fusion-positive patients.[1]

C22H22FN7O

419.46

C, 63.00; H, 5.29; F, 4.53; N, 23.37; O, 3.81

2739829-00-4

2739829-00-4

166560233

Typically exists as white to off-white solids at room temperature

2.8

CCN1C2=C(CC3=NN(N=C3C4=C(C=C(C=C4)F)[C@H](OC5=C(N=CC2=C5)N)C)C)C=N1

DTWUUAFTYSMNQX-GFCCVEGCSA-N

InChI=1S/C22H22FN7O/c1-4-30-21-13(11-26-30)7-18-20(28-29(3)27-18)16-6-5-15(23)9-17(16)12(2)31-19-8-14(21)10-25-22(19)24/h5-6,8-12H,4,7H2,1-3H3,(H2,24,25)/t12-/m1/s1

(19R)-3-ethyl-16-fluoro-10,19-dimethyl-20-oxa-3,4,9,10,11,23-hexazapentacyclo[19.3.1.02,6.08,12.013,18]pentacosa-1(25),2(6),4,8,11,13(18),14,16,21,23-decaen-22-amine

NVL520; NVL 520; NVL-520; Zidesamtinib; NVL-520; 2739829-00-4; NVL520; zidesamtinib [INN]; MX5KQV5XHC; Zidesamtinib [WHO-DD]; ZIDESAMTINIB [USAN];

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 (~119.20 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.98 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 12.5 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: ≥ 1.25 mg/mL (2.98 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 12.5 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: ≥ 1.25 mg/mL (2.98 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 12.5 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.)