FGFR; VHL E3 ligase

DGY-09-192 is a bivalent degrader that couples the pan-FGFR inhibitor BGJ398 to a CRL2VHL E3 ligase recruiting ligand, which preferentially induces FGFR1&2 degradation while largely sparing FGFR3&4. DGY-09-192 exhibited two-digit nanomolar DC50 s for both wildtype FGFR2 and several FGFR2-fusions, resulting in degradation-dependent antiproliferative activity in representative gastric cancer and cholangiocarcinoma cells.

Importantly, DGY-09-192 induced degradation of a clinically relevant FGFR2 fusion protein in a xenograft model.
Researchers conducted a pharmacokinetics (PK) study in mice following a single dose of DGY-09-192 via intravenous (IV, 1 mg/kg), intraperitoneal (IP, 3 mg/kg) injection or oral administration (10 mg/kg). DGY-09-192 exhibited a fairly long half-life (T1/2 of 5h) with low clearance via IV and IP administration, but negligible oral bioavailability (Table 4). We then chose to use a CCLP1-FGFR2-PHGDH xenograft model to assess in vivo degradation. Once tumors reached ~200 mm3, mice were administered DGY-09-192 (20 or 40 mg/kg, IP QD) for 6d. Immunoblot analysis of tumor samples isolated 4h after the last dose revealed reduction in both FGFR2-PHGDH protein levels and phosphorylation of downstream markers FRS2 and ERK1/2 in a dose-dependent manner[1].

TMT-based quantitative LCMS proteomics [1]
Kelly cells were treated with DMSO (biological triplicate) or DGY-09-192 at 1 µM for 5 hours and cells were harvested by centrifugation at 4 ºC. Cell lysis was performed by the addition of Urea buffer (8 M Urea, 50 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (EPPS) pH 8.5, Protease and Phosphatase inhibitors) followed by manual homogenization by 20 passes through a 21-gauge (1.25 in. long) needle. Lysate was clarified by centrifugation and protein quantified using bradford (Bio-Rad) assay. 200 µg of protein for each sample was reduced, alkylated and precipitated using methanol/chloroform as previously described. The resulting precipitated protein was resuspended in 4 M Urea, 50 mM HEPES pH 7.4, buffer for solubilization, followed by dilution to 1 M urea with the addition of 200 mM EPPS, pH 8. Proteins were digested for 12 hours at room temperature with LysC (1:50 ratio), followed by dilution to 0.5 M urea and a second digestion step was performed by addition of trypsin (1:50 ratio) for 6 hours at 37 ºC. Anhydrous ACN was added to each peptide sample to a final concentration of 30%, followed by addition of Tandem mass tag (TMT) reagents at a labelling ratio of 1:4 peptide:TMT label. TMT labelling occurred over a 1.5 hour incubation at room temperature followed by quenching with the addition of hydroxylamine to a final concentration of 0.3%. Each of the samples were combined using adjusted volumes and dried down in a speed vacuum followed by desalting with C18 SPE. The sample was offline fractionated into 96 fractions by high pH reverse-phase HPLC through an aeris peptide xb-c18 column with mobile phase A containing 5% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 5 mM NH4HCO3 in LC-MS grade H2O (both pH 8.0). The resulting 96 fractions were recombined in a non-contiguous manner into 24 fractions and desalted using C18 solid phase extraction plates, followed by subsequent mass spectrometry analysis. SUPPORTING INFORMATION 17 Data were collected using an Orbitrap Eclipse Tribrid mass spectrometer coupled with an UltiMate 3000 RSLCnano System. Peptides were separated on an EasySpray ES803a.rev2 75 μm inner diameter microcapillary column. Peptides were separated over a 190 min gradient of 6 - 27% acetonitrile in 1.0% formic acid with a flow rate of 300 nL/min. Quantification was performed using a MS3-based TMT method as described previously (McAlister et al., 2014). The data were acquired using a mass range of m/z 340 – 1350, resolution 120,000, AGC target 5 x 105, maximum injection time 100 ms, dynamic exclusion of 120 seconds for the peptide measurements in the Orbitrap. Data dependent MS2 spectra were acquired in the ion trap with a normalized collision energy (NCE) set at 35%, AGC target set to 1.8 104 and a maximum injection time of 120 ms. MS3 scans were acquired in the Orbitrap with HCD collision energy set to 55%, AGC target set to 2 105, maximum injection time of 150 ms, resolution at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10

Antiproliferation Assay [1]
SUPPORTING INFORMATION 16 Cells were plated in a density of 3000 cells in 96-well plate. 24 hours later, inhibitor drugs were added to the plate and cells were cultured for another 3 days. Cell viability was then assessed by MTT assay. Each dose point was normalized to DMSO controls to estimate relative viability. Dose-response curves were generated using non-linear regression curve fit in GraphPad Prism 9.0.0. Data are presented as mean ± SEM (n = 3, n represents biological replicates used for each treatment condition), as also indicated in the figure legends.

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Cellular VHL engagement assay [1]
Stable cells expressing the BRD4BD2-eGFP protein fusion and the mCherry reporter were seeded at a density of 1000-4000 cells/well in a 384-well plate. BRD4BD2-GFP reporter cells were treated with increasing concentration of lenalidomide or indicated compounds for 5 hrs in the presence of 0.25uM AT1. Relative abundance of BRD4BD2-GFP was measured by Laser Scanning Cytometry. Green fluorescent signal (Excitation laser: 488 nm; Filter: 500-530 nm) and Red florescent signal (Excitation laser: 561 nm; Filter: 575-640 nm) individually has been tested and exported for analysis. Data analysis can be performed using Cell Profiler and The values for the concentrations resulting in 50% degradation (DC50) were calculated using the nonlinear fit variable slope model. Data are represented as means ± s.d of three replicates (n = 3).

Pharmacokinetcs study We performed PK study in DMPK core facility at Scripps Florida (https://www.scripps.edu/science-and-medicine/cores-and-services/dmpk-core/index.html). Pharmacokinetics were tested in male C57Bl/6 mice via intravenous dosing in the tail vein, intraperitoneal injection and oral gavage. Triplicate mice were dosed at 1mg/Kg IV, 3mg/Kg IP, and 10mg/Kg PO formulated at 0.1mg/mL (IV), 0.3mg/ml (IP) and 1mg/mL (PO) in 5/95 v:v DMSO/10% w:v captisol. Approximately 20 µl blood is collected after 5, 15, 30, 60, 120, 240, 360, 480 and 1440 minutes. We use a micro-sampling technique where blood is collected in capillary hematocrit tubes to reduce the total blood taken from the mouse. This allows a single mouse to be used for the entire time course and reduced inter-individual variability. Additionally, because of the small sample volumes the health of the mouse is maintained. Plasma is generated by standard centrifugation techniques resulting in approximately 10 µl of plasma which is immediately frozen. Drug levels are determined by mass spectrometry using an ABSciex 5500 mass spectrometer using multiple reaction monitoring. Pharmacokinetic parameters were calculated using a non-compartmental model (Phoenix WinNonlin, Pharsight Inc.). All procedures are approved by the Scripps Florida IACUC and the Scripps vivarium is fully AAALAC accredited.

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Pharmacodynamics study Mice were housed in a specific pathogen-free environment. All experiments were conducted under protocol 2019N000116 approved by the Subcommittee on Research Animal Care at MGH. 6- to 8-week-old male NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, 00557, The Jackson Laboratory) were used in the experiments. CCLP-1 cells engineered with FGFR2-PHGDH fusion were subcutaneously injected into NSG mice (1x10^6 cells per mouse). Tumors were monitored and measured with a digital caliper. When tumor size was ~200 mm3, mice were randomized and treated with either vehicle (5% DMSO, 10% solutol in D5W), DGY09-192 20mg/kg or DGY-09-192 40mg/kg by intraperitoneal injection daily for continuous 6 days. Tumor samples were harvested at the last dose post 4 hours treatment and proceeded with signaling studies.

DGY-09-192 exhibits a fairly long half-life (T1/2 is 5 hours), low clearance rates via intravenous and intraperitoneal injection, but negligible oral bioavailability.

[1].Discovery of a Potent Degrader for Fibroblast Growth Factor Receptor 1/2. Angew Chem Int Ed Engl. 2021 Jul 12;60(29):15905-15911.

Aberrant activation of the FGFR signaling pathway occurs in various cancers, and ATP-competitive FGFR inhibitors have been approved by regulatory agencies. Although these inhibitors have demonstrated clinical efficacy, their lack of selectivity for FGFR family members leads to dose-limiting toxicity and poor tolerability. This article reports the discovery and characterization of DGY-09-192, a bivalent degrader that conjugates the pan-FGFR inhibitor BGJ398 to a CRL2VHL E3 ligase recruitment ligand, preferentially inducing the degradation of FGFR1 and 2, while having less effect on FGFR3 and 4. DGY-09-192 exhibits double-digit nanomolar DC50 values against wild-type FGFR2 and various FGFR2 fusion proteins, thus demonstrating degradation-dependent antiproliferative activity in representative gastric and cholangiocarcinoma cells. Importantly, DGY-09-192 induced the degradation of clinically significant FGFR2 fusion proteins in a xenograft model. In summary, we demonstrate that DGY-09-192 has the potential to be a prototype FGFR degrader. [1]

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Mutant FGFRs are effective targets for a variety of cancer types, but existing drugs not only lack selectivity for closely related FGFR1, 2 and 3, but also cannot distinguish between wild-type and mutant FGFRs. Therefore, this study aims to investigate whether FGFRs and their fusion variants are degradable targets; and whether degraders can achieve selectivity for specific FGFR subtypes. Our initial imide-based degrader was able to degrade TEL-FGFR2 expressed in mouse Ba/F3 cells, but failed to degrade the full-length FGFR2 protein in KATO III cells. This limitation was overcome by introducing a VHL E3 ligase, and DGY-09-192, a compound with low nanomolar degradative activity against both wild-type and fusion mutant FGFR2 proteins, was finally discovered. Furthermore, although DGY-09-192 maintained equivalent biochemical inhibitory activity against all four FGFR isoforms, it exhibited highly selective degradation of FGFR1 and FGFR2, and degradation of both full-length FGFR2 and FGFR2 fusion proteins produced significant degradation-dependent antiproliferative activity in various FGFR2-dependent cell lines. Additionally, our lead compound DGY-09-192 possesses a short two-carbon linker, which may enhance cell permeability and bioavailability. We confirmed that DGY-09-192 exhibits favorable pharmacokinetic characteristics and can induce the degradation of FGFR2 fusion proteins in vivo. However, as a prototype molecule, DGY-09-192 still has some limitations that need to be overcome. For example, DGY-09-192 is not superior to the FGFR parental inhibitor BGJ398 in terms of antiproliferative activity and is unlikely to overcome the resistance point mutations on the FGFR protein induced by BGJ398 (Table S3). Furthermore, DGY-09-192 still effectively inhibits all FGFR subtypes, so its antiproliferative activity cannot be attributed solely to degradation. Further optimization is needed to reduce its binding to FGFRs, improve oral bioavailability, enhance selectivity for specific FGFRs, reduce off-target binding to PDE6D, and improve its efficacy against drug-resistant mutants. In addition, we envision that further optimization could lead to the development of degradative agents that are selective for oncogenic mutants or fusions relative to wild-type FGFRs, thereby improving the therapeutic index. [1]

C49H59CL2N11O7S

1017.0332672596

1015.369

2504949-52-2

164889465

Typically exists as solid at room temperature

6.8

5

14

16

70

1720

4

CC1=C(SC=N1)C2=CC=C(C=C2)[C@H](C)NC(=O)[C@@H]3C[C@H](CN3C(=O)[C@H](C(C)(C)C)NC(=O)CN4CCN(CC4)C5=CC=C(C=C5)NC6=CC(=NC=N6)N(C)C(=O)NC7=C(C(=CC(=C7Cl)OC)OC)Cl)O

WDBUEMWHXKHQNB-AURZXHAXSA-N

InChI=1S/C49H59Cl2N11O7S/c1-28(30-9-11-31(12-10-30)44-29(2)54-27-70-44)55-46(65)35-21-34(63)24-62(35)47(66)45(49(3,4)5)57-40(64)25-60-17-19-61(20-18-60)33-15-13-32(14-16-33)56-38-23-39(53-26-52-38)59(6)48(67)58-43-41(50)36(68-7)22-37(69-8)42(43)51/h9-16,22-23,26-28,34-35,45,63H,17-21,24-25H2,1-8H3,(H,55,65)(H,57,64)(H,58,67)(H,52,53,56)/t28-,34+,35-,45+/m0/s1

(2S,4R)-1-[(2S)-2-[[2-[4-[4-[[6-[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl-methylamino]pyrimidin-4-yl]amino]phenyl]piperazin-1-yl]acetyl]amino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

DGY-09-192; 2504949-52-2; (2S,4R)-1-((S)-2-(2-(4-(4-((6-(3-(2,6-Dichloro-3,5-dimethoxyphenyl)-1-methylureido)pyrimidin-4-yl)amino)phenyl)piperazin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide; GLXC-26164; EX-A6660; (2S,4R)-1-[(2S)-2-[[2-[4-[4-[[6-[(2,6-dichloro-3,5-dimethoxyphenyl)carbamoyl-methylamino]pyrimidin-4-yl]amino]phenyl]piperazin-1-yl]acetyl]amino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)