Her3 (ErbB3) (IC50 = 23 nM)

TX1-85-1 has an anti-proliferative EC50 of 9.9, 11.5, and 16.9 μM on Ovcar8, HCC827 GR6, and PC9 GR4 cells, respectively[1].
After the iterative synthesis and assaying of approximately 100 pyrazolopyrimidine acrylamides, we developed the ‘lead’ compound TX1-85-1 (4) which possessed an IC50 of 23 nM in the binding assay (Fig. 1a, b). TX1-85-1 can be viewed as a molecular amalgam of the hinge binding moiety of KIN001-051 and the solubility enhancing tail of KIN001-111, combined with an acrylamide arm to reach Cys721 (Fig. 1c). As expected for a covalent inhibitor, the apparent IC50 decreases upon longer incubation with the protein and reaches a plateau at about 3 hours (Supplementary Fig. 2). Mass spectrometry was used to confirm the covalent addition of TX1-85-1 to recombinant Her3 kinase domain protein and subsequent proteolysis of Her3 with trypsin and MS2 analysis revealed unique modification of Cys721 (Supplementary Fig. 3). We next investigated whether TX1-85-1 could form a covalent bond with Her3 in cells using a cellular competition binding assay. To enable these experiments we synthesized a biotinylated derivative (TX1-85-1-biotin) (5) that still preserves the ability to covalently bind to Her3 (Fig. 1a, Supplementary Fig. 4, IC50 = 50.7 nM). Cells were incubated with TX1-85-1 and then lysates were labeled with TX1-85-1-biotin in order to quantify the amount of Her3 that had escaped labeling by the initial incubation with TX1-85-1. We determined that incubation of PC9 GR4 cells (EGFR E746_A750/T790M) with 5 μM of TX1-85-1 for 8 hours resulted in complete protection of Her3 from subsequent labeling with TX1-85-1-biotin (Fig. 1d). This result suggests that TX1-85-1 (MW = 580Da) can pass through the cell membrane and is capable of complete ‘target engagement’ with Her3 intracellularly. To examine the specificity with which TX1-85-1 modifies Her3 we performed a live cell chemical proteomics experiment using the KiNativ® approach which demonstrated potent binding to Her3 as well as to Lyn, Her2 and several other Src family kinases (Supplementary Table. 1). Binding to Lyn, Her2 and several other Src family kinases was expected as these kinases were equipotently bound by the non-acrylamide screening hit compound KIN001-111, suggesting that interaction with these targets is non-covalent. Collectively these results suggest that TX1-85-1 is capable of covalently modifying Cys721 of Her3 in vitro and in cells.[1]

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TX1-85-1 does not stop Her3 dependent signaling or growth [1]
We next evaluated the ability of TX1-85-1 to inhibit Her3-dependent signaling and growth. We utilized two established lung cancer cell lines, PC9 GR4 (EGFR E746_A750/T790M), HCC827 GR6 (EGFR E746_A750/MET amplification)18,37 and an ovarian cancer cell line, Ovcar8 that we reconfirmed to be ‘addicted’ to Her3 using siRNA mediated depletion of Her3 (Supplementary Fig. 5). TX1-85-1 possessed an anti-proliferation EC50 of greater than or equal to approximately 10 μM for all three cell lines (Fig. 2a). At a concentration of TX1-85-1 sufficient to fully label Her3 in cells (5 μM) there was no growth inhibition of PC9 GR4 cells and no inhibition of the phosphorylation of Akt, an important downstream effector of Her3 (Fig. 2b). These results suggest that despite successful target engagement of Her3 in cells by TX1-85-1, the compound is not capable of inhibiting Her3-dependent function under the conditions investigated.

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TX1-85-1-Adamantane conjugates induce Her3 degradation [1]
Recent reports have suggested that covalent modification of proteins with hydrophobic small molecules can result in their proteasome mediated degradation25,26,38,39. While a detailed mechanistic understanding for this phenomenon is unknown, there is considerable evidence that cells have evolved sophisticated protein homeostasis machinery that can eliminate unfolded and otherwise damaged proteins which are presumably engaged by this hydrophobic tagging strategy. Among the reported hydrophobic tags, we selected the adamantane group due to its ability to densely introduce hydrophobicity with a minimal increase in molecular weight. A series of compounds possessing various linkers connecting TX1-85-1 with an adamantane moiety were synthesized and confirmed to bind potently with Her3, identifying a representative molecule (TX2-121-1) (6) demonstrating an IC50 of 49.2 nM in the protein based binding assay. To confirm that the selectivity of TX2-121-1 remained similar to that exhibited by TX1-85-1 we performed KiNativ® profiling. The selectivity of TX2-121-1 was very similar to TX1-85-1 and displayed most potent binding to Her3 (90.3%). TX2-121-1 off-target labeling list included Her2, EGFR, several Src family kinases such as Src, Yes and Lyn which, as expected, is the same as the off-target list of TX1-85-1. (Supplementary Table 1). To investigate whether TX1-85-1 and TX2-121-1 could directly inhibit EGFR or Her2 we performed enzymatic assays (Z′-LYTE, Invitrogen, Supplementary Table 2) and cell proliferation assays using the Ba/F3 EGFR VIII cell line (Supplementary Fig. 6). TX2-121-1 does not directly inhibit the enzymatic activity of EGFR or Her2 nor inhibit EGFR-dependent proliferation at concentrations below 10 μM.

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To confirm that the observed pharmacology is Her3-dependent we engineered PC9 GR4 cells to possess either native Her3 containing the reactive Cys721 or Her3 containing a non-reactive serine mutation, C721S. The mutation was confirmed by Sanger DNA sequencing of ErbB3 (Supplementary Fig. 7). We verified target engagement using a TX1-85-1-biotin conjugate compound to perform pull-down assays. Indeed, TX1-85-1-biotin effectively pulled down Her3 WT, but not Her3 C721S suggesting that the acrylamide motif of TX1-85-1-biotin forms a specific covalent interaction with C721 but does not form a covalent bond with C721S Her3 (Supplementary Fig. 8).

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We performed cell proliferation assays against PC9 GR4 cells expressing either wild-type or C721S mutant Her3 with TX1-85-1 and TX2-121-1 and their corresponding propylamide-controls (TX1-85-3 (9) and TX2-135-2) in order to investigate the importance of the electrophilic acrylamide and the adamantane moiety. These experiments demonstrate a 6-fold rightward shift in EC50 for C721S Her3 (EC50= 5.5 μM for PC9 GR4 Her3 C721S) versus wild-type Her 3 expressing cells (EC50 =0.9 μM for PC9 GR4 WT Her3) only for TX2-121-1 which possesses both an electrophilic warhead and an adamantane moiety (Fig. 4a, Supplementary Fig. 9) and the downstream signaling western blot is consistent with the antiproliferation result (Fig. 4b). All other compounds: TX1-85-1, TX1-85-3 and TX2-135-2 have similar EC50 on both PC9 GR4 Her3 WT and PC9 GR4 Her3 C721S. These results demonstrate that in this cellular system, effective inhibition of Her3-dependent growth requires both covalent modification and the presence of the adamantane moiety. Incidentally, we also observe of the two reversible controls that TX2-135-2 bearing the adamantane group is more antiproliferative than TX1-85-3 [1].

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Inhibition of ErbB2 or ErbB3 suppressed the conversion of miPSCs into CSCs [2]
To assess the role of the ErbB2/ErbB3 signal in the conversion process, the hypothesis raised in the previous section, lapatinib as a tyrosine kinase inhibitor for ErbB2 and TX1-85-1 as ErbB3 inhibitor were used to interrupt tyrosine kinases. The MTT assay showed that the IC50 of lapatinib on miPSCs was approximately 12 µM while IC50 of TX1-85-1 was approximately 25 µM (Fig. 3A). The miPSCs are routinely cultured with leukemia inhibitory factor (LIF) to maintain stemness and miPSCs will start differentiation and stop growth namely fail to survive without LIF. The miPSCs cultured without LIF lost GFP expression and did not survive through passages. In contrast, in the presence of the CM of PK8 cells, miPSCs maintained GFP expression and kept proliferation without LIF. The CM with lapatinib in 1 µM or 5 µM appeared to maintain GFP while enhancement of differentiation in adhesive cells with 5 µM was observed (Fig. 3B). A portion of cells cultured with CM and 10 µM TX1-85-1 maintained GFP and differentiated cells were shown as GFP negative cells in the adhesive cell culture condition. The cells lost GFP expression completely and differentiated with 20 µM TX1-85-1 and could not survive beyond one week of the treatment (Fig. 3C).

Our group has demonstrated that iPSCs converted into CSCs maintain self-renewal and colony-forming abilities when culture in CM of cancer cells. The effect of the lapatinib and TX1-85-1 on the formation of colonies and spheroids was assessed during the conversion. The miPSCs cultured in the presence of 1 and 5 µM lapatinib showed a decrease in the number of colonies (Fig. 4A). While the decrease with 1 µM was insignificant, 5 µM exhibited a significant decrease in the number of colonies from 250 down to 17 (Fig. 4B). Cells cultured with TX1-85-1 showed a decrease in colony numbers in a dose-dependent manner. The average colony number were 217, 163 and 20 for 1, 5 and 10 µM TX1-85-1, respectively (Fig. 4A,C). One and 5 µM of lapatinib, 5 and 10 µM TX1-85-1 also inhibited the spheroid formation ability of the miPSCs cultured under low attachment culture conditions in serum-free media while the miPSCs cultured with CM maintained spheroid formation ability (Fig. 4C).

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Inhibition of ErbB2 or ErbB3 enhanced the activation of Erk and attenuated stemness during conversion [2]
To investigate the role of the ErbB2 and ErbB3 signaling in the conversion process, the effects of lapatinib and TX1-85-1 were assessed on miPSCs for the acquisition of CSC characteristics during conversion. The western blot showed that miPSCs cultured in CM maintained the Oct3/4 level while CD24, as a CSC marker, slightly increased when compared with that in miPSCs cultured with LIF. On the other hand, the level of Oct3/4 and CD24 were maintained in miPSCs cultured in the presence of 1 µM lapatinib. The Oct3/4 and CD24 levels were dramatically decreased in the presence of 5 µM of lapatinib compared to the levels in cells cultured without lapatinib (Fig. 6Aa,B). The data also showed overactivation of Erk along with attenuation of stemness in the miPSCs cultured in the presence of 5 µM of lapatinib when compared with the level in miPSCs cultured without lapatinib. One µM lapatinib decreased Erk activation in miPSCs cultured in CM when compared to that without lapatinib (Fig. 6Aa,B). Inhibition of ErbB3 with small interfering RNA (siRNA) or 10 µM TX1-85-1 decreased Oct3/4 and Nanog levels and overactivated Erk. Targeting ErbB3 with siRNA also showed a decrease in CD24 level compared with negative siRNA control. (Fig. 6Ab,c,C,D).

Her3 labeled with TX1-85-1 [1]
Labeling with TX1-85-1 was accomplished by incubating purified Her3 protein at a concentration of 15 μM (0.5 mg/ml) with 150 μM TX1-85-1 in buffer containing 50 mM MOPS pH 7, 50 mM ammonium sulfate, 1 mM TCEP and 5% glycerol at room temperature for 2 hours.

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LanthaScreen® technology kinase binding assay [1]
Binding potency (IC50) was measured by the Life Technologies LanthaScreen® Eu kinase binding assay, which is based on the binding and displacement of an Alexa Fluor 647 labeled ATP-competitive kinase inhibitor scaffold. Binding of the tracer to the kinase is detected using a europium-labeled anti-tag antibody (anti-GST). Purified ErbB3 (665-1001) protein was obtained from Life Technologies at a concentration of 1.19 mg/ml in 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.02% Triton® X-100, 2 mM DTT, 50% Glycerol. The typical experimental procedure was to dilute ErbB3 protein to 30 nM with Kinase Buffer A (50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35) followed by mixing the ErbB3 protein 1:1 with 12 nM LanthaScreen® Eu-anti-GST antibody solution which was also diluted in Kinase Buffer A. Prior to use, the antibody tube was thawed and centrifuged at approximately 10,000 g for 5 minutes and the amount used for the assay was aspirated from the top of the solution. This centrifugation step will eliminate spurious data points that can arise on occasion due to any particulates in the product. Next, 5 μl of the mixture of ErbB3 protein and Eu-anti-GST was added to 5 μl of test compound solution per well in Corning 384 plates. Then, 5 μl of Kinase Tracer 178 was added at a concentration of 39 nM per well. The plates were then incubated for 3 hours at 4 °C and read with a Perkin Elmer EnVision®.

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Mass spectrometry analysis of intact Her3 [1]
TX1-85-1 treated Her3 (~1 μg) was loaded onto self-packed reverse-phase column (1/32 in. outer diameter x 500 μm inner diameter, with 5 cm of POROS 10R2 resin). After desalting, protein was eluted with an HPLC gradient (0%–100% B in 4 min, A = 0.2 M acetic acid in water, B = 0.2 M acetic acid in acetonitrile, flow rate = 10 μl/min) into a QSTAR Elite mass spectrometer scanning m/z 330–1500. Mass spectra were deconvoluted using MagTran1.03b2 software.

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Protease digestion and nano LC/MS analysis of peptide fragments [1]
TX1-85-1 treated Her3 (~1 μg) was diluted with ammonium bicarbonate buffer (pH 8.0), reduced with 10 mM DTT for 30 min at 56 °C, alkylated with 22.5 mM iodoacetamide for 30 min (room temperature in the dark), and digested overnight with trypsin at 37 °C. Digested peptides (~2 pmol) were injected onto a self-packed pre-column (4 cm POROS10R2) and eluted into the mass spectrometer using an HPLC gradient (0%~35% B in 20 min, A = 0.2 M acetic acid in water, B = 0.2 M acetic acid in acetonitrile, flow rate ~30 nl/min)52. The top 10 ions in each MS scan (image current detection; resolution=30K) were subjected to CAD (electron multiplier detection, relative collision energy 35%, q = 0.25). Dynamic exclusion was enabled with a repeat count of 1 and exclusion duration of 15 seconds.

MTT assay [2]
The miPSCs were seeded in 96-well plates at 5000 cells/well density with miPS media supplemented with 50% CM and incubated at 37 °C and 5% CO2. After 24 h of seeding, cells were incubated with a different concentration of lapatinib or TX1-85-1 and after 24 h of incubation, cell viability was evaluated by thiazolyl blue tetrazolium blue. MTT solution at 0.5 mg/mL concentration was added into wells and plates were incubated for 4 h at 37 °C in 5% CO2. After incubation, media were discarded, and 100 µL of DMSO was used to dissolve the formazan crystals and the absorbance was measured at 570 nm by the MTP-800 Lab microplate reader. The IC50 value was estimated from the survival curve.

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Colony and sphere formation assays [2]
For colony assay, one thousand miPSCs were cultured in 60 mm-dishes with miPS media with 1000U/ml LIF. After 24 h of seeding, media changed to miPS media without LIF supplemented with either 50% CM, 50% CM and 1 µM lapatinib, 50% CM and 5 µM lapatinib or with 50% CM and either 1,5 or 10 µM TX1-85-1. Cells were incubated at 37 °C with 5% CO2 for 7 days. After incubation, cells were fixed with 75% methanol and stained with Giemsa stain. The dishes were photographed and ImageJ software was used to count colonies with a diameter ≥ 50 µm. To assess cell’s ability to form spheroids, miPSCs cultured in miPS media supplemented with above conditions for one week were seeded at density of 1000 cells/well into 24-well ultra-low attachment plates with serum-free media consisting of DMEM, 2 mM L-glutamine, 0.1 mM β-ME, 0.1 mM NEAA, and ITS-X supplemented with penicillin/streptomycin with 0,1 or 5 µM lapatinib or 1, 5, 10 µM TX1-85-1. The cells were then incubated at 37 °C in 5% CO2 for 7 days and the formed spheres were photographed using the Olympus IX81 microscope equipped with a light fluorescence device.

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Western blotting [2]
The miPSCs cultured in miPS media with either 50% CM, 50% CM and 1 µM lapatinib, or 50% CM with 5 µM lapatinib or with 50% CM and 10 µM TX1-85-1 for one week and cells transfected with either negative control siRNA or ErbB3’s siRNA were lysed with a lysis buffer consisting of 50 mM Tris pH 8.0, 150 mM sodium chloride, 0.05% Triton X-100, and 0.5 mM ethylenediaminetetraacetic acid (EDTA). The protease and phosphatase inhibitors were also added to the lysis buffer. The lysed cells were further sonicated, centrifuged at 15000xg for 30 min at 4 °C and supernatants were collected and kept at − 80 °C. The total protein concentration was assessed by Pierce™ BCA Protein Assay Kit and 30 µg of total protein were loaded on SDS-PAGE, transferred to Immobilon-FL transfer membrane.

[1]. Pharmacological targeting of the pseudokinase Her3. Nat Chem Biol. 2014 Dec;10(12):1006-12.

[2]. The significance of ErbB2/3 in the conversion of induced pluripotent stem cells into cancer stem cells. Sci Rep. 2022 Feb 17;12(1):2711.

TX1-85-1 is an aromatic ether.

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Here we report the development of the first small molecule Her3 ATP-competitive binder TX1-85-1, which can covalently modify Her3 via conjugate addition to Cys721. Treatment of cells with TX1-85-1 at single digit micromolar concentrations results in covalent modification of Her3 with kinome selectivity consistent with the chemotype from which the compound was derived. Despite successful target engagement by TX1-85-1, proliferation and Her3-dependent functions including phosphorylation of downstream effector Akt are not affected in PC9 lung carcinoma cell lines. Nevertheless, despite being a very poor inhibitor of the growth of Her3-dependent cells lines such as Ovcar8 and HCC2935 (EC50>10 μM), TX1-85-1 is capable of inducing partial degradation of Her3 protein and attenuating Her3-dependent signaling. These results are consistent with studies that have shown that some non-covalent kinase inhibitors of Her2 (lapatinib) and b-Raf (vemurafenib) can also lead to partial degradation of their kinases targets. [1]

C32H36N8O3

580.680046081543

580.291

C, 66.19; H, 6.25; N, 19.30; O, 8.27

1603845-32-4

78243752

White to light yellow solid powder

3.3

2

8

7

43

962

0

C(NC1=CC(C2C3=C(N)N=CN=C3N(C3CCC(N4CCN(C(C)=O)CC4)CC3)N=2)=CC=C1OC1=CC=CC=C1)(=O)C=C

FYICDSWKKFSYOM-UHFFFAOYSA-N

InChI=1S/C32H36N8O3/c1-3-28(42)36-26-19-22(9-14-27(26)43-25-7-5-4-6-8-25)30-29-31(33)34-20-35-32(29)40(37-30)24-12-10-23(11-13-24)39-17-15-38(16-18-39)21(2)41/h3-9,14,19-20,23-24H,1,10-13,15-18H2,2H3,(H,36,42)(H2,33,34,35)

N-[5-[1-[4-(4-acetylpiperazin-1-yl)cyclohexyl]-4-aminopyrazolo[3,4-d]pyrimidin-3-yl]-2-phenoxyphenyl]prop-2-enamide

TX185-1 TX1 85-1 TX1-85-1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~3.85 mg/mL (~6.63 mM)

Solubility in Formulation 1: ≥ 0.38 mg/mL (0.65 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 3.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.38 mg/mL (0.65 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 3.8 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.)