Mitochondrial protease OMA1

BTM compounds (e.g. BTM-3566 and BMT-3528) induce BAX-dependent DLBCL cell death in vitro.[1]
BTM-3566 and BMT-3528 induce activation of the ATF4-linked ISR.[1]
BTM-3566 induces OMA1-dependent mitochondrial fragmentation.[1]
In agreement with these findings, deletion of either OMA1 or DELE1 protected BJAB cells from BTM-3566 and BTM-3528 induced apoptosis, whereas OPA1ΔS1/ΔS1 BJAB cells remained fully sensitive.[1]
Following treatment with BTM-3528 or BTM-3566 compounds, neither phosphorylation of eIF2α nor increased ATF4 protein was observed in HRI−/− and eIF2αS49A/S52A cells.[1]
The mitochondrial protein FAM210B suppresses BTM-3528 and BTM-3566 activity.[1]
FAM210B-tGFP expression completely suppressed the activity of BTM-3528 and BTM-3566 but had no effect on bortezomib or FCCP-induced cell death.[1]
As expected, L-OPA1 cleavage in WT HCT-116 cells was observed in the presence of BTM-3528 or BTM-3566 but not BTM-3532.[1]

Once-daily oral dosing of BTM-3566 resulted in complete regression of xenografted human DLBCL SU-DHL-10 cells and complete regression in 6 of 9 DLBCL patient-derived xenografts. BTM-3566 represents a first-of-its kind approach of selectively hyperactivating the mitochondrial ISR for treating DLBCL.[1]

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BTM-3566 has favorable pharmacokinetic properties and potent in vivo activity in human cell line and patient-derived xenograft models. To investigate the pharmacokinetic properties of BTM-3566, we performed intravenous/oral crossover studies in mice (Fig 2A–C). Bioavailability was >90% and the terminal half-life of 4.4 to 6.6 hours was acceptable for oral, once daily dosing.[1]

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The therapeutic activity of BTM-3566 was assessed in a human xenograft model using the double-hit lymphoma DLBCL tumor line SU-DHL-10 (Fig 2D). At the 10 mg/kg dose, researchers observed delayed tumor growth that was lost with further dosing (Fig. 2E, top). At doses at or above 20 mg/kg, BTM-3566 treatment resulted in complete responses (CR; defined as no palpable tumor) in all animals by 10 days of dosing and maintained for 21 days of dosing. To assess whether BTM treatment would induce durable responses, animals were followed for 30 additional days after cessation of dosing. Thirty-day tumor-free survival was maintained in 40% of animals dosed with 20 mg/kg and 60% of animals dosed with 30 mg/kg BTM-3566 (Fig. 2E top). Body weight loss was dose-dependent but <10% at the 20 mpk dose level (Fig. 2E bottom). In the 30 mg/kg dose group, two of 10 mice exceeded 20% body weight loss, necessitating an unscheduled dose holiday. Weight loss in both groups was reversible with cessation of dosing. (Fig. 2E bottom).[1]
Having established the 20 mg/kg dose as effective and well tolerated in the SU-DHL10 model, researchers next tested BTM-3566 in 9 human DLBCL patient-derived xenograft (PDX) models representing ABC and GCB DLBCL-subtypes with high-risk genotypes (Fig. 2F-​-H).H). Treatment with 20 mg/kg BTM-3566 resulted in CR in all 3 mice from 6 of 9 PDX models. When pooling the 27 mice in the treatment arms of the nine models, CR was observed in 66% (19/27), partial response (PR) occurred in another four mice, 2 mice had stable tumors, and two had progressive disease. In summary, the single-agent overall response rate (CR + PR) was 85.2% with all models having at least one animal exhibiting full or partial regression (Fig. 2H).

Caspase Analysis[1]
Caspase 3/7 activity was determined following treatment of cells with BTM-3528 or BTM-3566. using the Caspase-Glo® 3/7 assay. All cells were treated with compound for 24 hours then processed for Caspase activity as per the manufacturer’s instructions.[1]

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Image analysis[1]
Mitochondrial morphology was assessed using Fiji/ImageJ software and a Trainable Weka Segmentation plugin. Mitochondrial membrane potential, TMRE/MTG fluorescence ratio was calculated from segmented mitochondrial structures obtained by MTG channel. One-way ANOVA and Tukey multiple comparison test were used for statistical analysis; P values ≤0.05 (*) were considered significantly different.[1]

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Mitochondrial respirometry[1]
Respirometry assays were run on a Seahorse Extracellular Flux Analyzer. HCT-116 cells were seeded at 14,000 cells/well using XF96 well microplates and incubated overnight (37°C and 5% CO2) in McCoy's 5a Modified Medium culture medium with 10% FBS. Before the respirometry assay, cells were washed with assay medium: DMEM with 10 mmol/L glucose, 2 mmol/L glutamine, 1 mmol/L pyruvate, 5 mmol/L HEPES, and 10% FBS (pH 7.4). BTM compounds were tested at a final concentration of 3 μmol/L, and cells were either acutely treated during the assay or pretreated for 4 hours before the assay. In pretreatment experiments, compounds were added in complete medium and incubated at 37°C and 5% CO2. Compounds injected during the assay included 2 μmol/L oligomycin, 1 μmol/L FCCP, and 2 μmol/L of antimycin A and rotenone. Upon completion of each respirometry assay, the cells were stained with 1 μg/mL Hoechst and cell number was measured with an Operetta High-Content Imaging System. The respirometry well level data (pmol O2/min) was normalized to cell number per well (pmol O2/min/103 cells) in each assay.

Cell line compound testing[1]
Screening of tumor cell lines was performed by Crown Bioscience. Cells were plated at a starting density of 4 × 103 cells/well and incubated for 24 hours. BTM-3528 was prepared as a 10× solution of test article with a final working concentration of 30 μmol/L of test article in media with nine 3.16-fold serial dilutions. Following the addition of BTM-3528, the plates were incubated for an additional 96 hours at 37°C with 5% CO2. Final cell numbers were determined using the Cell-Titre Glo assay. The absolute IC50 curve was fitted using a nonlinear regression model with a sigmoidal dose response. Activity area (AUC) for each compound was determined by calculating the integrated area bounded for each dose response curve fit. The AUC reflects both the magnitude of effect (maximal inhibition) and potency (IC50).[1]

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Annexin V apoptosis assay[1]
To quantify apoptosis, BJAB were cultivated using RPMI with 15% fetal bovine serum in a 96-well format and treated with BTM compounds for the indicated time intervals. Cells were washed twice with ice-cold PBS and resuspended in 1× Binding Buffer at a concentration of 1 × 106 cells/ml. To 0.5 × 105 cells, 2.5 μL of Annexin V-APC or Annexin V-FITC were added and incubated for 15 minutes at room temperature in the dark. Cells were washed once with Binding Buffer and the pellets were resuspended in 100 μL Binding Buffer containing either 2 μL Propidium Iodide (50 μg/mL) or 2 μL DAPI (1 mg/mL). Cells were analyzed on a Cytoflex S.[1]

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Transcriptomic profiling[1]
The human colon adenocarcinoma cell line HCT-116 was used to evaluate the effects of BTM compounds on gene expression. To fully evaluate the effects of the compound on cell-cycle controlled genes, cells were synchronized prior to BTM compound treatment. HCT-116 cells grown in McCoys 5a Media supplemented with 10% fetal bovine serum and penicillin/streptomycin were first blocked in the S-phase by treatment with thymidine. After 24 hours, the thymidine containing media was removed and replaced with media containing nocodazole to block cells in the M-phase in a high degree of synchrony. Cells were then released into G1 either in complete medium or complete medium plus 10, 1, or 0.1 μmol/L BTM-3528. Cells were harvested at five time points: 1, 2, 4, 6, and 8 hours after release into the G1 phase, and mRNA extracted for Illumina RNA sequencing (RNA-seq). Three replicates of each concentration and time point along with time point specific controls (i.e., cells without compound) were collected for RNA-seq.

In vivo efficacy of BTM-3566[1]
Human cell line xenograft models were established using SU-DHL-10 cells. Cells were grown in RPMI1640 supplemented with 15% fetal bovine serum and penicillin/streptomycin. Cells were harvested by centrifugation and resuspended in cold 50% serum-free medium: 50% Matrigel to generate a final concentration of 2.50E+07 trypan-excluding cells/mL. Female Envigo SCID beige mice (C.B-17/IcrHsd Prkdcscidlystbg-j) were implanted subcutaneously high in the right axilla on day 0 with 5 × 106 cells/mouse. Mice were randomized into groups based on tumor volume with a mean tumor burden for each group of 150 mm3. BTM-3566 was prepared as a solution in dosing vehicle containing 5% NMP, 15% PEG400, 10% Solutol, and 70% D5W. The final dose concentration was 4 mg/mL, and the dose volume was 5 μL/gram. All mice were dosed by oral gavage once daily for 21 days. Tumor volume and body weights were determined every third day. All mice were dosed according to individual body weight on the day of treatment.[1]

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For patient derived xenograft models, all tumors were sourced from Crown Bio. Models were established in female mice with an average body weight of 25 grams. Balb/c nude,NOD SCID mice, or NPG/NOD/SCID were used. Each mouse was inoculated subcutaneously in the right flank region with fresh tumor derived from mice bearing established primary human cancer tissue. Mice were randomized into vehicle or treatment groups with a mean tumor burden of 200 mm3. All mice were dosed once daily by oral gavage for 21 days. Tumor volume and body weights were determined three times per week. [1]

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Pharmacokinetic Analysis of BTM-3566 in mouse blood[1]
Blood was collected from a tail vein snip into K2-EDTA tubes. Plasma was isolated and a 20 mL sample was protein precipitated with 200 L of acetonitrile containing 100 ng/mL diclofenac, tolbutamide and labetalol as internal standards. The mixture was vortex-mixed and centrifuged at 13000 rpm for 15 min, 4 ℃. An 80 mL aliquot of the supernatant was transferred to a sample plate and mixed with 80 μL water, then the plate was shaken at 800 rpm for 10 min. A 1 L aliquot was injected on to a Waters ACQUITY UPLC BEH C18 2.1*50mm, 1.7µm reverse phase column using a two-component mobile phase gradient. Mobile phase A was 0.1% trifluoracetic acid in water and mobile phase B 0.1% TFA in acetonitrile. Plasma BTM-3566 was detected using electrospray ionization and multiple reaction monitoring mass spectrometry (BTM-3528 [M+H]+ m/z 522.10>203.1. All data were analyzed using single compartment analysis in WinNonLin.[1]

To investigate the pharmacokinetic properties of BTM-3566, researchers performed intravenous/oral crossover studies in mice. Bioavailability was >90% and the terminal half-life of 4.4 to 6.6 hours was acceptable for oral, once daily dosing.[1]

[1]. Targeting aggressive B-cell lymphomas through pharmacological activation of the mitochondrial protease OMA1. Mol Cancer Ther. 2023 Aug 30.

[2]. BTM-3566, a Novel Activator of the Mitochondrial Stress Response Promotes Robust Therapeutic Responses in Vitro and In Vivo in Diffuse Large B-Cell Lymphoma. Blood. Volume 138, Supplement 1, 23 November 2021, Page 684.

[3]. Pharmacokinetics and pharmacodynamics of the novel OMA1 activator BTM 3566 in a mouse model of diffuse large B-cell lymphoma. Cancer Research, 2023, 83(7_Supplement): 2803-2803.

DLBCL are aggressive, rapidly proliferating tumors that critically depend on the ATF4-mediated integrated stress response (ISR) to adapt to stress caused by uncontrolled growth, such as hypoxia, amino acid deprivation, and accumulation of misfolded proteins. Here, we show that ISR hyperactivation is a targetable liability in DLBCL. We describe a novel class of compounds represented by BTM-3528 and BTM-3566, which activate the ISR through the mitochondrial protease OMA1. Treatment of tumor cells with compound leads to OMA1-dependent cleavage of DELE1 and OPA1, mitochondrial fragmentation, activation of the eIF2α-kinase HRI, cell growth arrest, and apoptosis. Activation of OMA1 by BTM-3528 and BTM-3566 is mechanistically distinct from inhibitors of mitochondrial electron transport, as the compounds induce OMA1 activity in the absence of acute changes in respiration. We further identify the mitochondrial protein FAM210B as a negative regulator of BTM-3528 and BTM-3566 activity. Overexpression of FAM210B prevents both OMA1 activation and apoptosis. Notably, FAM210B expression is nearly absent in healthy germinal center B-lymphocytes and in derived B-cell malignancies, revealing a fundamental molecular vulnerability which is targeted by BTM compounds. Both compounds induce rapid apoptosis across diverse DLBCL lines derived from activated B-cell, germinal center B-cell, and MYC-rearranged lymphomas. Once-daily oral dosing of BTM-3566 resulted in complete regression of xenografted human DLBCL SU-DHL-10 cells and complete regression in 6 of 9 DLBCL patient-derived xenografts. BTM-3566 represents a first-of-its kind approach of selectively hyperactivating the mitochondrial ISR for treating DLBCL.[1]

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Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge, especially in patients not suitable for high dose chemotherapy, stem cell transplantation or patients who fail CAR-T-cell therapy. r/r-DLBCLs are highly heterogeneous both clinically and molecularly, which imposes a pressing need to develop novel therapies to improve outcomes in patients independently of the molecular subtype. We describe here BTM-3566, a first-in-class compound with activity against a variety of B-cell malignancies but with greatest effect in DLBCL. BTM-3566 activates the mitochondrial integrated stress response (ISR) through a novel mechanism regulated by the mitochondrial protein FAM210B. BTM-3566 induces apoptosis in DLBCL lines in vitro and complete tumor regression in vivo in DLBCL PDX mouse models harboring genetic alterations associated with poor prognosis.[2]

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BTM-3566 is an oral small molecule based on a pyrazolothiazol-backbone, developed for treatment of diffuse large B-cell lymphoma (DLBCL). BTM-3566 induces apoptosis and complete cell killing in DLBCL lines a with an IC 50 of ~200 - 500 nM. Responsive DLBCL cell lines include ABC, GCB, and double-hit and triple-hit lymphoma lines. Pharmacokinetic studies in mice showed suitability for once daily dosing, with > 50% of oral bioavailability and close to 6 hours of serum half-life. 14-day dosing in mice and dogs demonstrated excellent tolerability at therapeutic doses. BTM-3566 showed stability in human hepatocytes (IC < 5 ml/min*kg) as well and a favorable in vitro safety profile. In xenograft models using the double-hit DLBCL line SU-DHL-10, BTM-3566 treatment resulted in complete regression in all tumor-bearing animals. Most importantly, no subsequent tumor growth occurred for 2 weeks after cessation of therapy, indicating that treatment with BTM-3566 resulted in a durable complete remission in this model of double-hit DLBCL. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations, including Myd88 mutations and MYC and BCL2 rearrangements, demonstrated response in 100% of the lines with complete tumor regression in 6 of 8 PDX models tested (Table 1).[2]

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Transcriptome and proteome analyses revealed that BTM-3566 strongly activated the ATF4-integrated stress response (ISR), indicated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and subsequent upregulation of the transcription factor ATF4. Of the four eIF2a-kinases in the human genome we determined, using CRISPR-Cas 9 gene depletion, that HRI was uniquely required for BTM-3566 eIF2a phosphorylation, induction of ATF4 ISR and apoptosis. HRI is described as being activated by mitochondrial-related stress, including heme depletion, increased ROS generation or blockage of mitochondrial ATP synthesis which result in an increase in mitochondrial proteostasis including activation of mitochondrial protease OMA1. We determined that BTM-3566 activates OMA1 without acting as a classical mitochondrial toxin. Treatment with BTM-3566 induced OMA1-dependent OPA1 processing and mitochondrial fragmentation in as little as 30 minutes of treatment, in the absence of any reduction in mitochondrial oxygen consumption or membrane depolarization. This data indicates that BTM-3566 represents a new class of compounds that activate the mitochondrial protease OMA1.[2]

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Gene expression-based profiling of BTM-3566 sensitivity in over 400 cancer cell lines showed that FAM210B, a mitochondrial membrane protein, negatively correlated with response to BTM-3566. Notably, overexpression of FAM210B completely prevents OMA1 activation and causes complete resistance to BTM-3566-induced apoptosis in DLBCL cell line BJAB and the Burkitt lymphoma cell line Ramos. Thus, FAM210B serves as a strong predictor of BTM-3566 sensitivity, as well as revealing a novel mechanism of regulation of OMA1 activation.[2]

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In summary, we describe here a novel, highly potent activator of the mitochondrial ISR, which is well tolerated in mice and dogs, has favorable pharmacokinetics and induces robust DLBCL regression in-vivo. An IND application in B-cell malignancies will be completed by early Q1 2022 with initiation of first in human clinical trials the first half of 2022.[2]

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C24H23F4N3O2S2

525.581937074661

525.116

2228857-70-1

134564305

Light yellow to yellow solid powder

7.3

1

10

6

35

799

0

LDSVVHIOWFIJNE-UHFFFAOYSA-N

InChI=1S/C24H23F4N3O2S2/c1-12(2)34-22-19(14-7-9-16(10-8-14)24(26,27)28)29-23(35-22)31-20(21(32)33)18(13(3)30-31)15-5-4-6-17(25)11-15/h4-7,11-12,16H,8-10H2,1-3H3,(H,32,33)

4-(3-fluorophenyl)-5-methyl-2-[5-propan-2-ylsulfanyl-4-[4-(trifluoromethyl)cyclohexen-1-yl]-1,3-thiazol-2-yl]pyrazole-3-carboxylic acid

BTM-3566; 2228857-70-1; BTM3566; SCHEMBL20214005; LDSVVHIOWFIJNE-UHFFFAOYSA-N;

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.)