Hsp90 (mitochondrial-localized)
TRAP-1 (mitochondrial Hsp90-related chaperone) [1]

The Hsp90 ATPase inhibitory module of 17-allylaminogeldanamycin (17-AAG) and the mitochondrial moiety of tri-aldehyde binding are combined in gamitrinib TPP (G-TPP), a little molecule. TPP effectively destroys mitochondria and has no effect on the induction of Hsp90 outside of the organelle. Within 16 hours of exposure, patient-derived and cultivated cytoblastoma cell lines were indiscriminately destroyed by gamatrinib TPP at doses of 15–20 μM. With loss of membrane potential within the organelle, cytochrome c release in the cytoplasm, activation of promoter caspase-9 and effectors caspase-3 and caspase-7, and annexin V. Reactivity, this cell death response is typical of mitochondria[1].

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At concentrations of 15–20 μM, Gamitrinib TPP hexafluorophosphate (G-TPP) induces mitochondrial apoptosis in glioblastoma cell lines (U87, LN229, U251) and patient-derived glioblastoma cells (GS620, GS48, AS515), characterized by loss of mitochondrial inner membrane potential, cytosolic release of cytochrome c, activation of caspase-9, -3, -7, and annexin V reactivity; it does not kill normal fetal human astrocytes (FHAS) [1]
At suboptimal concentrations of 5–10 μM, G-TPP triggers autophagy in glioblastoma cells, leading to cytoplasmic vacuolization containing mitochondria (confirmed by COX-IV immunogold labeling), lipidation of LC3 (conversion of LC3-I to LC3-II), and punctate fluorescence of LC3-GFP; pharmacologic inhibition (bafilomycin A, 3-methyladenine) or genetic silencing (atg5 siRNA) of autophagy enhances G-TPP-induced tumor cell death [1]
G-TPP disrupts mitochondrial protein folding, resulting in accumulation of detergent-insoluble (unfolded) mitochondrial proteins and downregulation of mitochondrial superoxide dismutase (SOD2); it upregulates UPR-related transcription factors CHOP and C/EBPβ at subcytotoxic concentrations (5–10 μM), but not at mitochondriotoxic concentrations (10–20 μM) [1]
G-TPP completely abrogates constitutive and TNF-α-induced NF-κB-dependent gene expression, downregulating FLIP (protein and promoter activity), RelB, and Bcl-3; it does not affect p53 promoter activity induced by etoposide [1]
In combination with TRAIL, subcytotoxic concentrations of G-TPP (5 μM) synergistically kills glioblastoma, breast (MCF-7), and prostate (PC3) cancer cells, accelerating loss of mitochondrial membrane potential, cytochrome c release, annexin V reactivity, and activation of caspase-8, -9, -3, -7 [1]

The potential for stellate anti-astroblastoma action of TRAIL plus Gamitrinib TPP (G-TPP) combo treatment was examined. The right brain striatum of immunocompromised mice has U87 astroblastoma cells that contain luciferase. These cells use bioluminescence to form tumors quickly. It is not ideal to treat these animals with a vehicle, TRAIL three-dimensional gradient descent, or systemic administration. Gamitrinib TPP concentrations had no effect on tumor development in vivo. Similarly, orthotopic astroblast tumor growth was unaffected by systemic monotherapy with gamitrinib TPP at a dose that reduces tumor growth in subcutaneous xenografts in mice (20 mg/kg, daily intraperitoneal injection). On the other hand, two rounds of intracranial tumor growth produced no results. Without significantly reducing the animal's body weight over the course of treatment, TRAIL in combination with systemic gamitrinib TPP inhibits the growth of established astroblastomas [1].

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In nude mice bearing intracranial U87-Luc glioblastomas, combined treatment with systemic Gamitrinib TPP hexafluorophosphate (10 mg/kg daily i.p. on days 6, 7, 9, 10) and intracranial TRAIL (2 ng on days 7, 10) suppresses tumor growth (assessed by bioluminescence imaging), extends survival (35 days vs. 24–27 days in monotherapy/vehicle groups), and shows no significant animal weight loss [1]
Tumor tissues from combined treatment mice exhibit increased nuclear CHOP expression (indicative of mitochondrial UPR activation), reduced cell proliferation (Ki67+ cells), and enhanced apoptosis (TUNEL+ cells, cleaved caspase-3+ cells); normal brain regions (hippocampus, cortex) show no CHOP reactivity [1]
Systemic monotherapy with G-TPP (20 mg/kg daily i.p.) has no effect on orthotopic glioblastoma growth [1]
In human WHO grade IV glioblastoma specimens, nuclear CHOP is strongly expressed in tumor cells (especially adjacent to necrosis), but not in normal brain tissue [1]

Cell viability assay: Seed various cancer cells or normal FHAS in 96-well plates (2×10³ cells/well), treat with G-TPP (0–20 μM) or combination with TRAIL (100–200 ng/ml depending on cell type) for up to 24 hours, and quantify metabolic activity by MTT assay (absorbance at 405 nm) [1]
Apoptosis assay: Treat tumor cells (1×10⁶) with G-TPP or combination with TRAIL, stain with annexin V and PI, analyze by flow cytometry; prepare mitochondrial/cytosolic extracts and detect cytochrome c release, caspase cleavage, and Bcl-2 family protein expression by Western blot [1]
Autophagy assay: Transfect glioblastoma cells with LC3-GFP cDNA, treat with G-TPP, observe punctate GFP fluorescence by fluorescence microscopy (score >10 dots/cell as autophagic); detect LC3 lipidation by Western blot; visualize autophagic vacuoles and mitochondrial localization by electron microscopy (EM) and immunoelectron microscopy (COX-IV antibody) [1]
Mitochondrial membrane potential assay: Label U251 cells with TMRM (0.1 μM) or JC-1, treat with G-TPP alone or in combination with TRAIL, monitor changes in fluorescence (red/green ratio) by confocal microscopy or flow cytometry [1]
Mitochondrial protein folding assay: Treat LN229 cells with G-TPP (5 μM) for 16 hours, extract mitochondrial proteins with increasing concentrations of NP40 (0%–0.5%), separate detergent-insoluble proteins by SDS-PAGE, and visualize by silver staining [1]
Promoter activity assay: Transfect tumor cells with NF-κB, p53, or FLIP luciferase promoter constructs, treat with G-TPP (0–10 μM) or stimuli (TNF-α, CCCP, STS) for 6 hours, measure β-galactosidase-normalized luciferase activity by luminometry [1]
Genetic silencing assay: Transfect glioblastoma cells with siRNA (CHOP, TRAP-1, caspase-8, atg5) or stable shRNA (CypD, TAK1), confirm protein knockdown by Western blot, treat with G-TPP alone or in combination with TRAIL, analyze cell viability (MTT), apoptosis (PI staining/flow cytometry), or NF-κB activity [1]

Intracranial glioblastoma model establishment: Suspend U87-Luc cells (1×10⁵) in sterile PBS, stereotactically implant into the right cerebral striatum of immunocompromised nude mice [1]
Combination treatment protocol: Randomize mice into 4 groups (4 mice/group), administer sterile vehicle (cremophor), TRAIL alone, G-TPP alone, or TRAIL+G-TPP; TRAIL is stereotactically injected into the right cerebral striatum (2 ng on days 7 and 10 post-implantation), and G-TPP is given systemically (10 mg/kg daily i.p. on days 6, 7, 9, and 10 post-implantation); suspend treatment on day 10, monitor tumor growth weekly by bioluminescence imaging (110 mg/kg D-luciferin i.p.), record animal weight and survival, harvest brains for histology at study end [1]
G-TPP monotherapy protocol: Treat nude mice with established intracranial U87-Luc glioblastomas with systemic G-TPP (20 mg/kg daily i.p.), monitor tumor growth by bioluminescence imaging [1]
Histological analysis: Stain brain sections with H&E, Ki-67 (proliferation), cleaved caspase-3, TUNEL (apoptosis), or CHOP (UPR activation) antibodies; quantify positive cells by microscopy [1]

In vitro experiments showed that concentrations of up to 20 μM Gamitrinib TPP hexafluorophosphate did not kill normal fetal human astrocytes (FHAS) or SV40-transformed FHAS, demonstrating tumor-selective cytotoxicity [1]. In vivo experiments showed that combined treatment with G-TPP and TRAIL did not cause significant weight loss in animals, indicating extremely low systemic or organ toxicity [1]. No significant toxicity was observed in nude mice treated with G-TPP monotherapy (20 mg/kg, daily intraperitoneal injection) [1].

[1]. Exploiting the mitochondrial unfolded protein response for cancer therapy in mice and human cells. J Clin Invest. 2011 Apr 1; 121(4): 1349–1360.

Gamitrinib TPP (G-TPP) is a mitochondrial-targeted Hsp90 inhibitor composed of the Hsp90 ATPase inhibitory module 17-AAG and the mitochondrial-targeting triphenylphosphine; it selectively accumulates in mitochondria without affecting the homeostasis of cytoplasmic Hsp90[1]. Its mechanism of action involves a dual effect: high concentrations (15–20 μM) trigger Bcl-2-independent mitochondrial disintegration and apoptosis, while subcytotoxic concentrations (5–10 μM) induce mitochondrial UPR and autophagy, inhibit NF-κB-dependent survival signaling pathways, and make tumor cells more sensitive to TRAIL-induced apoptosis[1]. G-TPP targets mitochondrial Hsp90/TRAP-1, which is selectively expressed in tumor cell mitochondria rather than in normal tissues, contributing to its tumor selectivity[1].
Human glioblastoma specimens showed high expression of nuclear CHOP (a marker of mitochondrial UPR) in tumor cells near necrotic areas, indicating that this pathway is activated in clinical tumors and supports its potential therapeutic applications. [1]

C52H65F6N3O8P2

1036.02575755119

1035.415

1131626-47-5

Gamitrinib TPP;1131626-46-4

25232581

Pale purple to purple solid powder

4

16

15

71

1750

6

[P+](C1C=CC=CC=1)(C1C=CC=CC=1)(C1C=CC=CC=1)CCCCCCNC1C(C=C2C(C=1C[C@@H](C)C[C@@H]([C@@H]([C@@H](C)C=C(C)[C@@H]([C@H](C=CC=C(C)C(N2)=O)OC)OC(N)=O)O)OC)=O)=O.[P-](F)(F)(F)(F)(F)F |c:44,49,51|

NFIBTCZSRLMDOD-WLXHSWTPSA-O

InChI=1S/C52H64N3O8P.F6P/c1-35-31-42-47(54-29-18-7-8-19-30-64(39-22-12-9-13-23-39,40-24-14-10-15-25-40)41-26-16-11-17-27-41)44(56)34-43(49(42)58)55-51(59)36(2)21-20-28-45(61-5)50(63-52(53)60)38(4)33-37(3)48(57)46(32-35)62-6;1-7(2,3,4,5)6/h9-17,20-28,33-35,37,45-46,48,50,57H,7-8,18-19,29-32H2,1-6H3,(H3-,53,54,55,56,58,59,60);/q;-1/p+1/b28-20-,36-21+,38-33+;/t35-,37+,45+,46+,48-,50+;/m1./s1

6-[[(4E,6Z,8S,9S,10E,12S,13R,14S,16R)-9-carbamoyloxy-13-hydroxy-8,14-dimethoxy-4,10,12,16-tetramethyl-3,20,22-trioxo-2-azabicyclo[16.3.1]docosa-1(21),4,6,10,18-pentaen-19-yl]amino]hexyl-triphenylphosphanium;hexafluorophosphate

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 (~48.26 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.41 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 2: ≥ 2.5 mg/mL (2.41 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 3: 2.5 mg/mL (2.41 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)