RapaLink-1 is the third-generation mTOR inhibitor which can overcome mTOR resistance mutations. It exploits the unique juxtaposition of two drug-binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants, and reverses resistance due to mTOR FRB (resistant to Rapamycin) and kinase domain (resistant to AZD8055) mutations. RapaLink-1 is a potent mTOR inhibitor evidenced by suppression of downstream signaling, including p-AKT, p-p70S6K, p-S6 and p-4EBP1, in MCF-7 cells treated with RapaLink-1 at concentration>3nM post 4h. RapaLink-1 at low doses (3–10 nM) was the only drug regimen capable of inhibiting mTOR signalling in both F2108L mTOR- and M2327I mTOR-expressing cells. Administration of 1.5mg/kg RapaLink-1, i.p., weekly, significantly inhibited tumor growth in mice bearing RR1 or TKi-R xenograft tumors[3]. RapaLink-1 crosses the blood-brain barrier. RapaLink-1 drove regression of intracranial brain cancers in vivo, improving survival compared with earlier-generation inhibitors, first-generation allosteric mTOR inhibitor rapamycin and second-generation TORKi[4].
作用机制
RapaLink-1 is consist of MLN0128 targeting the ATP-site and Rapamycin targeting FRB/FKBP12-site, which facilitates it to exploit the unique juxtaposition of two drug-binding pockets to create a bivalent interaction that allows inhibition of these resistant mutants.[3]
RapaLink-1 细胞实验
Cell Line
Concentration
Treated Time
Description
References
K562 cells
3 nM
24 hours
Evaluate the effect of IFITM expression on the intracellular activity of RapaLink-1, results show that IFITM expression promotes the intracellular activity of RapaLink-1
Science. 2022 Dec 9;378(6624):1097-1104
Beta-TC-6 cells
10 nM
24 hours
To investigate the effects of RapaLink-1 on amino acid metabolism and mitochondrial functions. Results showed that RapaLink-1 significantly decreased L-aspartate, L-asparagine, L-glutamate, and glycine levels, and reduced ATP production and non-mitochondrial oxygen consumption.
RL1 showed weaker inhibitory effects on non-cancerous neural stem cells, indicating selectivity for cancer cells.
Cancers (Basel). 2020 Dec 21;12(12):3859
Glioblastoma stem cells (GSCs)
1.5, 6, 12, 24, 48 nM
4 days
RL1 significantly inhibited cell growth, proliferation, migration, and clonogenic potential of GSCs, and reduced the expression of stem cell markers (CD133, SOX2) and EMT markers (CD44, ZEB1).
Cancers (Basel). 2020 Dec 21;12(12):3859
3T3-L1 adipocytes
3 nM, 10 nM and 20 nM
4 hours
High-dose RapaLink1 substantially inhibited insulin-induced glucose uptake, indicating a major role for mTORC2, but not mTORC1, in acute insulin-regulated glucose uptake.
Sci Signal. 2021 Sep 21;14(701):eabe0161
MCF7 cells
10 nM
4 hours
To monitor mTOR signaling by western blot, results showed that RapaLink-1 significantly reduced the levels of phospho-S6 and phospho-4EBP, while the addition of FK506 completely blocked this effect
Nature. 2022 Sep;609(7928):822-828
HEK-293E cells
3 nM
4 hours
Selectively and almost completely blocked mTORC1 activity toward both rapamycin-sensitive and rapamycin-resistant sites without any demonstrable effect on mTORC2 targets.
Sci Signal. 2021 Sep 21;14(701):eabe0161
SU-DHL-6
12.5 nM and 50 nM
48 hours
Evaluate the sensitivity of RapaLink-1 in EZH2 wild-type and mutant lymphoma cells
Sci Transl Med. 2017 Jun 28;9(396):eaak9969
SU-DHL-4
12.5 nM and 50 nM
48 hours
Evaluate the sensitivity of RapaLink-1 in EZH2 wild-type and mutant lymphoma cells
Sci Transl Med. 2017 Jun 28;9(396):eaak9969
SU-DHL-10
12.5 nM and 50 nM
48 hours
Evaluate the sensitivity of RapaLink-1 in EZH2 wild-type and mutant lymphoma cells
Sci Transl Med. 2017 Jun 28;9(396):eaak9969
DoHH2
12.5 nM and 50 nM
48 hours
Evaluate the sensitivity of RapaLink-1 in EZH2 wild-type and mutant lymphoma cells
Sci Transl Med. 2017 Jun 28;9(396):eaak9969
Toledo
12.5 nM and 50 nM
48 hours
Evaluate the sensitivity of RapaLink-1 in EZH2 wild-type and mutant lymphoma cells
Sci Transl Med. 2017 Jun 28;9(396):eaak9969
OCI-LY19
12.5 nM and 50 nM
48 hours
Evaluate the sensitivity of RapaLink-1 in EZH2 wild-type and mutant lymphoma cells
Sci Transl Med. 2017 Jun 28;9(396):eaak9969
Normal human lung fibroblasts (NHLFs)
2 nM
48 hours
RapaLink-1 completely inhibited TGF-β-induced ATF4 accumulation and metabolic reprogramming, including increases in glycolysis and mitochondrial oxygen consumption.
Am J Respir Cell Mol Biol. 2020 Nov;63(5):601-612
BM18 PDX organoids
0.0003 µM (IC50)
48 hours
To evaluate the cytotoxic effect of RapaLink-1 on BM18 PDX organoids, results showed that RapaLink-1 significantly reduced organoid viability.
Front Oncol. 2020 Jun 23;10:1012
LAPC9 PDX organoids
0.0046 µM (IC50)
48 hours
To evaluate the cytotoxic effect of RapaLink-1 on LAPC9 PDX organoids, results showed that RapaLink-1 significantly reduced organoid viability.
Front Oncol. 2020 Jun 23;10:1012
HeLa cells
1 nM and 3 nM
48 hours
RapaLink1 significantly inhibited cell proliferation, showing better efficacy than rapamycin.
Sci Signal. 2021 Sep 21;14(701):eabe0161
HMLER cells
5 nM
6 days
Inhibition of mTOR signaling, reduction of CD24lowCD44high stem cell population and mammosphere formation
Sci Signal. 2019 Feb 26;12(570):eaau8544
K562 cells
10 nM
72 hours
To evaluate the effect of RapaLink-1 on cell proliferation, results showed that CRISPRi-mediated knockdown of FKBP12 gene significantly impeded the cellular activity of RapaLink-1
Nature. 2022 Sep;609(7928):822-828
RapaLink-1 动物实验
Species
Animal Model
Administration
Dosage
Frequency
Description
References
Male mice
Alcohol Use Disorder model
Intraperitoneal injection
1 mg/kg
Three times a week for 4 weeks
RapaLink-1 blocked mTORC1 activation in the liver, while RapaBlock protected mTORC1 activity in the periphery. Co-administration of RapaLink-1 and RapaBlock inhibited alcohol-dependent mTORC1 activation in the nucleus accumbens and attenuated alcohol seeking and drinking.
Nat Commun. 2021 Jul 27;12(1):4407
BALB/cnu/nu mice
Glioblastoma xenograft model
Intraperitoneal injection
1 mg/kg RapaLink-1 and 40 mg/kg RapaBlock
Every 5 days for 25-30 days
To evaluate the therapeutic effect of the combination of RapaLink-1 and RapaBlock, results showed that the combination significantly suppressed tumor growth and improved survival without detectable systemic toxicity
Nature. 2022 Sep;609(7928):822-828
NSG mice
Orthotopic mammary fat pad implantation model
Intraperitoneal injection
1.5 mg/kg
Every 5 days for 20 days, then once a week for 2 weeks
RapaLink-1 almost completely inhibited tumor formation
Sci Signal. 2019 Feb 26;12(570):eaau8544
CB17/SCID mice
LAPC9 PDX model
Intraperitoneal injection
1.5 mg/kg
Every 5-7 days, during the experiment period
To evaluate the anti-tumor effect of RapaLink-1 in vivo on LAPC9 PDX model, results showed that RapaLink-1 significantly delayed tumor growth.
Front Oncol. 2020 Jun 23;10:1012
Rats
Middle cerebral artery occlusion (MCAO) model
Intraperitoneal injection
2 mg/kg
Single dose, 10 minutes after MCAO
To investigate the effects of Rapalink-1 on neuronal survival and BBB disruption in the early stage of cerebral ischemia–reperfusion. Results showed that Rapalink-1 increased infarct size and BBB disruption.
Front Physiol. 2021 Jul 20;12:706528
Drosophila melanogaster
Adult flies
Food supplemented
6 μM
For three days
In contrast to rapamycin, RapaLink1 significantly shortened fly survival under starvation conditions.
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