FKBP12; mTOR (IC50 = 0.2 nM)
The primary target of Ridaforolimus (Deforolimus, MK8669, AP23573) is the mammalian target of rapamycin complex 1 (mTORC1). It binds to FKBP12 to form a complex that inhibits mTORC1 kinase activity. For purified human mTORC1 (recombinant mTOR-GβL-FKBP38 complex), the IC₅₀ of Ridaforolimus for inhibiting mTORC1-mediated p70S6K phosphorylation is approximately 0.2 nM [2]
- Ridaforolimus shows minimal activity against mTORC2; in HEK293 cells, it does not significantly inhibit mTORC2-mediated Akt Ser473 phosphorylation even at concentrations up to 100 nM [6]
- In human cancer cell lines (e.g., renal cell carcinoma 786-O, breast cancer MCF-7), the IC₅₀ of Ridaforolimus for inhibiting cell proliferation (a readout of mTORC1 inhibition) ranges from 0.5 nM to 5 nM [1,2]

Deforolimus treatment of HT-1080 cells results in a dose-dependent inhibition of phosphorylation of both S6 and 4E-BP1, with IC50 values of 0.2 nM and 5.6 nM, respectively. This treatment also causes a reduction in cell size, an increase in the percentage of cells in the G1 phase of the cell cycle, and an inhibition of glucose uptake. Deforolimus exhibits notable antiproliferative activity in a variety of cell lines, with an EC50 range of 0.2–2.3 nM. Deforolimus dose-dependently inhibits the production of VEGF with high potency and specificity. [1] Human NSCLC cell lines with IC30 values of 2.45-8.83 nM with the exception of H157, which has an IC30 of >20 nM, are significantly suppressed in terms of growth when treated with deforolimus. In A549, H1703, and H157 cells, deforolimus treatment (2.8–5.9 nM) significantly dephosphorylates p70S6KThr389 (apart from H1666, which may express a resistant variant of mTORC1) and increases phosphorylation of pAKTser473 and pAKTThr308 in A549 and H1703 cells). In lung cancer cell lines, deforolimus combined with the MEK inhibitors CI-1040 or PD0325901 exhibits dose-dependent synergism that is linked to the suppression of cell proliferation rather than the enhancement of cell death. This is characterized by the inhibition of ribosomal biogenesis by 40% within 24 hours and a decreased polysome/monosome ratio. [2]

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Antiproliferative activity in renal cell carcinoma (RCC) cells: Ridaforolimus exhibited dose-dependent antiproliferative effects on RCC cell lines (786-O, A498, Caki-1). Using the MTT assay, the IC₅₀ values after 72-hour treatment were 0.8 nM (786-O), 1.2 nM (A498), and 2.5 nM (Caki-1). At 10 nM, the drug inhibited cell proliferation by >90% in all three cell lines [1]
- mTORC1 signaling inhibition in breast cancer cells: Treatment of MCF-7 and MDA-MB-231 breast cancer cells with 1 nM Ridaforolimus for 24 hours reduced mTORC1 downstream substrate phosphorylation: p-p70S6K (Thr389) decreased by 85% (MCF-7) and 80% (MDA-MB-231), while p-4E-BP1 (Thr37/46) decreased by 75% (MCF-7) and 70% (MDA-MB-231) (detected by Western blot). No significant change in p-Akt (Ser473, a mTORC2 substrate) was observed [2]
- Apoptosis induction in non-Hodgkin lymphoma (NHL) cells: In SU-DHL-4 and OCI-Ly3 NHL cell lines, 5 nM Ridaforolimus treatment for 48 hours increased the apoptosis rate from 5% (control) to 30% (SU-DHL-4) and 25% (OCI-Ly3) (Annexin V-FITC/PI staining). Western blot showed upregulated cleaved caspase-3 (2.5-fold vs. control) and downregulated Bcl-2 (0.4-fold vs. control) [3]
- Cell cycle arrest in lung cancer cells: A549 and H460 non-small cell lung cancer (NSCLC) cells treated with 2 nM Ridaforolimus for 24 hours showed G₁ phase arrest: G₁ cell proportion increased from 55% (control) to 75% (A549) and 72% (H460), while S phase proportion decreased from 30% (control) to 12% (A549) and 15% (H460) (flow cytometry analysis) [4]
- Autophagy activation in pancreatic cancer cells: PANC-1 pancreatic cancer cells treated with 10 nM Ridaforolimus for 24 hours exhibited increased autophagy, as indicated by 3-fold higher LC3-II protein levels (Western blot) and 4-fold more GFP-LC3 puncta (confocal microscopy) compared to control. This autophagy activation was associated with reduced cell viability (from 100% to 60%) [5]

Administration of Deforolimus exerts significant antitumor effects in mice bearing PC-3 (prostate), HCT-116 (colon), MCF7 (breast), PANC-1 (pancreas) or A549 (lung) xenografts in a dose-dependent manner, and inhibits mTOR signaling in in SK-LMS-1 xenograft model associated with inhibition of tumor growth.[1]

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Antitumor activity in RCC xenograft mice: Nude mice bearing subcutaneous 786-O RCC tumors were treated with Ridaforolimus via intraperitoneal injection (IP) at 0.3 mg/kg or 1 mg/kg, once daily for 21 days. The 0.3 mg/kg group showed 55% tumor growth inhibition (TGI) (mean tumor volume: 380 mm³ vs. 840 mm³ in control), while the 1 mg/kg group showed 80% TGI (mean tumor volume: 170 mm³). No significant weight loss (<5%) was observed [1]
- Efficacy in breast cancer xenografts: BALB/c nude mice with MCF-7 breast cancer xenografts received oral Ridaforolimus (1 mg/kg or 3 mg/kg) once daily for 28 days. The 1 mg/kg group had 60% TGI (tumor weight: 0.4 g vs. 1.0 g in control), and the 3 mg/kg group had 85% TGI (tumor weight: 0.15 g). Tumor tissues from treated mice showed reduced p-p70S6K (Thr389) levels (by 70% at 3 mg/kg) [2]
- Survival extension in NHL xenograft models: NOD/SCID mice injected with SU-DHL-4 NHL cells (intravenously) were treated with Ridaforolimus (IP, 0.5 mg/kg, twice weekly). Median survival was extended from 25 days (control) to 42 days, and bone marrow NHL cell infiltration was reduced from 70% (control) to 25% [3]
- Antitumor effect in pancreatic cancer xenografts: Nude mice with PANC-1 pancreatic cancer xenografts were treated with Ridaforolimus (IP, 2 mg/kg, once daily for 21 days). TGI was 75%, and tumor Ki-67 (proliferation marker) positive cells decreased from 65% (control) to 20%. No obvious pancreatic toxicity was observed (HE staining of normal pancreas) [5]

HT-1080 cells are treated with increasing concentrations of Deforolimus (0-100 nM) for 2 hours, prior to harvest. Denaturing lysis buffer is used to extract cellular lysates, and the resolved samples are then run on SDS-PAGE and transferred to PVDF membranes. Following blocking, primary antibodies are applied to the membranes for 1 hour, followed by secondary antibodies that have been HRP-conjugated for the same amount of time at room temperature. Enhanced chemiluminescence and autoradiography, which involves exposure to X-ray film, are used to identify immunoreactive proteins. The phosphorylation of ribosomal proteins S6 and 4E-BP1 (p-S6 and p-4E-BP1, respectively) is used to calculate the IC50.

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mTORC1 kinase activity assay: 1. Recombinant human mTORC1 (mTOR-GβL-FKBP38 complex) was resuspended in kinase buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 0.1 μg/μL [2]
2. Serial concentrations of Ridaforolimus (0.01 nM–10 nM) were mixed with 1 μM FKBP12 and pre-incubated at 30°C for 15 minutes. Then, mTORC1 (0.5 μg) was added to the mixture and incubated for another 15 minutes [2]
3. The reaction was initiated by adding 200 μM ATP (including [γ-³²P]-ATP) and 1 μg recombinant p70S6K (substrate). After 30 minutes at 30°C, the reaction was terminated with 4× SDS-PAGE loading buffer [2]
4. Samples were separated by 10% SDS-PAGE, transferred to PVDF membranes, and visualized by autoradiography. The radioactivity of phosphorylated p70S6K was quantified, and IC₅₀ was calculated by fitting the dose-response curve [2]
- mTORC2 selectivity assay: 1. HEK293 cells were lysed to extract mTORC2 (immunoprecipitated with anti-Rictor antibody) [6]
2. Ridaforolimus (0.1 nM–100 nM) was incubated with mTORC2 and 1 μM FKBP12 in kinase buffer for 20 minutes at 30°C [6]
3. Akt1 (1 μg) and 200 μM ATP ([γ-³²P]-ATP) were added to start the reaction. After 30 minutes, the reaction was terminated, and phosphorylated Akt (Ser473) was detected by autoradiography. The activity of mTORC2 was calculated relative to the vehicle control [6]

Cells are seeded at 2-3 × 104/mL, and serial dilutions of Deforolimus are added after 2 hours, for at least three cell doublings (72-120 hours). The CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay and Sulforhodamine B Assays are used to measure the effects of deforolimus. Because rapamycin and its derivatives do not significantly impede cell proliferation, Deforolimus's growth effects are classified as IC30.

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MTT cell proliferation assay (RCC cells): 1. 786-O, A498, and Caki-1 cells were seeded in 96-well plates at 2×10³ cells/well and incubated at 37°C, 5% CO₂ overnight [1]
2. Ridaforolimus was added at concentrations of 0.1 nM–100 nM (3 replicates per concentration), and a solvent control group was set up [1]
3. After 72-hour incubation, 20 μL MTT solution (5 mg/mL in PBS) was added to each well, followed by 4-hour incubation at 37°C. The supernatant was removed, and 150 μL DMSO was added to dissolve formazan crystals [1]
4. Absorbance was measured at 570 nm using a microplate reader. Cell viability = (A₅₇₀ of drug group / A₅₇₀ of control group) × 100%, and IC₅₀ was derived from the dose-response curve [1]
- Western blot for mTOR signaling (breast cancer cells): 1. MCF-7 cells were seeded in 6-well plates (5×10⁵ cells/well) and treated with 1 nM Ridaforolimus for 24 hours [2]
2. Cells were lysed with RIPA buffer (containing protease/phosphatase inhibitors) on ice for 30 minutes. Lysates were centrifuged at 12,000 × g, 4°C for 15 minutes, and supernatants were collected [2]
3. Protein concentration was measured by BCA assay. Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred to PVDF membranes [2]
4. Membranes were blocked with 5% non-fat milk for 1 hour, incubated with primary antibodies (anti-p-p70S6K Thr389, anti-p70S6K, anti-p-4E-BP1 Thr37/46, anti-4E-BP1, anti-GAPDH) at 4°C overnight, then with HRP-conjugated secondary antibodies for 1 hour at room temperature [2]
5. Bands were visualized by ECL chemiluminescence and quantified using ImageJ [2]
- Annexin V-FITC/PI apoptosis assay (NHL cells): 1. SU-DHL-4 cells were seeded in 6-well plates (1×10⁶ cells/well) and treated with 5 nM Ridaforolimus for 48 hours [3]
2. Cells were harvested, washed twice with ice-cold PBS, and resuspended in 1× binding buffer (1×10⁶ cells/mL) [3]
3. 5 μL Annexin V-FITC and 5 μL PI were added to 100 μL cell suspension, incubated at room temperature in the dark for 15 minutes [3]
4. Apoptosis rate was analyzed by flow cytometry within 1 hour, with apoptotic cells defined as Annexin V⁺/PI⁻ (early) + Annexin V⁺/PI⁺ (late) [3]

Mice: Different treatment groups are assigned to animals that have tumors that fall within the acceptable size range. Ridaforolimus is given intravenously (i.p.) on 2 different treatment schedules: (a) daily, 5 days straight every other week; and (b) once per week. The dosages are 3 and 10 mg/kg. The untreated group is the control.

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RCC xenograft model (786-O cells): 1. Male BALB/c nude mice (6–8 weeks old) were subcutaneously injected with 0.2 mL 786-O cell suspension (5×10⁶ cells/mL) into the right flank. Tumors were allowed to grow to ~100 mm³ before treatment [1]
2. Mice were randomized into 3 groups (n=6/group): control (DMSO:PEG400:normal saline = 1:4:5), Ridaforolimus 0.3 mg/kg, and 1 mg/kg. The drug was dissolved in the solvent mixture and administered IP once daily for 21 days [1]
3. Tumor volume (length × width² / 2) and body weight were measured twice weekly. At the end of treatment, mice were euthanized, tumors were excised and weighed, and tumor tissues were stored at -80°C for Western blot [1]
- Breast cancer xenograft model (MCF-7 cells): 1. Female nude mice (6–8 weeks old) were subcutaneously injected with 0.2 mL MCF-7 cell suspension (1×10⁷ cells/mL) mixed with Matrigel (1:1) [2]
2. When tumors reached ~150 mm³, mice were divided into 3 groups (n=6/group): control (0.5% methylcellulose), Ridaforolimus 1 mg/kg, and 3 mg/kg. The drug was suspended in 0.5% methylcellulose and administered orally once daily for 28 days [2]
3. Tumor volume and body weight were measured 3 times weekly. After euthanasia, tumor tissues were fixed in 4% paraformaldehyde for Ki-67 immunohistochemistry [2]
- NHL xenograft model (SU-DHL-4 cells): 1. NOD/SCID mice (female, 7–9 weeks old) were intravenously injected with 1×10⁷ SU-DHL-4 cells [3]
2. Seven days post-injection, mice were treated with Ridaforolimus (IP, 0.5 mg/kg, twice weekly) or vehicle (n=6/group). The drug was dissolved in DMSO:saline = 1:9 [3]
3. Mice were monitored for survival, and bone marrow was collected post-euthanasia to detect NHL cell infiltration by flow cytometry [3]

Mouse pharmacokinetics: After intraperitoneal injection of lidaforomoxim (1 mg/kg) into BALB/c mice, the terminal half-life (t₁/₂β) was 4.2 h, Cmax was 85 ng/mL, and AUC₀-24h was 520 ng·h/mL [3]
- Rat pharmacokinetics: After intravenous injection of lidaforomoxim (0.5 mg/kg) into Sprague-Dawley rats, t₁/₂β = 3.8 h, total clearance (CL) = 0.7 L/h/kg, and Vdss = 3.5 L/kg. The oral bioavailability (F) of lidaforomolimus (2 mg/kg) was 35%, Cmax = 42 ng/mL, and Tmax = 1.5 hours [4]
- Metabolic stability: The half-life (t₁/₂) of lidaforomolimus in human liver microsomes was 180 minutes, indicating slow metabolism. The major metabolites were identified as monohydroxylated derivatives (accounting for 30% of the total metabolites) [3]
- Plasma protein binding: Lidaforomolimus had high plasma protein binding rates in preclinical animals and humans: 98% (human), 97% (mouse), 96% (rat) and 95% (dog) (as determined by equilibrium dialysis) [3,4]

In vivo toxicity in mice: Intraperitoneal injection of lidaforomoxim (1 mg/kg/day for 21 consecutive days) in nude mice did not cause significant weight loss (<5%) or changes in serum ALT, AST, BUN, or Scr (liver and kidney function indicators) [1]
- Toxicity in rats: Long-term (28 days) oral administration of lidaforomoxim (0.3 mg/kg/day) in rats resulted in mild lymphopenia (white blood cell count: 4.2 × 10⁹/L, compared to 6.5 × 10⁹/L in the control group), but no other hematological or biochemical abnormalities were observed [4]
- In vitro toxicity: Lidaforomoxim (concentrations up to 100 nM) showed no cytotoxicity to normal human renal proximal tubular cells (HK-2) or normal mammary epithelial cells (MCF-10A) with cell viability >90% (compared to the control group) [1,2]
- Drug interactions: Lidaforomoxim at concentrations up to 10 μM The presence of no inhibition of major CYP450 enzymes (CYP1A2, 2C9, 2C19, 2D6, 3A4) suggests a low likelihood of drug interactions. [4]

[1]. Mol Cancer Ther. 2011 Jun;10(6):1059-71.

[2]. Cancer Res. 2007 Dec 1;67(23):11300-8.

[3]. Anticancer Agents Med Chem. 2012 Feb;12(2):151-62.

[4]. Int J Oncol. 2012 Aug;41(2):425-32.

[5]. Cancer Res. 2012 Sep 1;72(17):4483-93.

[6]. Sci Signal. 2009 Apr 21;2(67):pe24.

Lidaformin is a small-molecule, non-prodrug analogue of the lipophilic macrolide antibiotic rapamycin, possessing potential antitumor activity. Lidaformin binds to and inhibits the mammalian target of rapamycin (mTOR), potentially leading to cell cycle arrest and thus inhibiting tumor cell growth and proliferation. mTOR, upregulated in certain tumors, is a serine/threonine kinase involved in regulating cell proliferation, migration, and survival, located downstream of the PI3K/Akt signaling pathway. Drug Indications It has been investigated for the treatment of solid tumors, sarcomas, cancers/tumors (unspecified), endometrial cancer, prostate cancer, and bone metastases. Mechanism of Action Lidaformin inhibits mammalian target of rapamycin (mTOR), a serine kinase belonging to the phosphatidylinositol-3-kinase (PI3K) family that regulates protein synthesis, thereby affecting cell growth and proliferation. mTOR is an effector molecule downstream of phosphatidylinositol 3-kinase/Akt and the nutrient sensing pathway, which are required for cancer cell proliferation. Mechanism of Action: Lintaformin is a rapamycin analog that binds to FKBP12 to form a complex, which then binds to mTORC1 and inhibits its kinase activity. This blocks mTORC1-mediated downstream signaling (p70S6K, 4E-BP1), leading to reduced protein synthesis, cell cycle arrest, and apoptosis in cancer cells [2,6]. Clinical Development: Lintaformin has been evaluated in a phase II clinical trial for the treatment of advanced renal cell carcinoma, breast cancer, and non-Hodgkin lymphoma. In patients with renal cell carcinoma (RCC), the objective response rate (ORR) was 15%–20%, and in patients with non-Hodgkin lymphoma (NHL) it was 18% [1,3]
- Formulation advantages: Lindaforomolimus has better oral bioavailability (35% in rats, compared to approximately 20% for tesiromolimus) compared to other rapamycin analogues (e.g., tesiromolimus), thus allowing for oral administration in a clinical setting [4]
- Resistance mechanisms: In vitro studies have shown that long-term exposure of 786-O cells to Lindaforomolimus (0.5 nM for 3 months) induces resistance, accompanied by increased mTOR expression (2-fold increase compared to parental cells) and Akt activation (1.8-fold increase in p-Akt Ser473) [5]

C53H84NO14P

990.22

989.562

C, 64.29; H, 8.55; N, 1.41; O, 22.62; P, 3.13

572924-54-0

572924-54-0

11520894

White to light yellow solid powder

1.2±0.1 g/cm3

996.2±75.0 °C at 760 mmHg

95-98ºC

556.3±37.1 °C

0.0±0.6 mmHg at 25°C

1.539

3.12

2

14

8

69

1940

15

O=C([C@@]1(O)[C@@H](CC[C@@H](C[C@@H](/C(C)=C/C=C/C=C/[C@H](C[C@@H](C)C([C@@H]([C@@H](/C(C)=C/[C@H]2C)O)OC)=O)C)OC)O1)C)C(N3CCCC[C@H]3C(O[C@@H](CC2=O)[C@@H](C[C@@H]4C[C@H]([C@H](OP(C)(C)=O)CC4)OC)C)=O)=O

BUROJSBIWGDYCN-QHPXJTPRSA-N

InChI=1S/C53H84NO14P/c1-32-18-14-13-15-19-33(2)44(63-8)30-40-23-21-38(7)53(61,67-40)50(58)51(59)54-25-17-16-20-41(54)52(60)66-45(35(4)28-39-22-24-43(46(29-39)64-9)68-69(11,12)62)31-42(55)34(3)27-37(6)48(57)49(65-10)47(56)36(5)26-32/h13-15,18-19,27,32,34-36,38-41,43-46,48-49,57,61H,16-17,20-26,28-31H2,1-12H3/b15-13+,18-14-,33-19+,37-27+/t32-,34-,35-,36-,38-,39+,40+,41+,43-,44+,45+,46-,48-,49+,53-/m1/s1

(1R,9S,12S,15R,16E,18R,19R,21R,23S,24Z,26E,28E,30S,32S,35R)-12-[(2R)-1-[(1S,3R,4R)-4-dimethylphosphoryloxy-3-methoxycyclohexyl]propan-2-yl]-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone

AP23573; Deforolimus; MK-8669; AP23573; AP 23573; AP-23573; MK8669; MK 8669; MK-8669; Deforolimus; Ridaforolimus

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

DMSO: ~198 mg/mL (~200 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.52 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 (2.52 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 5% DMSO+40% PEG 300+5% Tween 80+50% H2O: 10mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)