Melanoma cells accumulate autophagic vesicles when exposed to DC661 at concentrations of 0.1 and 10 µM [1].

Melanoma cells accumulate autophagic vesicles when exposed to DC661 at concentrations of 0.1 and 10 µM [1].

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DC661 treatment of A375P melanoma cells resulted in accumulation of the autophagic vesicle marker LC3BII at concentrations as low as 0.1 µM, with complete cell death at concentrations above 10 µM. In contrast, Lys05 and HCQ did not cause cell death at these concentrations.[1]
DC661 induced significantly more potent inhibition of autophagic flux in melanoma cells expressing an mCherry-eGFP-LC3B reporter compared to HCQ or Lys05.[1]
DC661 caused significantly greater lysosomal deacidification compared to either HCQ or Lys05 in A375P cells, as measured by LysoSensor staining.[1]
DC661 induced lysosomal membrane permeabilization in a significantly higher percentage of cancer cells relative to Lys05 or HCQ, as indicated by galectin-3 puncta formation.[1]
The IC₅₀ of DC661 in 72-hour MTT assays was approximately 100-fold lower than that of HCQ across multiple cancer cell lines including colon and pancreatic cancer lines.[1]
DC661 suppressed long-term clonogenic growth of melanoma cells more effectively than HCQ or Lys05 in a colony formation assay.[1]
DC661 induced significantly more apoptosis than Lys05, HCQ, or combined BRAF- and MEK-inhibition in BRAF-mutant melanoma cells.[1]
When cancer cells were cultured under acidic conditions (mimicking the tumor microenvironment), DC661 was the only compound tested (vs. HCQ and Lys05) capable of inhibiting autophagy (as shown by LC3BII accumulation) and decreasing cell viability.[1]
Treatment of A375P cells with DC661 resulted in accumulation of the palmitoylated form of CD44, indicating inhibition of PPT1-dependent depalmitoylation.[1]
CRISPR-Cas9 knockout of PPT1 in A375P cells (KO PPT1) significantly blunted the ability of DC661 to cause lysosomal deacidification, LC3II lipidation (autophagosome accumulation), and inhibit cell proliferation compared to wild-type (WT PPT1) cells. KO PPT1 cells were significantly less sensitive to the anti-proliferative effects of DC661.[1]
Similar resistance to DC661's effects on LC3B lipidation was observed in mouse melanoma B16 cells with CRISPR-Cas9 knockout of Ppt1.[1]
In 3D spheroid culture, A375P KO PPT1 cells formed significantly smaller spheroids than WT PPT1 cells. Loss of Ppt1 in B16 cells severely limited spheroid viability.[1]

In HT29 colorectal cancer xenograft models established in NSG mice, two daily intraperitoneal doses of DC661 (10 mg/kg) resulted in significant and sustained tumor growth impairment, whereas two doses of Lys05 (10 mg/kg) had only a transient effect. However, all mice treated with DC661 at 10 mg/kg were euthanized due to lethargy.[1]
In a subsequent HT29 xenograft experiment, daily intraperitoneal administration of DC661 at a reduced dose of 3 mg/kg resulted in a significant reduction in tumor volume and almost complete suppression of daily tumor growth rate compared with vehicle control, without significantly affecting mouse weight. Immunoblotting of tumor lysates showed evidence of in vivo autophagy inhibition and apoptosis induction.[1]
Xenograft tumors generated from A375P KO PPT1 cells displayed significantly blunted tumor growth compared to tumors from WT PPT1 cells.[1]

Differential scanning calorimetry (DSC) was performed using purified recombinant PPT1 protein with and without a 4-fold molar excess of DC661. A statistically significant decrease in the PPT1 melting temperature was observed in the presence of DC661, consistent with direct binding.[1]
PPT1 enzymatic activity assays were performed in A375P cells treated with DC661 (0–100 µM) for 1 hour. DC661 inhibited PPT1 enzymatic activity in a dose-dependent manner, with increasing potency compared to HCQ and Lys05.[1]

For autophagy flux analysis using the mCherry-eGFP-LC3B reporter: A375P cells expressing the reporter were treated with DC661 (0–100 µM) for 1 hour and analyzed by fluorescence microscopy to quantify autophagic flux inhibition.[1]
For lysosomal pH measurement: A375P cells were treated with DC661 (3 µM) for 6 hours, stained with LysoSensor dye, and imaged via fluorescence microscopy. Fluorescence intensity was quantified to assess lysosomal deacidification.[1]
For lysosomal membrane permeabilization assay: A375P cells were treated with DC661 (3 µM) for 6 hours, fixed, and immunostained for galectin-3. Cells were imaged via fluorescence microscopy and the percentage of cells with galectin-3 puncta was quantified.[1]
For immunoblot analysis of autophagy markers: Cells were treated with DC661 at indicated concentrations and times, lysed, and proteins were separated by SDS-PAGE. Membranes were immunoblotted with antibodies against LC3B to assess LC3II accumulation.[1]
For MTT cell viability assay: Cells were plated in 96-well plates and treated with DC661 at indicated concentrations for 72 hours. MTT reagent was added, incubated, and formazan crystals were dissolved before measuring absorbance.[1]
For clonogenic assay: Cells were treated chronically with DC661 (0–1000 nM) for 2 weeks, with fresh drug and medium changes every 3-4 days. Colonies were stained with crystal violet and imaged.[1]
For rescue experiments with thioesterase mimetic: A375P cells expressing the mCherry-eGFP-LC3B reporter were treated with DC661 (3 µM) in the presence or absence of N-tert-Butylhydroxylamine (NTBHA, 2 mM) for 1 hour and analyzed by microscopy to assess rescue of autophagy inhibition.[1]
For acyl biotin exchange (ABE) assay: A375P cells were treated with DC661 (3 µM) for 1 hour. The assay was performed to detect palmitoylated proteins (e.g., CD44) as previously described, indicating inhibition of depalmitoylation.[1]

For the HT29 xenograft efficacy study: HT29 colorectal cancer cells (1 × 10⁶) were subcutaneously injected with Matrigel over the right flank of NSG mice. Treatment commenced once tumors became palpable. DC661 was administered intraperitoneally (i.p.) daily at a dose of 3 mg/kg. The vehicle control was water. Tumor volumes were measured with calipers and calculated as (Length × Width² × 0.5). The study duration was until tumors reached endpoint criteria.[1]
For the PPT1 knockout tumor growth study: A375P WT PPT1 or KO PPT1 cells (1 × 10⁶ cells/mouse) were injected into the flanks of NSG mice. Tumor volumes were measured every three days. No drug treatment was administered in this genetic model experiment.[1]

In the initial HT29 xenograft study, all mice treated with 10 mg/kg (intraperitoneal injection, twice) of DC661 were euthanized due to lethargy, indicating that this dose was significantly toxic. [1]
In subsequent studies, treatment with a reduced dose of 3 mg/kg (intraperitoneal injection, once daily) of DC661 did not have a significant effect on mouse body weight. [1]

[1]. PPT1 Promotes Tumor Growth and Is the Molecular Target of Chloroquine Derivatives in Cancer. ancer Discov. 2019 Feb;9(2):220-229.

DC661 is a novel dimeric chloroquine (CQ) derivative. Its design is based on the previously reported structure of the dimeric CQ (Lys05), with modifications such as extended linker length (six carbon atoms linked between nitrogen atoms) and methylation of the central nitrogen atom, which have been shown to enhance lysosomal localization and potency. [1]
DC661 has been identified as a potent inhibitor of autophagy and lysosomal function, exhibiting superior activity compared to the monomeric HCQ and the dimeric precursor Lys05. [1]
The molecular target of DC661, along with HCQ and Lys05, has been identified as PPT1, a lysosomal depalmitoylase. This reclassifies these chloroquine derivatives as targeted therapies rather than nonspecific lysosomal targeted therapies. [1]
Inhibition of PPT1 by DC661 disrupts the interaction between the Ragulator complex (via LAMTOR1/p18) and v-ATPase (via ATP6V1A), leading to the translocation of mTORC1 from the lysosomal membrane and inhibition of mTORC1 signaling. [1]
Analysis of Cancer Genome Atlas (TCGA) data showed that PPT1 expression was elevated in many tumor types compared to normal tissues and was associated with poorer overall survival in cancers such as esophageal cancer, hepatocellular carcinoma, clear cell renal cell carcinoma, and head and neck cancer. [1]
PPT1 is also a defective enzyme in the neurodegenerative disease infantile neuronal cerebrospinal deposition (INCL). Chloroquine derivatives have been observed to cause retinopathy with symptoms similar to some of the symptoms of INCL retinopathy, providing a mechanistic link to the known toxicity profile of these drugs. [1]

C31H39CL2N5

552.58

551.258

1872387-43-3

130467298

White to yellow solid powder

8.3

2

5

16

38

587

0

ClC1C=CC2C(C=1)=NC=CC=2NCCCCCCN(C)CCCCCCNC1C=CN=C2C=C(C=CC=12)Cl

VJKCWFZTSDXOBS-UHFFFAOYSA-N

InChI=1S/C31H39Cl2N5/c1-38(20-8-4-2-6-16-34-28-14-18-36-30-22-24(32)10-12-26(28)30)21-9-5-3-7-17-35-29-15-19-37-31-23-25(33)11-13-27(29)31/h10-15,18-19,22-23H,2-9,16-17,20-21H2,1H3,(H,34,36)(H,35,37)

N-(7-chloroquinolin-4-yl)-N'-[6-[(7-chloroquinolin-4-yl)amino]hexyl]-N'-methylhexane-1,6-diamine

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 : ~62.5 mg/mL (~113.11 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.76 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 20.8 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.)