QX77 is a novel and potent chaperone-mediated autophagy (CMA) activator. The lysosomal storage disease cystinosis, caused by cystinosin deficiency, is characterized by cell malfunction, tissue failure, and progressive renal injury despite cystine-depletion therapies. Cystinosis is associated with defects in chaperone-mediated autophagy (CMA), but the molecular mechanisms are incompletely understood. Treatment of cystinotic cells with a CMA activator increased LAMP2A localization at the lysosome and increased cell survival. Altogether, we show that LAMP2A trafficking is regulated by cystinosin, Rab11, and RILP and that CMA up-regulation is a potential clinically relevant mechanism to increase cell survival in cystinosis.

Physicochemical Properties

Molecular Formula	C16H13CLN2O2
Molecular Weight	300.739622831345
Exact Mass	300.066
CAS #	1798331-92-6
Related CAS #	1798331-92-6
PubChem CID	118129505
Appearance	Light yellow to yellow solid powder
LogP	2.9
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	2
Heavy Atom Count	21
Complexity	419
Defined Atom Stereocenter Count	0
SMILES	ClC1C=CC2=C(C=1)OCC(C1C=CC(=CC=1)NC(C)=O)=N2
InChi Key	PYCTUCLCTVWILY-UHFFFAOYSA-N
InChi Code	InChI=1S/C16H13ClN2O2/c1-10(20)18-13-5-2-11(3-6-13)15-9-21-16-8-12(17)4-7-14(16)19-15/h2-8H,9H2,1H3,(H,18,20)
Chemical Name	N-(4-(7-Chloro-2H-benzo[b][1,4]oxazin-3-yl)phenyl)acetamide
Synonyms	QX-77;QX 77;QX77
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	In Ctns-/- MEFs, QX77 (48 hours) increases the expression levels of Rab11 [1]. Rab11-positive vector vesicles restored the high-motility transport phenotype of wild-type cells, and QX77 restored downregulated LAMP2A expression in Ctns-/-MEFs [1]. In cystine cells, treatment with the CMA activator QX77 prevented Rab11 downregulation and trafficking abnormalities. Additionally, LAMP2A's location on the lysosomal membrane is enhanced by QX77 treatment [1]. QX77 corrects the location of LAMP2A on the lysosomal membrane of cystine cells and dramatically promotes its relocalization to LAMP1-positive lysosomes in cystine cells [1]. QX77 restores the levels of resistance seen in wild-type cells while shielding cystine cells from oxidative damage caused by tert-butyl hydroperoxide. LAMP2A expression is required for QX77 to have an impact on cystine cell survival [1]. In D3 and E14 ES cells, QX77 (10 μM; 0, 3 or 6 days) downregulates pluripotency factors and AP responsiveness, increases LAMP2A expression, activates CMA, and partially loses typical ES cell shape [2].
Cell Assay	Western Blot analysis [2] Cell Types: ES D3 cell line; E14TG2a (E14) Cell line Tested Concentrations: 10 μM Incubation Duration: 3 or 6 days Experimental Results: LAMP2A expression increased, SOX2 and Oct4 protein expression diminished.
References	[1]. Cystinosin, the small GTPase Rab11, and the Rab7 effector RILP regulate intracellular trafficking of the chaperone-mediated autophagy receptor LAMP2A. J Biol Chem. 2017 Jun 23;292(25):10328-10346. [2]. Chaperone-mediated autophagy regulates the pluripotency of embryonic stem cells. Science. 2020 Jul 24;369(6502):397-403.

Solubility Data

Solubility (In Vitro)	DMSO:≥ 60mg/mL Water:N/A Ethanol:N/A
Solubility (In Vivo)	Solubility in Formulation 1: 10 mg/mL (33.25 mM) in 50% PEG300 +50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.3251 mL	16.6257 mL	33.2513 mL
	5 mM	0.6650 mL	3.3251 mL	6.6503 mL
	10 mM	0.3325 mL	1.6626 mL	3.3251 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.