Discovery of the novel autophagy inhibitor aumitin that targets mitochondrial complex I

Macroautophagy is a conserved eukaryotic process for degradation of cellular components in response to lack of nutrients. It is involved in the development of diseases, notably cancer and neurological disorders including Parkinson's disease. Small molecule autophagy modulators have proven to be valuable tools to dissect and interrogate this crucial metabolic pathway and are in high demand. Phenotypic screening for autophagy inhibitors led to the discovery of the novel autophagy inhibitor aumitin. Target identification and confirmation revealed that aumitin inhibits mitochondrial respiration by targeting complex I. We show that inhibition of autophagy by impairment of mitochondrial respiration is general for several mitochondrial inhibitors that target different mitochondrial complexes. Our findings highlight the importance of mitochondrial respiration for autophagy regulation.


SI-
: Structure activity relationship of the di-aminopyrimidines. Starvation = starvation induced autophagy assay; Rapamycin = Rapamycin induced autophagy assay; Viability = survival assessed by means of an ADP-glow assay. > 10 = no inhibition at a test concentration of 10 µM. Data is mean; SD = ± standard deviation; n ≥ 3 for all IC 50 Table 2: Biochemical Inhibition of kinases by Aumitin. The detection of biochemical inhibition of overall 419 kinases was carried out by Life Technologies. The screen were performed in three different assays formats: Adapta (activity based), Z-Lyte (activity based) and Lantha (binding based) at a concentration of 1 µM for Aumitin. Only PI4KB was inhibited more than 40 % in the screen and thus tested for in dose response. PIK3C2G was not tested in the initial screen but tested later on directly in dose response. [ Lantha NA Lantha NA Lantha NA Lantha NA Lantha NA DMPK 12 5 9 Lantha NA DYRK2 13 3 8 Lantha NA Lantha NA Lantha NA Lantha NA Lantha NA Lantha NA Lantha NA SI- Figure 1: Synthesis of Aumitin. The regioselective replacement of the 4-chloride substituent in the first reaction was achieved by changing the solvent to n-butanol. The synthesis of Aumitin started from commercially available 2,4-dichloro-6-methylpyrimidine, which was regioselectively reacted with aniline to replace the 4-chloro substituent. A screen of different bases and solvents revealed n-butanol as the superior solvent for the employment of DIPEA as the base. The second nucleophilic substitution on the pyrimidine occurred neatly with p-phenylendiamine in ethanol in the microwave. The amidecoupling succeeded following standard conditions using PyBOP.

Thawing and Freezing Cryo-Conserved Cells
Thawing of frozen cells was performed by transferring the vial with the cells into a 37 °C warm water bath. The thawed cells were then diluted with 10 mL of the respective medium and the resulting suspension was transferred into a T-75 flask.
The cells were incubated as described above. On the next day the DMSO-containing medium was replaced by DMSO-free medium.
For cryo-conservation of mammalian cells, these were grown until reaching 80-95%

Determination of Cell Number
The concentration or number of cells were determined with the help of the

Cell Treatment with Small Molecules
If not stated otherwise all small molecules were dissolved in DMSO (Bioreagent, Sigma Aldrich, cat# D8418). Cells were seeded into vessels in a cell number that allowed 75-90% confluence at the end of the experiment. Cells were incubated for 12-24 hours as described above and then treated with compounds, which were prediluted in the given medium. The final DMSO concentration did not exceed 0.5% (v/v).

Low-throughput Screening for Autophagy Inhibitors
The low-throughput version utilizes the same MCF7-eGFP-LC3 cell line as the above

Protein Concentration Determination
Routine determination of protein concentration was performed using the Bradford method with the Bradford reagent (Bio-Rad laboratories) in the linear range of a BSA control (0.3 -10 mg/mL). The absorbance was measured at 595 nm using a BioPhotometer (Eppendorf).
For a large number of samples or low protein concentrations, a DC protein assay (Bio-Rad laboratories, cat# 500-0116) was performed in 96-well plates according to the manufacturer's instructions. The absorbance was measured at 650-750 nm using a Tecan microplate reader (Tecan Infinite M200) and the concentration calculated using a BSA standard curve.

Detection of p62 conversion and LC3-I to LC3-II ratios by means of immunoblotting
200,000 MCF7-eGFP-LC3 cells in 2 ml media were seeded in 6-well plates and incubated (37 °C, 5% CO 2 ) overnight. The media was removed and the cells were

CellTiter-Glo® Luminescent Cell Viability Assay
The assay is a Luciferin based measurement of ATP-levels. The ATP-levels are considered to be proportional to the amount of viable and metabolically active cells.

Measurement of Mitochondrial Respiration
The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured by the Seahorse XFe96 analyzer (Agilent

Semi-intact Assay for Mitochondrial Respiration
The semi-intact assay for mitochondrial respiration was carried out by the established method 6 with some modifications in the concentrations of digitonin and substrates.
Afterwards the ethanol was removed in vacuo and the product used without further purification.

PIK3C2G inhibitors
All PIK3C2G inhibitors were synthesized as described in the