The highly conserved p97 ATPase functions in endoplasmic reticulum-associated degradation (ERAD) by hydrolyzing ATP needed to export ubiquitinated substrates to the cytosol for degradation by the proteasome. Inhibition of p97 leads to unfolded protein accumulation, and ultimately, cell death. The discovery of p97 missense mutations in a genetic form of human dementia, in addition to the localization of p97 in ubiquitylated inclusions in affected neurons of amyotrophic lateral sclerosis and Parkinson's disease, and the overproduction of p97 in multiple cancers suggests that p97 has diverse and essential cellular roles. Thus, the identification of probes that selectively target p97 activity would be useful for providing insights into the biological roles of P97. The probe ML080 (CID-25110544) is the first cell active, reversible inhibitor of P97. This compound in the ATP-depletion-based assay, is cell penetrable as demonstrated by activity in the cell-based ubiquitin-GFP turnover assay.

Probe Structure & Characteristics

This compound CID-25110544 has a 2-amino thiazole core and met the probe definition criteria for this P97 Inhibitor project. This compound in the ATP-depletion-based assay, is cell penetrant as demonstrated by activity in the cell-based Ub-GFP turnover assay. The structure of the compound, CID-25110544 (CYM-5654), which met probe selection criteria, is indicated in the box below.

Recommendations for the scientific use of this probe

This compound is useful for biochemical and cell-based assays in which it is desirable to specifically block p97 activity. Inhibition of p97 leads to unfolded protein accumulation, and ultimately cell death. This probe is the first cell active, reversible inhibitor of P97.

1. Scientific Rationale for Project

Misfolded proteins accumulate in the endoplasmic reticulum (ER) in response to environmental stress(1). To reduce the burden these proteins place on the secretory pathway, eukaryotic cells have evolved a process, known as ER-associated degradation (ERAD), to recognize and eliminate these proteins (1, 2). The highly conserved p97 ATPase functions in ERAD by hydrolyzing ATP needed to export ubiquitinated substrates to the cytosol for degradation by the proteasome (2, 3). The discovery of p97 missense mutations in a genetic form of human dementia (4–6), the localization of p97 in ubiquitylated inclusions in affected neurons of amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (8–9), and the overproduction of p97 in multiple cancers (10–14), suggests that p97 has diverse and essential cellular roles. Thus, the identification of probes that selectively target p97 activity may provide insights into the biological roles of P97.

2. Project Description

3. Probe

b. Chemical structure

g. Detailed synthetic pathway for making probe

4. Appendices

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R1	R2	MLSMR SID	MLSMR CID	MLS	Vendor	IC50 (M) p97 activity	IC50 (M) Ub-GFP turnover
		56432228	25110136	None	Roberts Lab, TSRI	4.7e-6	NA
		56432230	25110138	None	Roberts Lab, TSRI	>50e-6	ND
		56432232	25110139	None	Roberts Lab, TSRI	>50e-6	ND
		56432234	25110140	None	Roberts Lab, TSRI	>50e-6	ND
		56432236	25110141	None	Roberts Lab, TSRI	>50e-6	ND
		56432238	25110143	None	Roberts Lab, TSRI	>50e-6	ND
		56432668	25110543	None	Roberts Lab, TSRI	3.7e-6	7e-6
		56432376	25110268	None	Roberts Lab, TSRI	>50e-6	ND

NA: Not Active, ND Not Determined

Appendix 1. Endoplasmic reticulum-associated degradation (ERAD) Assay (Ub-_G76V-GFP/HeLa Titration Assay)

In order to determine whether the identified p97 inhibitors were cell permeable and could inhibit endogenous p97 in vivo, ERAD assays were performed for selected compounds. Ub-_G76V-GFP/HeLa cells were treated with MG132 (2 μM) for 1h and washed with PBS three times. DMEM containing cycloheximide (50 μg/mL) and test compounds (0 ~ 10 μM) was added to wells. 8 of 96 well plates were prepared and one of the plates was imaged at 25, 50, 70, 100, 110,125, 145, or 170 minutes after washing with PBS 3 times.

5. Bibliography

1.

Raasi S, Wolf DH. Ubiquitin receptors and ERAD: a network of pathways to the proteasome. Seminars in cell & developmental biology. 2007;18:780–791. [PubMed: 17942349]

2.

Halawani D, Latterich M. p97: The cell’s molecular purgatory? Molecular cell. 2006;22:713–717. [PubMed: 16793541]

3.

Wang Q, Li L, Ye Y. Inhibition of p97-dependent protein degradation by Eeyarestatin I. The Journal of biological chemistry. 2008;283:7445–7454. [PMC free article: PMC2276333] [PubMed: 18199748]

4.

Mizuno Y, Hori S, Kakizuka A, Okamoto K. Vacuole-creating protein in neurodegenerative diseases in humans. Neuroscience letters. 2003;343:77–80. [PubMed: 12759168]

5.

Watts GD, Wymer J, Kovach MJ, Mehta SG, Mumm S, Darvish D, Pestronk A, Whyte MP, Kimonis VE. Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nature genetics. 2004;36:377–381. [PubMed: 15034582]

6.

Weihl CC, Dalal S, Pestronk A, Hanson PI. Inclusion body myopathy-associated mutations in p97/VCP impair endoplasmic reticulum-associated degradation. Human molecular genetics. 2006;15:189–199. [PubMed: 16321991]

7.

Tetko IV, Tanchuk VY, Kasheva TN, Villa AE. Estimation of aqueous solubility of chemical compounds using E-state indices. J Chem Inf Comput Sci. 2001;41:1488–1493. [PubMed: 11749573]

8.

Ishigaki S, Hishikawa N, Niwa J, Iemura S, Natsume T, Hori S, Kakizuka A, Tanaka K, Sobue G. Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders. J Biol Chem. 2004 2004 Dec 3;279(49:):51376–85. [PubMed: 15456787]

9.

Hirabayashi M, Inoue K, Tanaka K, Nakadate K, Ohsawa Y, Kamei Y, Popiel AH, Sinohara A, Iwamatsu A, Kimura Y, Uchiyama Y, Hori S, Kakizuka A. VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration. Cell Death Differ. 2001;8:977–984. [PubMed: 11598795]