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Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling


Chemical genomics aims to discover small molecules that affect biological processes through the perturbation of protein function1,2. However, determining the protein targets of bioactive compounds remains a formidable challenge3. We address this problem here through the creation of a natural product–inspired small-molecule library bearing protein-reactive elements. Cell-based screening identified a compound, MJE3, that inhibits breast cancer cell proliferation. In situ proteome reactivity profiling revealed that MJE3, but not other library members, covalently labeled the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), resulting in enzyme inhibition. Interestingly, MJE3 labeling and inhibition of PGAM1 were observed exclusively in intact cells. These results support the hypothesis that cancer cells depend on glycolysis for viability and promote PGAM1 as a potential therapeutic target. More generally, the incorporation of protein-reactive compounds into chemical genomics screens offers a means to discover targets of bioactive small molecules in living systems, thereby enabling downstream mechanistic investigations.

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Figure 1: A natural product-inspired library of protein-reactive chemical genomics probes.
Figure 2: Evaluation of the antiproliferation effects and in situ proteome reactivity profiles of spiroepoxide probes.
Figure 3: Identification and characterization of PGAM1 as the 26-kDa MJE3 target.
Figure 4: MJE3 inhibits PGAM1 activity in MDA-MB-231 cells.


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We thank J. Tamiya for valuable contributions to the initial design of the probe library and A. Speers for assistance with the identification of the probe labeling site on PGAM1. This work was supported by the National Institutes of Health grant CA087660 (to B.F.C.), the California Breast Cancer Research Foundation (B.F.C.), a Merck Fellowship of the Life Sciences Research Foundation (A.S.) and the Skaggs Institute for Chemical Biology.

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Correspondence to Benjamin F Cravatt.

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Supplementary information

Supplementary Fig. 1

Synthesis of a natural products-inspired spiroepoxide probe library. (PDF 168 kb)

Supplementary Fig. 2

Structures of the amine substituents of various MJE probes. (PDF 94 kb)

Supplementary Fig. 3

Evaluation of the anti-proliferation effects and in situ proteome reactivity profiles of spiroepoxide probes. (PDF 688 kb)

Supplementary Fig. 4

MJE3 inhibition of PGAM1 catalytic activity in living cells. (PDF 82 kb)

Supplementary Fig. 5

Characterization of the site of MJE3 labeling in PGAM1. (PDF 398 kb)

Supplementary Fig. 6

Structure-activity relationship of the anti-proliferative effects and PGAM1 labeling by MJE3. (PDF 195 kb)

Supplementary Methods (PDF 279 kb)

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Evans, M., Saghatelian, A., Sorensen, E. et al. Target discovery in small-molecule cell-based screens by in situ proteome reactivity profiling. Nat Biotechnol 23, 1303–1307 (2005).

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