Fig 1.
ENTPD5 is an ER-resident UDPase important for proper glycoprotein folding.
Schematic diagram highlighting the role ENTPD5 plays in the glycoprotein refolding cycle in the ER. For proper glycoprotein folding to occur, UDP-glucose is brought into the ER by an antiporter that uses UMP as the counter molecule. ENTPD5 activity produces UMP, leading to increased levels of UDP-glucose entering the ER for glycoprotein refolding. ENTPD5 expression is upregulated through two independent pathways: PI3K-AKT axis signaling and mutant p53 interactions.
Fig 2.
ENTPD5 construct, purification, and activity.
A) Diagram of ENTPD5 construct used for recombinant protein production, highlighting the expected internal disulfide bonds. B) SDS-PAGE gel of ENTPD5 purified from insect cells (lane I) and from bacteria (lane B). Molecular weight marker (lane MW) sizes in kDa are shown to the left of the gel. BSA, the additional band in lane B at 66 kDa, was added post-purification to stabilize B.ENTPD5. C) Gel filtration traces of recombinant ENTPD5 proteins. D) Michaelis-Menten plot of purified I.ENTPD5 activity with substrate UDP using the Malachite Green assay. Km: 480 ± 111 μM; kcat: 783 ± 68 s-1. Reactions were performed in triplicate. Error bars designate standard deviation.
Fig 3.
ENTPD5 HTS assay and screening results.
A) Schematic of ENTPD5 HTS assay. Following a 1-hour coupled reaction of ENTPD5 and UMPK, the residual ATP is measured indirectly using luciferase. B) Replicate plot of ENTPD5 HTS, showing percent ENTPD5 inhibition by compounds in each replicate set. Compounds were screened at 20 μM in duplicate. Compounds producing >25% inhibition (blue shaded box in B) were selected for follow-up analysis and are shown in C). Percent ENTPD5 inhibition by each hit is shown in parentheses.
Fig 4.
Structure activity relationship of select ENTPD5 inhibitors identified by HTS.
Close analogs of hits 1a (A) and 2a (B) were assayed with B.ENTPD5 to assess the effect of conservative substitutions to the scaffolds. Compounds were assayed at 5, 10, 20, and 40 μM using the coupled enzyme HTS assay to determine IC50 values. Data were fit by nonlinear regression.
Fig 5.
ENTPD5 HTS hit characterization.
A) Structures of compounds 1a, 1b, and 2f. B) ENTPD5 inhibition by compounds 1a, 1b, and 2f using the MG assay. 1a IC50: 3.1 ± 1.4 μM; 1b EC50 >100 μM; 2f EC50: 1.5 ± 1.3 μM. Error bars represent standard deviation from triplicate measurements.
Fig 6.
Treatment of prostate cancer cell lines with ENTPD5 inhibitors.
A) Growth of LNCaP cells treated with compound 1a, 1b, or 2f for 48h. 1a EC50: 0.47 ± 1.28 μM; 1b EC50: 12.9 ± 3.3 μM; 2f EC50: 24 ± 3 μM. B) Growth of LNCaP and DU145 cells treated with compound 1a for 48h. 1a EC50 in DU145: 3.6 ± 1.2 μM; 1a EC50 in LNCaP: 0.47 ± 1.28 μM. Relative cell count was normalized to DMSO-treated cell counts. Error bars represent standard error of the mean between three independent experiments.
Fig 7.
O-glycan levels of cells treated with ENTPD5 inhibitors.
A) Baseline levels of ENTPD5, PTEN, Sp1, and O-glycans in untreated LNCaP and DU145 cells. B) Levels of O-glycan in LNCaP and DU145 cells treated with 10 μM 1a or 2f for 24h. Relative protein amounts were normalized to DMSO-treated cells from each cell line. Error bars represent standard deviation between 2 experiments.