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. 2016 Jan 15;24(2):160-78.
doi: 10.1016/j.bmc.2015.11.043. Epub 2015 Nov 30.

8-Hydroxyquinoline-based inhibitors of the Rce1 protease disrupt Ras membrane localization in human cells

Idrees Mohammed  1 , Shahienaz E Hampton  1 , Louise Ashall  1 , Emily R Hildebrandt  2 , Robert A Kutlik  3 , Surya P Manandhar  2 , Brandon J Floyd  3 , Haley E Smith  1 , Jonathan K Dozier  4 , Mark D Distefano  4 , Walter K Schmidt  2 , Timothy M Dore  5
Affiliations

8-Hydroxyquinoline-based inhibitors of the Rce1 protease disrupt Ras membrane localization in human cells

Idrees Mohammed et al. Bioorg Med Chem. .

Abstract

Ras converting enzyme 1 (Rce1) is an endoprotease that catalyzes processing of the C-terminus of Ras protein by removing -aaX from the CaaX motif. The activity of Rce1 is crucial for proper localization of Ras to the plasma membrane where it functions. Ras is responsible for transmitting signals related to cell proliferation, cell cycle progression, and apoptosis. The disregulation of these pathways due to constitutively active oncogenic Ras can ultimately lead to cancer. Ras, its effectors and regulators, and the enzymes that are involved in its maturation process are all targets for anti-cancer therapeutics. Key enzymes required for Ras maturation and localization are the farnesyltransferase (FTase), Rce1, and isoprenylcysteine carboxyl methyltransferase (ICMT). Among these proteins, the physiological role of Rce1 in regulating Ras and other CaaX proteins has not been fully explored. Small-molecule inhibitors of Rce1 could be useful as chemical biology tools to understand further the downstream impact of Rce1 on Ras function and serve as potential leads for cancer therapeutics. Structure-activity relationship (SAR) analysis of a previously reported Rce1 inhibitor, NSC1011, has been performed to generate a new library of Rce1 inhibitors. The new inhibitors caused a reduction in Rce1 in vitro activity, exhibited low cell toxicity, and induced mislocalization of EGFP-Ras from the plasma membrane in human colon carcinoma cells giving rise to a phenotype similar to that observed with siRNA knockdowns of Rce1 expression. Several of the new inhibitors were more effective at mislocalizing K-Ras compared to a potent farnesyltransferase inhibitor (FTI), which is significant because of the preponderance of K-Ras mutations in cancer.

Keywords: Protease inhibitors; Ras converting enzyme (Rce1); Ras mislocalization.

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Figures

Figure 1
Figure 1. NSC1011 (1) and ring identification used to describe the SAR
Figure 2
Figure 2. HsRce1 IC50 values
Inhibitors were evaluated using the fluorescence-based CaaX proteolysis assay measuring the percent activity remaining.
Figure 3
Figure 3. Rce1 inhibitor toxicity in human cells
Compounds were added to cells at 25 μM then incubated for 20 hours. UT = untreated, DMSO = DMSO (0.4%) treated cells. Cell viability was measured using the CellTiter-Blue assay (Promega). Each column is the average of three independent experiments. Error bars indicate SEM.
Figure 4
Figure 4. Rce1 siRNA knock-down mislocalizes EGFP-Ras isoforms in human cells
(A) HCT-116 cells were transfected with either control (CT) or Rce1 siRNA and with either EGFP-H-, N-, or K-Ras. Cells were imaged by confocal microscopy 24 hours post-transfection. Images show representative cells. Scale bars = 10 μm. (B) Quantification of EGFP-H-, N-, and K-Ras mislocalization (see Experimental Section for method). ∼30 cells per condition were analyzed. (C) HCT-116 cells were transfected with either CT or Rce1 siRNA and with either EGFP-H-, N-, or K-Ras. Cells were imaged by confocal microscopy 24 hours post-transfection, then lysed for mRNA analysis by semi-quantitative RT-PCR for Rce1 and cyclophilin A expression. Graphs report the densitometric analysis of the gels.
Figure 5
Figure 5. Rce1 inhibitors mislocalize H-, N-, and K-Ras in human cells
(A) HCT-116 cells were transiently transfected with EGFP fusion constructs of H-, N-, or K-Ras, and treated with either DMSO, FTI (2 μM), or Rce1 inhibitors (25 μM) for 20 hours. Images show representative cells. Scale bars = 10 μm. (B-D) Quantification of H-, N-, and K-Ras mislocalization (see supporting information for method). Percent PM association is reported with SEM bars.
Figure 6
Figure 6. Efficacies of Rce1 inhibitors for Ste24 and FTase
A) Compounds were evaluated at 10 μM using the fluorescence-based CaaX proteolysis assay measuring the percent activity remaining. Yeast cells expressed either HsRce1 or HsSte24. Error bars show SD. B) Indicated compounds were evaluated at 10, 25, and 50 μM using the fluorescence-based FTase assay. Error bars show SD.
Scheme 1
Scheme 1. General synthetic approach to NSC1011 analogsa
aReagents and conditions: (i) See the experimental section for details of various reaction conditions.
Scheme 2
Scheme 2. Synthesis of intermediates 26a and 39aa
aReagents and conditions: (i) K2CO3, cat. KI, acetone, reflux; (ii) PPh3, DIAD, THF, 0 °C to rt, 1 hr.
Scheme 3
Scheme 3. Synthetic approach to 43a
aReagents and conditions: (i) neat, 60 °C; (ii) n-BuLi, THF, -78 °C; (iii) 43a, THF, -78 °C-rt, 3 h.
Scheme 4
Scheme 4. Synthesis of analogs 48-58
Reagents and conditions: (i) (a) i. TiCl4, DCM; 0 °C - rt; (b) NaCNBH3, MeOH; (ii), 5N NaOH (aq), dioxane/EtOH, rt; (iii) R-NH2, COMU, DIPEA, DMF, 0 °C - rt
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