The isoprenoid substrate specificity of isoprenylcysteine carboxylmethyltransferase: development of novel inhibitors

Abstract

Isoprenylcysteine carboxylmethyltransferase (Icmt) is an integral membrane protein localized to the endoplasmic reticulum of eukaryotic cells that catalyzes the post-translational alpha-carboxylmethylesterification of CAAX motif proteins, including the oncoprotein Ras. Prior to methylation, these protein substrates all contain an isoprenylcysteine residue at the C terminus. In this study, we developed a variety of substrates and inhibitors of Icmt that vary in the isoprene moiety in order to gain information about the nature of the lipophilic substrate binding site. These isoprenoid-modified analogs of the minimal Icmt substrate N-acetyl-S-farnesyl-L-cysteine (AFC) were synthesized from newly and previously prepared farnesol analogs. Using both yeast and human Icmt enzymes, these compounds were found to vary widely in their ability to act as substrates, supporting the isoprenoid moiety as a key substrate recognition element for Icmt. Compound 3 is a competitive inhibitor of overexpressed yeast Icmt (K(I) = 17.1 +/- 1.7 microm). Compound 4 shows a mix of competitive and uncompetitive inhibition for both the yeast and the human Icmt proteins (yeast K(IC) = 35.4 +/- 3.4 microm, K(IU) = 614.4 +/- 148 microm; human K(IC) = 119.3 +/- 18.1 microm, K(IU) = 377.2 +/- 42.5 microm). These data further suggest that differences in substrate specificity exist between the human and yeast enzymes. Biological studies suggest that inhibition of Icmt results in Ras mislocalization and loss of cellular transformation ability, making Icmt an attractive and novel anticancer target. Further elaboration of the lead compounds synthesized and assayed here may lead to clinically useful higher potency inhibitors.

Figure 1

(A) CaaX protein processing in eukaryotes. (B) Isoprene-modified analogs of N-acetyl-S-farnesylcysteine (AFC, 1). Compound 3, an isobutenyl derivative of AFC (1). Compound 4, an isobutenyl biphenyl derivative of AFC (1).

Figure 2

(A) Synthesis of compound 3. (B) Synthesis of compound 4.

Figure 3

(A) Expression level of CH2766 and CH2704 One μg total crude membrane protein from CH2704 (His₁₀myc₃N-Ste14p) and 2 μg total crude membrane protein from the empty vector negative control CH2714 (Δste14) and CH2766 (His₁₀myc₃N-hIcmt) crude were subjected to SDS-PAGE analysis and the proteins were visualized by immunodetection with an α-myc monoclonal antibody (1:10,000). Protein bands were visualized by enhanced chemiluminescence following incubation with an HRP conjugated secondary antibody (α-mouse 1:4000). (B) In vitro methyltransferase activity of Ste14p and hIcmt in crude membranes. Five μg total protein from crude membrane preparations were incubated with 20 μM ¹⁴C-SAM (S-adenosyl-L-[¹⁴C-methyl]methionine) and 200 μM AFC (N-acetyl-S-farnesyl-L-cysteine) for 30 min at 30°C and activity quantified by the in vitro vapor diffusion methyltransferase assay as described in Experimental Procedures. CH2714 is a Δste14 strain containing the empty vector, CH2704 is His₁₀myc₃N-Ste14p expressed behind the 3′-phosphoglycerate kinase promoter, and CH2766 is His₁₀myc₃N-hIcmt expressed behind the 3′-phosphoglycerate kinase promoter. Data are the average of three experiments done in duplicate. Error bars represent ± 1 SEM.

Figure 4. Activity of AFC Analogs as Icmt Substrates

(A) Total protein (5 μg) from the crude His-Ste14p membranes were incubated with 20 μM ¹⁴C-SAM (S-adenosyl-L-[¹⁴C-methyl]methionine) and increasing concentrations of compounds 1, 3, and 4 for 30 min at 30°C and activity quantified by the in vitro vapor diffusion methyltransferase assay as described in Experimental Procedures. (B) Total protein (5 μg) from the crude His-hIcmt membranes were incubated with 20 μM ¹⁴C-SAM (S-adenosyl-L-[¹⁴C-methyl]methionine) and increasing concentrations of compounds 1, 3, or 4 for 30 min at 30°C and activity quantified by the in vitro vapor diffusion methyltransferase assay as described in Experimental Procedures. Data are the average of three experiments done in duplicate. Error bars represent ± 1 SEM. For both panels, ■ – 1, ▼ – 3, ●– 4.

Figure 5. AFC analogs as purified His-Ste14p substrates

Purified His-Ste14p (0.089 μg) was reconstituted in 100 μg E. coli 100 nm liposomes in the presence of increasing concentrations of compounds 1, 3, or 4 as described in Experimental Procedures. Reactions were carried out for 30 min at 30°C following addition of 20 μM ¹⁴C-SAM (S-adenosyl-L-[¹⁴C-methyl]methionine). Activity was quantified by the in vitro vapor diffusion methyltransferase assay as described in Experimental Procedures. Data are the average of three experiments performed in duplicate. Error bars represent ± 1 SEM. ■ – 1, ● – 3, ▼ – 4.

Figure 6. AFC Analogs as Icmt Inhibitors

(A) Total protein (5 μg) from the crude His-Ste14p membranes were incubated with increasing concentrations of compounds 3 or 4, 33 μM AFC (N-acetyl-S-farnesyl-L-cysteine), and 20 μM ¹⁴C-SAM (S-adenosyl-L-[¹⁴C-methyl]methionine) for 30 min at 30°C and activity quantified by the in vitro vapor diffusion methyltransferase assay as described in Experimental Procedures. Data are the average of three experiments done in duplicate. Error bars represent ± 1 SEM. ■ – 3, ▼– 4. (B) Total protein (5 μg) from crude His-hIcmt membranes were incubated with increasing concentrations of compound 4, 33 μM AFC (N-acetyl-S-farnesyl-L-cysteine), and 20 μM ¹⁴C-SAM (S-adenosyl-L-[¹⁴C-methyl]methionine) for 30 min at 30°C and activity quantified by the in vitro vapor diffusion methyltransferase assay as described in Experimental Procedures. Data are the average of three experiments done in duplicate. Error bars represent ± 1 SEM. ■ – 4.

Figure 7. AFC analogs as purified His-Ste14p Inhibitors

Purified His-Ste14p (0.089 μg) was reconstituted in 100 μg E. coli 100 nm liposomes in the presence of increasing concentrations of compounds 3 or 4, 50 μM AFC (N-acetyl-S-farnesyl-L-cysteine), and 20 μM ¹⁴C-SAM (S-adenosyl-L-[¹⁴C-methyl]methionine) for 30 min at 30°C and activity quantified by the in vitro vapor diffusion methyltransferase assay as described in Experimental Procedures. Data are the average of three experiments done in duplicate. Error bars represent ± 1 SEM. ■ – 3, ▼ – 4.

Figure 8. Determination of K_I Values for Compounds 3 and 4

Activity was determined by the in vitro vapor diffusion methyltransferase assay as described in Experimental Procedures. AFC (N-acetyl-S-farnesyl-L-cysteine) substrate curves over a 0–200 μM range were performed in the presence of increasing concentrations of 3 or 4 as shown. (A) Lineweaver-Burk plot for compound 3 with crude His-Ste14p membranes. (B) Lineweaver-Burk plot for compound 4 with crude His-Ste14p membranes. (C) Lineweaver-Burk plot for compound 4 with crude His-hIcmt membranes.