Protein farnesyltransferase inhibitors interfere with farnesyl diphosphate binding by rubber transferase

Abstract

Rubber transferase, a cis-prenyltransferase, catalyzes the addition of thousands of isopentenyl diphosphate (IPP) molecules to an allylic diphosphate initiator, such as farnesyl diphosphate (FPP, 1), in the presence of a divalent metal cofactor. In an effort to characterize the catalytic site of rubber transferase, the effects of two types of protein farnesyltransferase inhibitors, several chaetomellic acid A analogs (2, 4-7) and a-hydroxyfarnesylphosphonic acid (3), on the ability of rubber transferase to add IPP to the allylic diphosphate initiator were determined. Both types of compounds inhibited the activity of rubber transferases from Hevea brasiliensis and Parthenium argentatum, but there were species-specific differences in the inhibition of rubber transferases by these compounds. Several shorter analogs of chaetomellic acid A did not inhibit rubber transferase activity, even though the analogs contained chemical features that are present in an elongating rubber molecule. These results indicate that the initiator-binding site in rubber transferase shares similar features to FPP binding sites in other enzymes.

Initiation of rubber biosynthesis: In vitro comparisons of benzophenone-modified diphosphate analogues in three rubber-producing species

“…Based on this possibility and the results presented herein, high-affinity inhibitors may be preferred to substrates as probe molecules. Rubber transferases are effectively inhibited by protein farnesyltransferase inhibitors in vitro with species-specific differences (Mau et al, 2003). Interspecies differences, observed here as well, suggest that selection of probes for photo-labeling experiments might be tailored to the species for best results.…”

“…As noted in earlier work with natural isoprenoid diphosphates (Cornish and Siler, 1995;Cornish et al, 1998) our results with Bz-modified initiators demonstrate that biosynthetic rate increases with increasing length of allylic pyrophosphate molecule. This has been shown to be true even for protein farnesyltransferase inhibitors (Mau et al, 2003) where lengthening the hydrophobic moiety of analogues increased the binding affinity of the inhibitor for the substrate binding site. We demonstrate that additional structural or stereochemical factors also play a role in recognition by rubber transferases.…”

“…Photoaffinity labeling studies have been used to identify binding regions in specific enzymes and to isolate a number of previously unidentified proteins, (Yokoyama et al, 1995;Gaon et al, 1996a;Turek et al, 1997Turek et al, , 2001Zhang et al, 1988Zhang et al, , 2004Webb et al, 1999) including protein prenyltransferases (Omer et al, 1993;Bukhtiyarov et al, 1995;Edelstein and Distefano, 1997) with remarkable specificity (Dorman and Prestwich, 1994). FPP binding to the rubber transferase active site occurs in a similar manner to the FPP-requiring enzymes mentioned above (Mau et al, 2003). Indeed, we have previously employed a Bz-containing inhibitor of rubber synthesis to label proteins found in enzymatically active rubber particles suggesting that this could be a valuable approach (DeGraw et al, 2007).…”

Transcriptome and gene expression analysis in cold-acclimated guayule (Parthenium argentatum) rubber-producing tissue

“…Transcripts encoding other proteins related to rubber biosynthesis (Table 3) were identified in guayule using the Hevea sequences for comparison, including farnesyl pyrophosphate synthase (FPPS), which synthesizes FPP, an initiator of rubber biosynthesis both in vivo and in vitro (Tanaka et al, 1996;Castillon and Cornish, 1999;Mau et al, 2003). Another Hevea protein homolog identified in our EST-deduced protein sequences, although not at as high abundance as in Hevea, is SRPP.…”

Novel route to chaetomellic acid A and analogues: Serendipitous discovery of a more competent FTase inhibitor

“…13 Vederas, instead, in an effort to characterize the FPP binding site of rubber transferase showed that 1 is able to inhibit, in vitro , rubber biosynthesis promoted by rubber transferase from Hevea Brasiliensis . 14 Besides, the same author found that 1 was also able to reduce the activity of the PBP1b (penicillin-binding protein 1b), albeit with modest potency. 15 Recently, Sabbatini has shown that the inhibition of the Ras/ERK1/2 pathway by 1 resulted in a beneficial effect on acute ischemia-reperfusion injury in rats, preserving either renal function and histology.…”