Tài liệu Báo cáo khoa học: Protein farnesyltransferase inhibitors interfere with farnesyl

Protein farnesyltransferase inhibitors interfere with farnesyl

diphosphate binding by rubber transferase

Christopher J. D. Mau1

, Sylvie Garneau2

, Andrew A. Scholte2

, Jennifer E. Van Fleet1

, John C. Vederas2

and Katrina Cornish1

1

USDA, Agricultural Research Service, Western Regional Research Center, Albany, CA, USA; 2

Department of Chemistry,

University of Alberta, Edmonton, Alberta, Canada

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 com￾pounds 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.

Keywords: Hevea brasiliensis; Parthenium argentatum;

chaetomellic acid A; hydroxyfarnesylphosphonic acid.

Rubber transferase catalyzes the biosynthesis of natural

rubber [1]. To form this polymer of cis-polyisoprene, rubber

transferase adds up to thousands of molecules of isopente￾nyl diphosphate (IPP) to a single initiating allylic diphos￾phate, usually considered to be farnesyl diphosphate (FPP,

1, Fig. 1) as the in vivo substrate. However, rubber

transferase can also use other allylic diphosphates as

initiators; this substrate flexibility is probably a reflection

on the manner in which the catalytic site deals with the

elongating rubber polymer. In addition, a divalent metal

cofactor, such as Mg2+, is required. In spite of the

dependence of modern industrial society on natural rubber,

the biochemical properties of rubber transferase are only

partially understood [1–6].

Several compounds are known to bind to FPP sites in

other enzymes that use FPP as a substrate. Most of these

substances have been discovered as a result of oncogenesis

studies involving protein farnesyltransferases. Chaetomel￾lic acid A (2) (Fig. 1), made by Chaetomella acutiseta, is an

inhibitor of protein farnesyltransferases (PFTs), such as

those that modify Ras, and competes for the FPP binding

site of PFTs with an IC50 of 55 nM [7]. Derivatives of

chaetomellic acid A have also been found to inhibit PFTs

[8]. a-Hydroxyfarnesylphosphonic acid (HFPA, 3) (Fig. 1)

is another compound shown to inhibit PFTs [9] with an

IC50 of 30 nM [7].

In an effort to characterize the FPP binding site of rubber

transferase, we have tested the ability of chaetomellic acid A

and several analogs, as well as HFPA, to inhibit rubber

biosynthesis in vitro. We have used rubber transferases from

Hevea brasiliensis and Parthenium argentatum to determine

if there are similarities in enzymatic behavior that might be

characteristic of rubber transferases in general, as well as

species-specific differences.

Materials and methods

Chemicals

Chemicals were purchased from Sigma Chemical Com￾pany unless otherwise noted. Farnesyl diphosphate (FPP),

dimethyl allyl diphosphate (DMAPP) and [1-14C]IPP (2.04

GBqÆmmol)1

) were purchased from American Radiolabe￾led Chemicals, Inc. (St. Louis, MO, USA). a-Hydroxy￾farnesylphosphonic acid (HFPA) was purchased from

Calbiochem-Novabiochem Corp. Washed rubber particles

(WRP) from P . argentatum and H. brasiliensis were

purified ([10,11], respectively) and stored in liquid nitrogen

[12].

Synthesis of chaetomellic acid A analogs

Several analogs of chaetomellic acid, purified as lithium

salts, were made according to Ratemi et al. [8]. The

structures of (Z)-2-octyl-3-methylbutenedioic acid dilithium

Correspondence to K. Cornish, Western Regional Research Center,

USDA-ARS 800 Buchanan Street, Albany, CA 94710, USA.

Fax: + 1 510 559 5663, Tel.: + 1 510 559 5950,

E-mail: [email protected]

Abbreviations: DMAPP, dimethyl allyl diphosphate; FPP, farnesyl

diphosphate; IPP, isopentenyl diphosphate; HFPA, a-hydroxyfarne￾sylphosphonic acid; PFT, protein farnesyltransferase; WRP, washed

rubber particles; UDP, undecaprenyl diphosphate.

(Received 5 June 2003, revised 8 July 2003, accepted 31 July 2003)

Eur. J. Biochem. 270, 3939–3945 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03775.x