Phosphonocarboxylates Inhibit the Second Geranylgeranyl Addition by Rab Geranylgeranyl Transferase

Abstract

Rab geranylgeranyl transferase (RGGT) catalyzes the posttranslational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((؉)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC 50 and K i . (؉)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (؉)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (؉)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases.Most proteins of the Ras-like GTPase superfamily need to be post-translationally modified by prenyl groups to associate with cellular membranes and to activate downstream effectors (1). Protein prenylation involves the formation of a thioether link between conserved C-terminal cysteines in protein substrates and farnesyl pyrophosphate (FPP) 7 or geranylgeranyl pyrophosphate (GGPP) (2, 3). These prenyl pyrophosphates, which originate from the mevalonate pathway are utilized by three different prenyltransferase enzymes (2, 3). Farnesyl transferase (FT) and geranylgeranyl transferase type I (GGT-I) transfer FPP or GGPP, respectively, to a cysteine residue in the context of a C-terminal CAAX motif, where C is a cysteine, A is an aliphatic residue, and X is any amino acid. X contributes significantly to substrate specificity in FT and GGT-I (2). Rab geranylgeranyl transferase (RGGT) is distinct from FT and GGT-I in that it specifically recognizes Rab proteins, which vary in their C terminus containing CCXX, XCXC, XXCC, CCXXX, CAAX, and other motifs (3). RGGT exhibits exquisite specificity for Rabs due to the strict requirement of Rab escort protein (REP), which is a general Rab-GDP binding protein (4). RGGT is a heterodimeric enzyme ...

RAB26 and RAB3D Are Direct Transcriptional Targets of MIST1 That Regulate Exocrine Granule Maturation

“…We transfected HGC-MIST1 cells with PGC-RFP in the presence of 5 mM 3-PEHPC (3,10), an inhibitor of RAB geranylgeranyl transferase (RGGT), the enzyme that catalyzes the addition of geranylgeranyl groups to the carboxy-terminal dicysteine (CC or CXC) motifs in RAB3D and RAB26 (55). We used the minimal concentration of 3-PEHPC that completely dispersed the normal cellular localization of an eGFP-RAB26 construct.…”

Prenyltransferase inhibitors: treating human ailments from cancer to parasitic infections

“…72 Substitution of the pyridine ring structure in 3-PEHPC with a imidazo[1,2-a]pyridine cyclic moiety led to the most potent inhibitor of RabGGTase in this compound class, with an IC 50 value of 1.3 μM, nearly 25-fold better than the initial inhibitor structure. 73 It was recently discovered that this class of inhibitors inhibits only the second geranylgeranylation step of RabGGTase, not the first, and it was suggested that the phosphonocarboxylate inhibitors bind in a site adjacent to the active site, preventing the mono-geranylgeranylated protein from translocating to provide room for the second prenylation step to occur. 73 Some Rab proteins are only mono-geranylgeranylated and as a consequence are not inhibited by the phosphonocarboxylates; the development of specific inhibitors of all Rab protein geranylgeranylation has become a high priority.…”

“…73 Additionally, the (+)-enantiomer of 3-IPEHPC has been found to be more potent than the (−)-enantiomer; however, the more active absolute configuration for the remaining bisphosphonates has yet to be determined. 73 …”

N-Oxide derivatives of 3-(3-pyridyl)-2-phosphonopropanoic acids as potential inhibitors of Rab geranylgeranylation

“…16 In contrast the phosphono carboxylate 4 , also known as IPEHPC and viewed as an analogue of the bisphosphonate minodronate ( 5 ), 12,17 shows significantly greater potency even as the racemate, and the (+)-enantiomer 4 is ~60-fold more active than the (−)-enantiomer. 15 However, the (+)-enantiomer also shows weak inhibition of at least one other enzyme involved in isoprenoid biosynthesis, probably geranylgeranyl diphosphate synthase (GGDPS). 16 …”