Synthesis and studies of enzyme inhibitors for protein prenylation

Abstract

A significant number of proteins including members of the Ras superfamily, which contain a CAAX motif (C denotes cysteine, A represents any aliphatic amino acid and X may be any amino acid) in their carboxyl terminus, undergo extensive posttranslational modification especially prenylation. Prenylation or isoprenylation regulates specific protein-protein interaction, protects the proteins from proteolytic degradation, and more importantly facilitates membrane attachment and determines their subcellular localization and function. The dysfunction of these proteins often leads to disease and the best-known are Ras proteins which have been reported to be mutated in different types of cancer. Mutational activation of Ras occurs in approximately 20% of human cancers. Consequently, the prenyltransferase can be targeted for pharmacological intervention to block transformation by Ras oncoproteins, and the inhibitors of prenyltransferase are sought because of their potential as chemotherapeutic agents. The prenylation (farnesylation and geranylgeranylation, respectively) includes the covalent attachment of a non-sterol isoprenoid from either farnesylpyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) to the cysteine residue of the CAAX motif by protein farnesyltransferase (FTase) and geranylgeranyltransferase type-I (GGTase-I) respectively. Both of the enzymes are heterodimers and catalyze the thio-ether bond formation with the involvement of an essential zinc ion. Whereas FTase recognizes proteins with CAAX motifs in which X is a serine, methionine, alanine, or glutamine residue, GGTase-I favors CAAX motifs in which the carboxy-terminal residue is a leucine. However, enzyme specificity is not absolutely stringent, and there are abundant examples of protein cross-prenylation. Farnesyltransferase inhibitors (FTIs) have been the subject of intensive development in the past decade and have shown efficacy in clinical trials. Most inhibitors described in the literature are highly selective over GGTase-I. Although several FTIs were tested in clinical trials, the results were negative for all of them. Cross-prenylation of KRAS and NRAS has been the most consistently cited reason for this clinical outcome. Overall, the antitumor activity of FTIs as single agents in solid tumors was far less than anticipated. Besides that, studies have revealed that administration of GGTI to cells can cause cell cycle arrest at G0/G1, which has led to increased interest in therapeutic targeting of GGTase-I. Indeed, most GGTIs also show high selectivity over FTase. Despite their potent antiproliferative and pro-apoptotic properties in both in vitro and in vivo models, clinical development of GGTIs has been problematic owing to toxicity. To overcome the side effects of FTIs and GGTIs, compounds that simultaneously inhibit FTase and GGTase-I (dual prenylation inhibitors, DPIs) have been developed. This strategy takes into consideration several lines of evidence that suggest a synergistic antigrowth activity of combining FTase and GGTase-I inhibition on tumor cell lines. To the best knowledge, development of DPIs remains challenging. According to the reports, there may be also a synergistic activity in combinations of statins with FTIs. Statin/FTI combinations can induce a more effective inhibition of prenylation modifications by both decreasing isoprenoid levels and by inhibiting geranylgeranylation and farnesylation. It is feasible to evaluate and find the better combinations of these agents exhibiting anticancer activity. The objective of this study is to design, synthesize new dual prenyltransferase inhibitors which could inhibit both FTase and GGTase-I simultaneously, and to study their ability on regulating cell proliferation and apoptosis to different cancer cell lines. In the present study, various potential DPIs based on the core of piperazinedione which plays an important role in hydrogen bonding with active site were designed and synthesized. According to the synthesis route, I totally synthesized 48 new compounds, which were divided into three types. Biological activity test of these compounds showed that (1) Most of compounds displayed moderate inhibition for both enzymes and gave better inhibition for FTase than GGTase-I. (2) Compounds 85 and 87 exhibited best inhibition for both of enzymes and IC50 values are 17.8 nM and 13.1 nM for FTase, 32.3 nM and 21.0 nM for GGTase-I respectively. Then Enzyme kinetic studies were carried out to investigate the relationship of inhibitors with substrates. The result showed that compounds 85 and 87 are competitive bisubstrate potential DPIs. To predict the structure and orientation of the DPIs in the binding cavity of these two enzymes, the binding interactions between small molecule inhibitors and enzymes are also investigated by using the computer molecular docking simulation. Molecular docking analysis illustrated that these compounds bind to the active site of the enzymes in a favorable position via hydrogen bonds, metal ions, hydrophobic and van der Waals interactions with the amino acid residues. These results indicated that the active compounds are likely to be competitive inhibitors. Characterization of enzyme inhibitors through kinetic studies is in good agreement with the predicted binding mode based on simulation study. The inhibitors were also evaluated against different cancer cell lines using MTT assay methods with high-throughput screening (HTS). Most of compounds showed better antiproliferation ability and induced more apoptosis to MCF-7 than other cell lines. Compounds 85 and 87 show best inhibition for the four cancer lines, which was in good agreement with the in vitro test. The IC50 values for 85 are 12.9 nM (MCF-7), 26.5 nM (HepG2), 29.4 nM (Hela) and 34.7 nM (DU-145). The IC50 values for 87 are very close to those of compound 85. Besides, α subunit and β subunit of the two enzymes were cloned successfully. However, their expression and purification need further exploration due to the formation of inclusion bodies. In conclusion, a series of dual prenyltransferase inhibitors based on piperazinedione analogs were designed and synthesized, which show potent inhibition for both enzymes. In vitro and In vivo tests of these compounds have been studied. Cell survival assay with new piperazinedione analogs demonstrated their potential usage for cancer treatment.

Date of Award	15 Jul 2010
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Zhengtao XU (Supervisor), Ding LI (Supervisor) & Kam Wing Kenneth LO (Supervisor)