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|Title: ||Synthesis and studies of enzyme inhibitors for protein prenylation|
|Other Titles: ||Mei yi wu xi hua yi zhi ji de he cheng yu yan jiu|
|Authors: ||Qiao, Yuqin (喬玉琴)|
|Department: ||Department of Biology and Chemistry|
|Degree: ||Doctor of Philosophy|
|Issue Date: ||2010|
|Publisher: ||City University of Hong Kong|
|Subjects: ||Enzyme inhibitors.|
|Notes: ||CityU Call Number: QP601.5 .Q25 2010|
xxvi, 271 leaves : ill. (some col.) 30 cm.
Thesis (Ph.D.)--City University of Hong Kong, 2010.
Includes bibliographical references (leaves 181-205)
|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.|
|Online Catalog Link: ||http://lib.cityu.edu.hk/record=b3947774|
|Appears in Collections:||BCH - Doctor of Philosophy |
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