A.N. Matralis et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
useful mechanisms for the treatment of some age-related cancers (e.g.
multiple myeloma)16 and tau-related pathologies, such as the Alzhei-
mer’s disease.17 In this study, we report the design of a new class of
compounds that induce intracellular accumulation of farnesylated
prelamin A in human cancer cells, including osteosarcoma (U-2 OS),
pancreatic adenocarcinoma (HPAF-II, SW1990, KP-4) and colon carci-
noma (HCT-116). Furthermore, a phosphinyl derivative was identified,
which not only induces accumulation of prelamin A in one of the most
highly tumorigenic pancreatic cancer cell lines (KP-4), but it also de-
creases the transmigration potential of these cells, without inducing any
significant toxicity to either the KP-4 cells or normal fibroblasts (IMR-
90). Optimization of this class of compounds could plausibly lead to the
identification of novel antitumor agents that can block both cancer cells
proliferation and migration/metastasis.
2. Results and discussion
To date, selective inhibitors of ZMPSTE24 have not been identified.
Structural information that could potentially guide the design of in-
hibitors for this target is also limited to only two structures of the
human recombinant enzyme2,18 and one of the S. cerevisiae19 enzyme.
Unfortunately, these structures provide insufficient information about
the molecular recognition elements that dictate binding of ligands to
the active site of ZMPSTE24 (i.e. high resolution data at the atomic level
of ZMPSTE24/ligand co-crystal structures). Consequently, these struc-
tures cannot effectively guide structure-based drug design. Identifica-
tion of hits via high throughput screening (HTS) has not been reported
for this target; it is anticipated that such efforts would be very chal-
lenging due to the biophysical nature of this protein. ZMPSTE24 is a
helical barrel structure,2,18 thus requiring high concentrations of de-
tergents and/or protein-lipid micelle-bound protein preparations in
order to be rendered soluble. Such conditions are not ideal for HTS nor
structure-activity relationship (SAR) studies, as in vitro testing would be
highly prone to false positive/negative results. To date, biochemical
studies on the inhibition of ZMPSTE24 have been conducted primarily
using indirect inhibition assays, most often coupled to the methylation
step catalyzed by ICMT, rather than direct in vitro assays of a soluble
form of the human recombinant ZMPSTE24. Therefore, the binding
affinity of any ligand for this target and the details on its interactions
with ZMPSTEP24 remain unclear.
Figure 1. Biogenesis of mature lamin A from prelamin A; key enzymes involved
in the last three steps of the post-translational modifications of prelamin A are
indicated, particularly the role of the zinc metalloprotease STE24 (ZMPSTE24),
which is highlighted in blue. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
Figure 2. Inhibitors of isoprenoid biosynthesis and/or protein prenylation;
pravastatin (1), zoledronic acid (2), and lonafarnib (3) inhibiting HMG-CoA
reductase, farnesyl pyrophosphate synthase (hFPPS), farnesyl transferase en-
zyme (FTPase), respectively.
that specifically target the mevalonate pathway, inhibiting HMG-CoA
reductase and farnesyl pyrophosphate synthase (hFPPS), respectively.
These drugs block isoprenoid biosynthesis and consequently, farnesy-
lation of prelamin A. Similarly, inhibition of the farnesyl transferase
enzyme (FTPase) with lonafarnib (3) in monotherapy,12 or in combi-
nation with 1 and 2 have been shown to ameliorate some aspects of the
cardiovascular and bone diseases afflicting children with HGPS.13 In-
terestingly, cancers are extremely rare in children with HGPS, despite
their accelerated aging and the typically expected increase in cancer
rates as a consequence of aging.14 A possible explanation for this ob-
servation could be that children with HGPS do not live long enough to
provide statistically reliable epidemiology. Alternatively, it has been
proposed that the accumulation of prenylated prelamin A may lead to
impaired cell cycle progression that halts cancer progression in these
children. In support of this hypothesis, genetically engineered Zmpste24
knockout mice were shown to be resistant to cancer invasion.15 Col-
lectively, all recent biochemical findings suggest that inhibition of
ZMPSTE24 will block maturation of lamin A and induce cell senes-
cence, providing a novel mechanism for the treatment of cancer.
However, in the absence of potent and selective inhibitors of
ZMPSTE24, validation of this enzyme as a therapeutic target remains a
speculation.
HIV protease inhibitors (HIV PIs), such as lopinavir (4), ritonavir
(5)18,20 and atazanavir (6),18,21 have been reported to weakly inhibit
ZMPSTE24, leading to the accumulation of prelamin A in cellular assays
(Fig. 3, compounds 4–6). It is noteworthy that clinical observations
contradictory to these findings.22 However, this discrepancy may be
due to the low affinity of these compounds for ZMPSTE24, their high
protein binding and the differences between protein concentrations in
cultured cells versus human plasma. Inhibition of ZMPSTE24 with the
only non-peptidic HIV PI, tipranavir18 and accumulation of prelamin A
I) inhibitors have also been reported.23 However, the structural features
of these latter compounds deviate significantly from those of the pep-
tidomimetics 4–6.
We initiated our medicinal chemistry efforts with the knowledge
that aspartate proteases (e.g. HIV protease) and the zinc metallopro-
teases (e.g. ZMPSTE24, thermolysin, angiotensin converting enzyme
and others) share a common mechanistic action. Both families of en-
zymes use a water molecule as the nucleophile that attacks the scissile
amide bond. This is in contrast to the serine/cysteine proteases, which
use the side chain of a nucleophilic amino acid residue to initiate amide
bond hydrolysis. Based on the mechanistic similarities of aspartate
proteases and the zinc metalloprotease, we designed a minimal con-
sensus scaffold of well-known inhibitors of such enzymes (Fig. 3). A
common structural motif of the peptidomimetic HIV PIs is the central
Over the last few years, we have been investigating the association
between age-related diseases and isoprenoid metabolism in mammals,
including humans. For example, we recently showed that down-
regulation of the intracellular levels of farnesyl pyrophosphate (FPP)
and geranylgeranyl pyrophosphate (GGPP) can be therapeutically
2