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Critical Reviews in Biochemistry and Molecular Biology
ISSN: 1040-9238 (Print) 1549-7798 (Online) Journal homepage: http://www.tandfonline.com/loi/ibmg20
Rce1: mechanism and inhibition
Shahienaz E. Hampton, Timothy M. Dore & Walter K. Schmidt
To cite this article: Shahienaz E. Hampton, Timothy M. Dore & Walter K. Schmidt (2018) Rce1:
mechanism and inhibition, Critical Reviews in Biochemistry and Molecular Biology, 53:2, 157-174,
DOI: 10.1080/10409238.2018.1431606
To link to this article: https://doi.org/10.1080/10409238.2018.1431606
© 2018 The Author(s). Published by Informa
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REVIEW ARTICLE
Rce1: mechanism and inhibition
Shahienaz E. Hampton
a
, Timothy M. Dore
a,b
and Walter K. Schmidt
c
a
New York University Abu Dhabi, Abu Dhabi, United Arab Emirates;
b
Department of Chemistry, University of Georgia, Athens, GA,
USA;
c
Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA, USA
ABSTRACT
Ras converting enzyme 1 (Rce1) is an integral membrane endoprotease localized to the endoplas-
mic reticulum that mediates the cleavage of the carboxyl-terminal three amino acids from CaaX
proteins, whose members play important roles in cell signaling processes. Examples include the
Ras family of small GTPases, the c-subunit of heterotrimeric GTPases, nuclear lamins, and protein
kinases and phosphatases. CaaX proteins, especially Ras, have been implicated in cancer, and
understanding the post-translational modifications of CaaX proteins would provide insight into
their biological function and regulation. Many proteolytic mechanisms have been proposed for
Rce1, but sequence alignment, mutational studies, topology, and recent crystallographic data
point to a novel mechanism involving a glutamate-activated water and an oxyanion hole. Studies
using in vivo and in vitro reporters of Rce1 activity have revealed that the enzyme cleaves only
prenylated substrates and the identity of the a
2
amino residue in the Ca
1
a
2
X sequence is most
critical for recognition, preferring Ile, Leu, or Val. Substrate mimetics can be somewhat effective
inhibitors of Rce1 in vitro. Small-molecule inhibitor discovery is currently limited by the lack of
structural information on a eukaryotic enzyme, but a set of 8-hydroxyquinoline derivatives has
demonstrated an ability to mislocalize all three mammalian Ras isoforms, giving optimism that
potent, selective inhibitors might be developed. Much remains to be discovered regarding cleav-
age specificity, the impact of chemical inhibition, and the potential of Rce1 as a therapeutic tar-
get, not only for cancer, but also for other diseases.
ARTICLE HISTORY
Received 16 November 2017
Revised 15 January 2018
Accepted 19 January 2018
KEYWORDS
Ras converting enzyme;
CaaX proteins; Ras;
proteases; cancer
Introduction
Many eukaryotic proteins are isoprenylated with either
farnesyl (C15) or geranylgeranyl (C20) groups. The
CaaX-type prenyl proteins are historically defined by a
carboxyl-terminal CaaX motif where C is cysteine, a is
an aliphatic amino acid, and X is one of several amino
acid residues. Proteins containing this motif are often
subject to an ordered series of post-translational modifi-
cations (PTMs): C15 or C20 isoprenylation of the cyst-
eine, endoproteolysis by Rce1 to remove aaX, and
carboxyl methylation (Figure 1). Deviations from this
standard pathway occur. Some proteins are isopreny-
lated, but not cleaved (i.e. shunt pathway), whereas
other proteins are subject to additional PTMs after the
canonical set of modifications (e.g. a-factor and S-acyl-
ation pathways). For the relatively few CaaX-type pro-
teins examined in detail, disruption of the PTMs has
profound effects on their function and localization. The
CaaX-type proteins play an important role in cell
signaling processes; their members comprise the Ras
family of small GTPases, the c-subunit of heterotrimeric
GTPases, nuclear lamins, and protein kinases and phos-
phatases (Gao et al. 2009; Wang and Casey 2016). The
CaaX proteins Ras, RhoA, RhoC, RheB, Rac proteins,
CDC42, and PTP4A3 have been implicated in cancer
(Winter-Vann and Casey 2005). Understanding the PTMs
of CaaX proteins is therefore expected to provide
insight into their regulation and biological function.
This review focuses on the activity, mechanism,
and inhibition of Ras converting enzyme (Rce1), which
mediates the endoproteolytic cleavage of the carboxyl-
terminal three amino acids from CaaX proteins.
Clinical relevance of Rce1
CaaX proteins are abundant eukaryotic proteins with
diverse biological functions. They are involved in cancer,
development, aging, parasitic growth, and many
CONTACT Timothy M. Dore timothy.dore@nyu.edu New York University Abu Dhabi, Saadiyat Island, PO Box 129188, Abu Dhabi, United Arab
Emirates
ß2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-
nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed,
or built upon in any way.
CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, 2018
VOL. 53, NO. 2, 157–174
https://doi.org/10.1080/10409238.2018.1431606
other biological pathways (Wang and Casey 2016). Ras
GTPases are some of the more prominently
studied CaaX proteins because of their involvement in
cancer. Because Ras function optimally requires post--
translational modification, there is intense interest for
developing therapeutic inhibitors for all modification
steps (Silvius 2002; Winter-Vann and Casey 2005;
Konstantinopoulos et al. 2007; Berndt et al. 2011; Cox
et al. 2015; Wang and Casey 2016). Such agents include
farnesyl and geranylgeranyl transferase inhibitors (FTIs
and GGTIs), dual prenyl transferase inhibitors (DPIs),
Rce1 protease inhibitors (RPIs), and carboxyl methyl
transferase inhibitors (MTIs). FTIs are the most advanced
of these agents and have reached clinical trials, but
their utility as anti-cancer agents has not been substan-
tiated (Gelb et al. 2006; Cox et al. 2015). The develop-
ment and testing of RPIs and MTIs substantially lag
that of prenyl transferase inhibitors. Rce1 is also being
investigated as a potential biomarker in various types of
cancer, but its utility as a disease predictor remains far
from being understood (Huang et al. 2016; Li et al.
2017; Shi et al. 2017).
Studies investigating the importance of Rce1 in the
cancer setting have largely relied on rce1
/
mouse
embryonic fibroblasts (MEFs; the mouse homozygous
knockout is inviable), tissue-specific mouse knockouts,
and small molecule agents that interfere with Rce1
activity (Chen 1998,1999; Bergo et al. 2002,2004;
Wahlstrom et al. 2007). Loss of Rce1 activity is associ-
ated with Ras mislocalization, diminished growth of
Ras-transformed fibroblasts in culture and in nude mice,
and hypersensitivity to FTIs. Chemical inhibition of Rce1
yields similar results. Collectively, these observations
suggest that Rce1-deficiency correlates with a reduced
ability to maintain the transformed state. It is unclear
though whether outcomes observed with knockout
approaches are directly due to defects in Ras signaling
alone, or more likely, due to a broad impact of Rce1
inhibition on multiple CaaX proteins. The latter likely
underlies the varied outcomes observed for tissue-
specific knockout studies. Loss of Rce1 in hematopoietic
tissue exacerbates K-Ras induced myeloproliferative
disease (Wahlstrom et al. 2007). Loss of Rce1 in cardiac
tissue results in high rates of lethal cardiomyopathy
Figure 1. Overview of post-translational modifications associated with CaaX proteins. Both farnesyl (C15) and geranylgeranyl
(C20) can be added to CaaX proteins. There are multiple classes of isoprenylated CaaX proteins: those with motifs that resist
cleavage (shunt), those with motifs that are cleaved (canonical), and those additionally modified by cleavage (a-factor) or S-acyl-
ation. Motifs shown are for indicated yeast (Ydj1p, Ste18p, a-factor) or human proteins (all others).
158 S. E. HAMPTON ET AL.
(Bergo et al. 2004). Loss of Rce1 in retina compromises
photoreceptor function and is associated with retinal
degeneration (Christiansen et al. 2011). Whether chem-
ical-based inhibition of Rce1 will yield similar outcomes
remains undetermined.
There is additional disease-related biology outside
of cancer in which Rce1 is involved (Gelb et al. 2006).
Rce1 is essential for growth of Plasmodium sp. and
Trypanosoma brucei, the causative agents of malaria
and African sleeping sickness, respectively (Gelb et al.
2003; Gillespie et al. 2007). Inhibitors that preferentially
target parasite Rce1 over human could thus have
potential clinical value in this setting (Mokry et al.
2009). In the case of certain bacterial infections, mam-
malian Rce1 regulates the modification and effective-
ness of bacterial effector proteins injected into the
eukaryotic host (Fueller et al. 2006; Price et al. 2010).
Prokaryotic Rce1 itself appears to regulate the patho-
genicity of bacterial species associated with high
mortality rates, such as Staphylococcus aureus and
Streptococcus pneumoniae (Frankel et al. 2010). Hence,
the clinical importance of Rce1 may extend beyond can-
cer to several forms of infectious disease. Multiple
developmental processes also appear to rely on Rce1.
Loss of mouse Rce1 activity (i.e. rce1
/
) results in late
embryonic lethality, and tissue specific knockouts result
in various disease states (Kim et al. 1999; Bergo et al.
2004; Wahlstrom et al. 2007; Christiansen et al. 2011).
Regulation of Rce1
Little is known about the regulation of Rce1 activity,
and it is likely to have a general house-keeping func-
tion. Consistent with the widespread tissue expression
of CaaX proteins, Rce1 is expressed in multiple tissues
with some variation in expression levels (Otto et al.
1999; Young et al. 2001; Uhlen et al. 2015; Tissue
Expression of RCE1, The Human Protein Atlas 2017).
There is evidence, however, that Rce1 levels can be
regulated by the USP17 deubiquitinating enzyme,
whose activity leads to proteasomal degradation of
Rce1 and hence reduced protein levels (Burrows et al.
2009; Jaworski et al. 2014). In wild-type MEF cells, the
presence of USP17 results in down regulation of Rce1
activity, resulting in relocalization of GFP H-Ras from the
plasma membrane to the cytosol and down regulation
of Ras/MEK/ERK pathway signaling. Interestingly, these
studies suggest that USP17 has differential impact on
distinct splice variant isoforms of Rce1 in mammals
(Rce1 Iso1 and Rce1 Iso2). Rce1 Iso2 protein levels
appear to be regulated by USP17, whereas Rce1 Iso1 is
unaffected. Nevertheless, both isoforms of Rce1 are
required to reconstitute GFP H-Ras to the plasma
membrane in rce1
/
MEF cells, and USP17 on its own
could not block cell proliferation in the absence of
Rce1, suggesting a strong and direct link between the
Rce1 and USP17 proteins. Further studies are required
to understand the roles of different isoforms of Rce1
and their effect on the oncogenic Ras signaling path-
way, as this would impact drug discovery efforts.
Identification and homologs of Rce1
The Ras converting enzyme (Rce1) is alternatively
known as FACE-2 and Type II CAAX prenyl endopeptid-
ase. Prior to its genetic identification, attempts to purify
Rce1 using traditional biochemical approaches did not
lead to its identification (Young et al. 2001). In retro-
spect, the main difficulties hindering the purification of
Rce1 were its membrane-associated nature and the con-
taminating presence of Ste24p (FACE-1/Type I CAAX
prenyl endopeptidase/Afc1), an evolutionarily unrelated
protease with partially overlapping CaaX protease activ-
ity (Tam et al. 1998; Trueblood et al. 2000). The inability
to biochemically purify or significantly enrich Rce1 is a
complicating issue that continues to this date.
The genes encoding Rce1 and Ste24 were eventually
both identified in Saccharomyces cerevisiae through an
elegant genetic screen involving the a-factor mating
pheromone as a reporter (Boyartchuk et al. 1997).
Functional orthologs have subsequently been identified
in other eukaryotic organisms (humans, mice, plant,
parasite, worm, pathogenic fungi, etc.) (Kim et al. 1999;
Otto et al. 1999; Cadi~
nanos, Schmidt, et al. 2003;
Cadi~
nanos, Varela, et al. 2003; Mokry et al. 2009; Esher
et al. 2016). Prokaryotic orthologs also exist, and while
not expected to possess farnesyl-dependent specificity,
at least one can cleave a synthetic farnesylated sub-
strate in vitro (Pei and Grishin 2001; Manolaridis et al.
2013).
Collectively, Rce1 orthologs vary in their primary
sequence identity (9–63%) and size (30–37 kDa) (Pei and
Grishin 2001; Dore and Schmidt 2013). They all are pre-
dicted to be multi-span membrane proteins. Sequence
divergence for transmembrane segments among homo-
logs of any type is a common occurrence, and this may
in part explain the low degree of sequence homology
between Rce1 orthologs. Despite their low homology,
Rce1 orthologs all have several conserved amino acids
that are proposed to serve as catalytic residues: two
adjacent glutamates and two separate histidines (Pei
and Grishin 2001; Manolaridis et al. 2013). Other highly
conserved residues include an arginine often positioned
four residues away from the pair of glutamates, an aro-
matic residue (Phe or Tyr) often positioned four residues
from the first histidine, and a asparagine/aspartate
CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY 159
positioned near the second histidine. A cysteine near
the second histidine is conserved in eukaryotic ortho-
logs, but not in prokaryotic ones. The importance of
conserved residues for the activity of eukaryotic Rce1
has been investigated for multiple eukaryotic orthologs,
which are all expected to be involved in the cleavage of
CaaX-type proteins (Dolence et al. 2000a; Plummer et al.
2006; Mokry et al. 2009). With exception of the pro-
posed catalytic residues, the roles of other conserved
residues are not yet fully understood and complicated
by different observations. For example, the cysteine
conserved in eukaryotic Rce1 orthologs has been
reported to be essential for activity in one study but
completely dispensable for activity in other studies
(Dolence et al. 2000a; Plummer et al. 2006; Hildebrandt
et al. 2013). Whether conserved residues are important
for the activities of the prokaryotic orthologs remains
an open question. Where examined, prokaryotic ortho-
logs also appear to be associated with cleavage activ-
ities, including protection against antimicrobial
peptides (Ellermeier and Losick 2006; Kjos et al. 2010).
Rce1 has distinct, but somewhat similar sequence
motifs to the PrsW family of proteases, the domain of
unknown function protein DUF2324 from the Pfam fam-
ily, and the 7-transmembrane-spanning APH-1 subunit
of c-secretase (Pei et al. 2011).
Topology and structure of Rce1
Rce1 is an integral membrane protein that is localized
to the endoplasmic reticulum (ER) in eukaryotes
(Schmidt et al. 1998; Bracha-Drori et al. 2008).
Prokaryotic orthologs are envisaged to be located to
the plasma membrane (Xiang et al. 2017). Hydropathy
and multiple-sequence alignment across Rce1 orthologs
from Homo sapiens (HsRce1), S. cerevisiae Rce1 (ScRce1),
and several other species predicted seven or eight
trans-membrane segments depending on species
(Figure 2) (Hildebrandt et al. 2013). Topology studies
carried out on ScRce1 and based on maleimide labeling
of cysteines in ScRce1 and in an HA tag placed at either
the N- or C-terminal end of the enzyme suggested that
the N-terminus projected into the ER lumen, whereas
the C-terminus was located on the cytosolic side. This
orientation was further supported by an N-linked glyco-
sylation assay. Such an orientation would require an
odd number of transmembrane spans (Figure 2(B)).
Maleimide labeling of natural and introduced cysteines
indicated that ScRce1 might contain several reentrant
helices, which do not span the bilayer, on the cytosolic
face of the membrane (Figure 2(C)). The topology study
relied heavily on mutants that introduced non-natural
cysteines into ScRce1, which could introduce subtle
changes that disrupt a-helices and helix hairpins lead-
ing to alternative topologies, misfolded protein, or both
(Monne et al. 1999; Norholm et al. 2011). The maleimide
reagent could also react with cysteines in large pockets
that extend deeply into the membrane, which is a struc-
tural feature of Rce1 from an archaeon (vide infra).
The crystal structure of an Rce1 homolog from the
archaea Methanococcus maripaludis (MmRce1) was elu-
cidated at 2.5-Å resolution in complex with a monoclo-
nal antibody Fab fragment (PDB accession number
4CAD) (Figure 3) (Manolaridis et al. 2013). Its structure,
measuring approximately 35 26 46 Å, is made up of
8 transmembrane a-helices linked by short loops plus
two peripheral membrane a-helices (topology shown in
Figure 2(A)). The C- and N-termini point into the ER
lumen, which is in agreement with the hydropathy pre-
dictions, but contradicts the topology data reported for
ScRce1 (Hildebrandt et al. 2013). At its core is a conical
Figure 2. Postulated topology models of Rce1. (A) Topology
with 8 transmembrane helices found in MmRce1 crystal struc-
ture and predicted for HsRce1; (B) 7 transmembrane helices
predicted for ScRce1; and (C) Proposed alternative topology
for ScRce1 based on substituted cysteine accessibility experi-
ments. Conserved catalytic glutamate and histidine residues
are located on blue-colored helices. (see color version of this
figure at www.tandfonline.com/ibmg).
160 S. E. HAMPTON ET AL.
cavity with a large volume of 1400 Å
3
that encompasses
the catalytic site and opens to the cytosol. Inside the
cavity, the conserved residues E140, E141, H173, H227,
and N231 reside within the span of the membrane, and
each project their side chains into the cavity, except
E141, suggesting that it might not be directly involved
in catalysis. The highly-conserved residue R145 sits at
the base of the cone, separating the active site from the
ER lumen. A water molecule is located in the cavity
approximately 10 Å from the cytosolic surface of the
membrane and is bridged by E140 and H173, which
are positioned opposite one another. Additionally, one
side of the cavity is open to the membrane via a gap
between two of the transmembrane helices (TM2 and
TM4).
Proteolytic mechanism
Early studies implicated Rce1 to be from the family of
cysteine proteases based on the fact that mechanism-
based cysteine protease inhibitors abolished activity
and mutational studies showing that catalytic activity
resulted when the only conserved cysteine among
eukaryotic orthologs was changed to alanine (Ma et al.
1993; Chen et al. 1996; Dolence et al. 2000a). More
recently, however, a study on ScRce1 found that the
only conserved cysteine among eukaryotic Rce1 ortho-
logs was not necessary for enzyme function, and a cyst-
eine-free Rce1 mutant was fully functional in vivo
(Plummer et al. 2006; Hildebrandt et al. 2013). Rce1 was
also putatively assigned as a membrane-bound metallo-
protease because of a conserved set of glutamates and
histidines found across eukaryotic and prokaryotic
orthologs (Pei and Grishin 2001). A mutational analysis
study revealed the importance of these residues and
the requirement of glutamate and histidine by ScRce1
in catalysis, suggesting either a zinc metalloenzyme or a
novel proteolytic mechanism (Plummer et al. 2006).
Indeed, several pieces of data implicate a novel proteo-
lytic mechanism, rather than the four established
canonical mechanisms of proteolysis: aspartate, cyst-
eine, serine/threonine, and metal-based. Rce1 has little
sequence similarity with other proteases and no con-
served cysteine across eukaryotes and prokaryotes (Pei
and Grishin 2001). EDTA does not affect the activities of
MmRce1 or ScRce1, although excess Zn
2þ
inactivates
ScRce1, but not MmRce1 (Plummer et al. 2006;
Manolaridis et al. 2013). Phenanthroline inactivation of
MmRce1 is the result of protein unfolding, and no Zn
2þ
was detected in MmRce1 in proton-induced X-ray emis-
sion (PIXE) spectroscopy, total reflection X-ray fluores-
cence (TXRF) spectroscopy, or X-ray crystallography
experiments.
The crystal structure of MmRce1 suggested proteoly-
sis through a novel mechanism, corroborating an earlier
proposal (Plummer et al. 2006), involving an active site
glutamate, two histidines, and an asparagine that are
essential for catalytic activity (Figure 4) (Manolaridis
et al. 2013). Computational docking studies, using a
decapeptide containing the farnesylated canonical CaaX
motif of RhoA (CLVS), suggested that the substrate
adopts a b-hairpin structure to insert itself into the cav-
ity. The farnesyl group fills the space between TM2 and
TM4, which are decorated with non-polar amino acids,
positioning the peptide bond between the cysteine and
the first aliphatic amino acid near the water bridging
H173 and E140 (Figure 3). The model does not provide
insight into the position of the C-terminal carboxylate
and any interactions that it might have with MmRce1.
The water nucleophile attacks the carbonyl of the
amide of the prenylated cysteine (Figure 5). H227 and
N231, which form a structural element called an oxyan-
ion hole, stabilize the resulting tetrahedral oxyanion
intermediate that collapses to the carboxylate, jettison-
ing the amine, which is facilitated by proton transfer
from H173 or E140, and completing the proteolysis.
Although the proteolysis mechanism of Rce1 is novel,
elements of the proposed mechanism, such as a glu-
tamate-activated water and an oxyanion hole, are found
in other membrane-associated proteases (Manolaridis
et al. 2013): S2P (Feng et al. 2007), Ste24 (Pryor et al.
2013; Quigley et al. 2013), SGP (Fujinaga et al. 2004),
Figure 3. Crystal structure of MmRce1 (PDB: 4CAD). The pro-
tein structure is oriented with the cytosolic face at the top and
that of the ER lumen on the bottom of the image. (see color
version of this figure at www.tandfonline.com/ibmg).
CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY 161
and GlpG rhomboid protease (Baker et al. 2007; Ben-
Shem et al. 2007; Wang and Ha 2007; Vinothkumar
et al. 2010). The crystal structure model provides a foun-
dation to gain coarse-level insight into the structure
and mechanism of the eukaryotic Rce1; nevertheless,
caution should be exercised with the mechanistic
hypothesis because of species specific differences in the
primary structure and topology of Rce1. For example,
the primary structure of MmRce1 is only 13% identical
to that of HsRce1 and 12% identical to ScRce1 based on
Clustal Omega analysis (UnitProtKB), and the N- and C-
termini of MmRce1 point into the ER lumen if adopting
an MmRce1 topology (Manolaridis et al. 2013), or are
located on opposite sides of the ER membrane if ori-
ented like ScRce1 (Hildebrandt et al. 2013). Solved crys-
tal structures of human and yeast Rce1 would
contribute significantly to a better understanding of the
catalytic mechanism of proteolysis.
Reporter substrates for measuring Rce1
activity
Various reporter substrates have been developed to
monitor Rce1 activity both in vivo and in vitro (Table 1).
These reporters invariably take the format of an isopre-
nylated polypeptide with an aaX extension.
In vivo reporters
Many in vivo studies of Rce1 activity have relied on the
yeast a-factor mating pheromone (36-mer polypeptide)
and the genetic tractability that the yeast system pro-
vides. This includes the study that ultimately uncovered
the identity of the RCE1 gene (Boyartchuk et al. 1997).
The a-factor reporter has since been used in various
studies to systematically explore Rce1 and Ste24 specifi-
city (Trueblood et al. 2000; Cadi~
nanos, Schmidt, et al.
2003; Cadi~
nanos, Varela, et al. 2003; Plummer et al.
2006; Bracha-Drori et al. 2008; Mokry et al. 2009).
Through use of this reporter, there has been extensive
but not exhaustive evaluation of CaaX protease specifi-
city; collectively only 75 CaaX combinations have
been evaluated out of the 8000 possible CaaX sequence
combinations (Boyartchuk and Rine 1998; Trueblood
et al. 2000; Cadi~
nanos et al. 2003b; Young et al. 2005;
Plummer et al. 2006; Krishnankutty et al. 2009; Mokry
Figure 5. Proposed basic catalytic mechanism of MmRce1 proteolysis.
Figure 4. Crystal structure of the MmRce1 catalytic site (PDB:
4CAD), showing the locations of the critical residues E140,
E141, R145, H173, H227, and N231. Selected N–O distances
between residues are given in Ångstr€
oms and marked by
black dashed lines. The hydrogen bond between H227 and
N231 reported by Manolaridis et al. is shown as a yellow line.
(see color version of this figure at www.tandfonline.com/
ibmg).
162 S. E. HAMPTON ET AL.
et al. 2009; Hildebrandt et al. 2016b). The utility of the
a-factor reporter is tempered by the high sensitivity of
the biological assays used to detect the reporter that
could potentially lead to false-positives for cleavable
CaaX motifs, an inability to distinguish between defect-
ive farnesylation and defective proteolysis for certain
CaaX motifs, and the evaluation of CaaX motifs outside
of their natural context. As an example, the CASQ motif
has been widely regarded as an example of a Ste24-spe-
cific motif, but the amount of a-factor produced in the
context of CASQ is only 1–2% relative to wild-type a-
factor (CVIA), and the motif is not cleaved in its natural
context as part of the yeast Hsp40 chaperone Ydj1
(Hildebrandt et al. 2016b). Despite these limitations, the
a-factor reporter system has been one of the more use-
ful genetic systems for exploring Rce1 activity and
specificity.
By comparison to a-factor, Ras-based genetic meth-
ods are not widely used to study Rce1 activity due to
their qualitative nature and reliance on rather compli-
cated genetic set ups. Nevertheless, such methods have
demonstrated that the absence of Rce1 modulates the
thermosensitivity of yeast (Boyartchuk et al. 1997). More
commonly, GFP-tagging of Ras and other Ras-related
GTPases is used to assess the role of Rce1 in regulating
small GTPase subcellular localization (Boyartchuk et al.
1997; Choy et al. 1999; Michaelson et al. 2005;
Manandhar et al. 2007; Bracha-Drori et al. 2008; Roberts
et al. 2008; Mokry et al. 2009; Hanker et al. 2010;
Manandhar et al. 2010; Gentry et al. 2015; Mohammed
et al. 2016). In the yeast system, GFP-Ras2 is cytosolic in
the absence of farnesylation and has diffuse membran-
ous patterns of intracellular distribution in the absence
of either Rce1p or the ICMT (Ste14p) (Boyartchuk et al.
1997; Manandhar et al. 2010); the absence of Ste24p
does not impact Ras2p localization. An altered response
to heat shock is observed for the rce1 deletion relative
to wild-type, suggesting that mislocalization of Ras dis-
rupts the Ras signaling circuit (Boyartchuk et al. 1997).
In mammalian cells, Rce1-dependent localization is
similarly observed for the H-, N-, and K-Ras isoforms
(Choy et al. 1999; Kim et al. 1999; Bergo et al. 2002;
Michaelson et al. 2005; Mohammed et al. 2016).
The Rheb family of small GTPases also rely on Rce1
for their proper localization to the ER and Golgi
(Hanker et al. 2010), as do the oncogenic Ras-like small
GTPases RalA and RalB to the plasma membrane
(Gentry et al. 2015). A reliance on Rce1 for proper local-
ization of small GTPases is, however, not generalizable.
It is often unclear whether perturbing Rce1 activity also
impacts other associated post-translational modifica-
tions that occur to CaaX proteins (e.g. palmitoylation of
H- and N-Ras (Hancock et al. 1990,1991)). Rho-family
proteins (i.e. Cdc42, Rac1, Rac2, and RhoA) are cytosolic,
and this localization is independent of Rce1 activity
(Michaelson et al. 2001,2005). The localization of non-
GTPase CaaX proteins can also vary in their dependency
on Rce1 activity (Christiansen et al. 2011). Recently,
GFP-Ras reporters have been useful for examining the
impact of RPIs on Ras localization (Manandhar et al.
2010; Mohammed et al. 2016). Where examined, these
inhibitors pharmacologically phenocopy effects
observed with Rce1 knockouts and siRNA knockdowns.
A major limitation of all localization studies, whether
using knockouts or inhibitor-based approaches, is the
possibility that altering the cleavage state of the entire
prenylome at once may result in pleiotropic effects that
confound interpretation of results. Moreover, delocaliza-
tion effects can be difficult to quantify, especially when
the signal is distributed across multiple locations.
Despite these issues, GFP-based reporters continue to
have great utility for studies of Rce1 and are potentially
useful for future high-content HTS in association with
inhibitor identification.
Recently, the yeast Hsp40 chaperone Ydj1p has been
developed as yet another reporter, in this case one that
is useful for both genetic and localization studies
(Hildebrandt et al. et al. 2016b). Whereas the endogen-
ous Ydj1p CaaX motif (i.e. CASQ) is subject only to iso-
prenylation and avoids subsequent PTMs, a process
referred to as shunting, it can be made susceptible to
Rce1 cleavage by mutation of the CaaX motif to those
Table 1. Reporter substrates used to monitor Rce1 activity in vivo and in vitro.
Assay type Reporter Sequence
a
Detection method
In vivo GFP-Ras (H, N, K4a, K4b) (45 kDa) GFP-H-Ras-CVLS GFP-N-Ras-CVVM GFP-K-Ras4a-CIIM GFP-K-Ras4b-CVIM Cell-based fluorescence
GFP-Ras2p (60 kDa) GFP-Ras2p-CIIS Cell-based fluorescence
a-factor precursor (36-mer) Mature a-factor peptide-CVIA Bioactivity
In vitro a-factor CaaX (15-mer) YIIKGVFWDPACVIA Bioactivity, coupled
a-factor (12-mer) KGVFWDPACVIA HPLC
K-Ras4b (9-mer) fluor-KSKTKCK
Q
IM Fluorescence quenching assay
a-factor CaaX (8-mer) dansyl-WDPACVIA HPLC
a-factor CaaX (8-mer) KWDPACV[
3
H]IA HPLC, radiography
Biopep 4-mer bio-CVIA Affinity purification, radiography
a
Cysteine in the CaaX sequence is farnesylated. Abbreviations: fluor: ortho-aminobenzoic acid; K
Q
:N
6
–(2,4-dinitrophenyl)-L-lysine; bio: 1-N-biotinyl-(13-N-suc-
cinimidyl-(S-farnesyl-l-cysteinyl)-l-valinyl-l-isoleucinyl-[
14
C]-l-alanine)-4,7,10-trioxatridecanediamine.
CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY 163
that are cleaved in the context of other reporters (e.g.
CVIA and CTLM). Ydj1p thus has the potential to report
on both cleavable and cleavage resistant CaaX motifs.
Determining the specificity of Rce1 through use of vari-
ous in vivo reporters is likely to help resolve how many
CaaX proteins are shunted and how many are regulated
by Rce1-mediated cleavage.
In vitro reporters
Rce1 in vitro activity can be monitored using a variety
of farnesylated substrates, ranging from synthetic pepti-
des modeled on CaaX proteins (e.g. Ras and a-factor) to
recombinant proteins (e.g. GST-fusions) that have to be
farnesylated in vitro prior to use. Early assays using syn-
thetic peptides incorporated a radiolabeled amino acid
within the aaX portion of the CaaX motif, enabling dir-
ect tracking of the liberated aaX tripeptide through
HPLC and thin-layer chromatography methods [exten-
sively reviewed in Young et al. 2001)]. Indirect tracking
of Rce1-mediated activity has involved use of synthetic
peptide substrates or recombinant protein substrates,
and coupled proteolysis-methylation assays. In these
types of assays, ICMT-dependent methylation of the
proteolyzed product is used to mark cleaved substrates.
Addition of a radioactive methyl group requires enrich-
ment and quantification of the marked products,
whereas addition of a non-radioactive methyl group to
an a-factor-based reporter can be monitored directly
using a biological assay (Tam et al. 2001).
A more utilitarian version of the direct assay moni-
tors cleavage of an internally quenched fluorogenic
(IQF) nonapeptide modeled on the farnesylated COOH-
terminus of K-Ras4b (Hollander et al. 2000). In this assay,
cleavage directly correlates with increased fluorescence,
which is monitored in real time using a standard micro-
plate reader having fluorescence detection capability.
This assay approach is convenient for kinetic studies,
HTS studies, and benefits from being non-radioactive.
The assay confirms many of the expected properties of
Rce1, ranging from isoprenoid dependence to tolerance
for certain substitutions within the CaaX motif
(Hollander et al. & Mallon 2003; Hildebrandt et al.
2016a). A major limitation of the IQF approach, how-
ever, is that either a fluorophore or quencher must be
accommodated within the aaX portion of the CaaX
motif, and such placement impacts Rce1 specificity and/
or activity (Porter et al. 2007). For example, yeast Rce1
can tolerate lysine e-dinitrophenyl (a quencher) at a
1
,
and to a much lesser extent at X, but not at a
2
in the
context of a farnesylated nonapeptide modeled on
K-Ras4b; a similar observation has been made for
human Rce1 (Porter and Schmidt, unpublished). The
requirement of a non-natural amino acid within the aaX
portion of the motif also limits the utility of this
approach for Rce1 specificity studies since the entire
combinatorial landscape of the CaaX motif cannot be
explored. Nonetheless, the mix-and-measure nature of
this assay continues to be useful and is a major reason
that HTS-based small molecule RPI identification has
been able to proceed (Manandhar et al. 2007).
Specificity of Rce1
Through use of the above in vivo and in vitro
approaches to examine Rce1 activity, a snapshot of
Rce1 sequence specificity is beginning to emerge. In
terms of isoprenylation, Rce1 cleaves substrates modi-
fied with either C15 and C20, but not unprenylated sub-
strates (Otto et al. 1999; Hildebrandt et al. 2016b).
To the best of our knowledge, the specificity of Rce1
toward substrates with shorter and longer isoprenoids
(e.g. C5 and C25) or alternative lipids (e.g. C14 and C16
acyl lipids) has not been examined. Earlier studies,
where this topic might have been explored, would have
also likely used membranes containing both Rce1 and
Ste24 making specificity interpretations difficult. In
terms of peptide length, a farnesylated tetrapeptide
CaaX motif is the minimally effective length for Rce1
recognition, although Rce1 can also cleave certain iso-
prenylated tripeptides in vitro with kinetics that rival
that of traditional farnesylated tetrapeptides (Hollander
et al. 2003). The biological relevance of shorter motifs
has not been explored in vivo.
In terms of amino acid specificity, the a
2
position
appears to be the most critical residue for ScRce1 recog-
nition as inferred from extensive substitution analysis of
the a-factor CVIA motif (Trueblood et al. 2000). In this
context, the most favored amino acids are Ile, Leu, and
Val (i.e. branched chain, aliphatic amino acids). This is
followed by Cys and Met, which are weakly tolerated.
The next most critical residue appears to be a
1
for
which there is more tolerance, with the best tolerances
observed with Ala, Cys, Ile, Leu, and Val and the small
polar residues Ser and Thr; some additional polar resi-
dues are weakly tolerated. These results suggest that
aliphatic is an oversimplification of allowable residues
at the a
1
position. The traditional X position can be
occupied by about half the amino acids without signifi-
cant impact on Rce1p activity: Ala, Val, Ser, Thr, Asn,
Gln, His, Phe, Tyr, Cys, and Met. Besides its high sensitiv-
ity, the a-factor-based assay used in the Trueblood
study is subject to false-positive readouts. Another limi-
tation is its coupled nature. Any sequence combination
that reports negatively or weakly in the assay could
result from incomplete farnesylation rather than
164 S. E. HAMPTON ET AL.
incomplete proteolysis. Thus, Rce1 may tolerate other
amino acids within the CaaX motif, but such substrates
were not biologically available if incompletely farnesy-
lated leading to a weak signal in the genetic-based
assay.
Additional specificity information is likely to come
from other assay approaches. These certainly could
involve more comprehensive genetic screens involving
a-factor to identify more cleavable CaaX combinations
or Ydj1 to identify both cleavable and non-cleavable
sequences, but these approaches will still be biased
toward sequences that are efficiently farnesylated, or
possibly even geranylgeranylated in some cases. Other
reporters such as Ras could also be utilized. Intriguingly,
the reported specificity preferences of ScRce1 observed
with the a-factor reporter are similar to those inferred
from a Ras-based yeast genetic screen intended to
interrogate farnesyl transferase specificity (Stein et al.
2015). The Ras-based screen evaluated the farnesylation
potential of all 8,000 possible CaaX variations. Analysis
of the top performing sequences reveals that a
2
is
enriched for Ile, Val, Leu, Met, and Cys (Figure 6). Thus,
both Ras and a-factor reporters identify similar enrich-
ment of amino acids at a
2
; considerable variation was
observed at a
1
and X. It is tempting to speculate that
the top performing sequences in the Ras-based study
are cleaved, but this was not directly assessed.
Moreover, the Ras-based screen was conducted in the
presence of both Rce1 and Ste24, making it unclear
which protease is responsible for cleavage of the top
Ras-based sequences. The Ras-based assay also suffers
from the same limitation as the a-factor based assay in
that incompletely farnesylated sequences could report
as false-negatives in the Ras-based assay despite having
amino acids compatible for cleavage. For a truly
unbiased identification of Rce1 cleavable sequences, it
is feasible to expect that a synthetic library representing
8000 farnesylated Cxxx tetrapeptides could be devel-
oped to explore specificity without concern for isopre-
nylation status. Such an approach currently has
technical and cost-related challenges.
Inhibitors of Rce1
Inhibitors of Rce1 that are not only potent but also
selective could be used to further investigate the
physiological role of Rce1 in regulating CaaX proteins
and downstream effects of its inhibition. There are a
number of Rce1 inhibitors reported in the literature
(Bergman et al. 2011; Schmidt and Dore 2011; Dore and
Schmidt 2013; Mohammed et al. 2016) that can be cate-
gorized as follows: anti-sense reagents to target the
Rce1 mRNA, general protease inhibitors, substrate mim-
etics, and small molecule inhibitors.
Anti-sense reagents
A cocktail of three siRNA oligonucelotides (19 bases)
knocks down human Rce1 expression in human colon
carcinoma (HCT-116) cells (Mohammed et al. 2016). A
60–75% reduction in Rce1 mRNA levels occurred with a
mixture of siRNA oligonucleotides targeting a translated
sequence (GUCAUCAAGCGACGCUUCA), the end of the
translated region (GCUCCUGACCUAUGCUCCU), and a
30-untranslated section of the mRNA (CCUCUGCCUCU
GAAAAGCU). Treatment of HCT-116 cells expressing
GFP tagged H-, N-, or K-Ras (K-Ras4b) with the siRNA
cocktail also mislocalized the Ras isoforms. N-Ras was
most affected followed by H-Ras, then K-Ras4b. While
not directly inhibiting Rce1 activity, siRNA represents a
powerful tool to explore the downstream impact of
Rce1 function.
General protease inhibitors
The general inhibitors cover a variety of structures.
These include chloromethyl ketones such as N
a
-tosyl-L-
phenylalanine chloromethyl ketone (TPCK), which is
considered a non-specific irreversible inhibitor of serine/
cysteine proteases (Figure 7). This inactivates ScRce1
with an approximate K
i
of 1 mM (Chen et al. 1996).
Interestingly TPCK inhibits ScRce1 in the presence of a
Ras reporter, but not in the presence of the a-factor
peptide. Therefore, it should be noted that differential
Figure 6. Weblogo amino acid frequency analysis of sequen-
ces identified by a Ras-based strategy (Stein et al. 2015). The
sequences analyzed were part of a set of sequences (n¼496)
having an enrichment score greater than 3, which is suggest-
ive of prenylation. The average enrichment score of the full
8,000 sequence set was 0.80, whereas the average score of
the sequences analyzed for frequency analysis was 9.42. Color
scheme: Cys is blue; polar charged amino acids are green
(Asp, Arg, Glu, His, and Lys); polar uncharged residues are
black (Asn, Gln, Ser, Thr, and Tyr); branched chain amino acids
are red (Ile, Leu, and Val); and all other residues are purple
(Ala, Gly, Met, Phe, Pro, and Trp). (see color version of this fig-
ure at www.tandfonline.com/ibmg).
CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY 165
inhibition of Rce1 can depend upon the reporter uti-
lized in biochemical activity assays. TPCK has compar-
able inhibitory activity against Rce1 from species other
than yeast, including Arabidopsis thaliana (AtRce1),
human (HsRce1), and trypanosome (TbRce1) (Porter
et al. 2007; Mokry et al. 2009). When applied at 250 lM,
TPCK inhibited ScRce1, AtRce1, and HsRce1 to 32, 0.17,
and 7%, respectively. A similar 200-lM concentration of
TPCK inhibited TbRce1 activity to 22% (Mokry et al.
2009). A more lipophilic chloromethyl ketone, N
a
-tosyl-
L-lysine chloromethyl ketone (TLCK), is a trypsin and
trypsin-like enzyme inhibitor. By comparison to TPCK,
TLCK does not inhibit Rce1 under the same conditions.
Other examples of general protease inhibitors reported
to inhibit Rce1 include the organomercurial compounds
para-hydroxymercuribenzoic acid (pHM), para-hydroxy-
mercuriphenylsulfonic acid (pHMS), and mersalyl acid
(MSA) (Dolence et al. 2000a). Non-specific organomercu-
rials and other general protease inhibitors have gener-
ally not been useful for probing the proteolytic
mechanism of Rce1 and have led to various erroneous
proposed mechanisms for Rce1.
Substrate mimetics
A number of inhibitors can be justifiably classified as
substrate mimetics based on structural elements that
mimic the natural substrate features. The first specific,
non-reversible inhibitor of Rce1 was created by modify-
ing TPCK and TLCK with a farnesyl group and replacing
the sulfonamide with a tert-butylcarbamate to create
BFCCMK (K
i
¼30 lM) (Figure 8) (Chen et al. 1996).
Replacement of the thiofarnesyl unit with a tridecyl
group gave a more stable and cell-permeable analog,
UM96001 (Chen 1998).
Peptidyl acyloxymethyl ketones (AOMKs) inhibit Rce1
(Porter et al. 2007). The peptidyl portion modulates the
inhibitory properties of these compounds, but signifi-
cant improvement of inhibitory properties is not likely
achievable through alteration of the peptidyl portion
alone (Dechert et al. 2010)(Figure 8). Because AOMKs
are known to be irreversible cysteine protease inhibi-
tors, the reactivity of these derivatives can also be
modulated by altering the acyloxy leaving group.
Various derivatives with differing benzyloxy groups
have been evaluated, but AOMKs surveyed had weak
inhibitory properties against ScRce1 (>40% ScRce1
activity remaining after 100-lM treatment). A substi-
tuted analog that did not contain a leaving group suit-
able for an irreversible binding mechanism (“warhead-
free”AOMK, Figure 8) proved to be the best inhibitory
compound (25% ScRce1 activity remaining after
100-lM treatment). These results suggest that AOMKs
do not likely work as mechanism-based inhibitors
and are most likely non-covalent, reversible inhibitors
of Rce1.
Figure 8. Structures of selected halo and acyloxymethyl
ketone inhibitors of Rce1.
Figure 9. Structures of RPI, SA, and selected RPI analogs.
Figure 7. Structures of general protease inhibitors of Rce1.
166 S. E. HAMPTON ET AL.
To date, the most potent of all Rce1 inhibitors are
the farnesylated peptide mimetics, RPI and a statine
analog (SA) (Figure 9) with K
i
¼86 and 64 nM, respect-
ively (Ma et al. 1993; Chen et al. 1996), and an IC
50
value
of 5 nM for RPI (Otto et al. 1999). RPI and SA are farne-
sylated CaaX motifs with a non-hydrolyzable linkage
between cysteine and the aaX sequence of K-Ras
(CVIM). SA is most likely a transition state analog inhibi-
tor of Rce1. RPI and SA remain the gold standard in
terms of inhibitory activity against HsRce1. In addition
to RPI and SA, other tetrapeptide inhibitors (peptides
1–5, Figure 9) have been developed. Many of these are
less potent by comparison to RPI and SA (ScRce1
IC
50
¼3.3–17.6 lM) (Dolence, Dolence, et al. 2000;
Dolence, Steward, et al. 2000; Dolence et al. 2001).
Non-peptidyl, non-prenylic inhibitors (NPNPIs) of the
farnesyl transferase also inhibit HsRce1 (Schlitzer et al.
2001). The three best compounds NPNPI-A, B, C (Figure
10) displayed moderate inhibitory activity with IC
50
val-
ues of 14, 7, and 7 mM, respectively. These are bi-sub-
strate analogs that contain a peptidomimetic
representing the CaaX motif and a lipophilic non-prenyl
moiety representing the farnesyl group. The fatty acid
lipid-like structure of these inhibitors invites speculation
that these might be promiscuous aggregate-based
inhibitors (McGovern et al. 2002 2003; Coan and
Shoichet 2008).
The marine natural products barangcadoic acid A
and rhopaloic acids A to G (Figure 11), isolated from
Hippospongia species, are terpenoids with IC
50
values of
approximately 26 lMinanHsRce1-proteolysis assay
(Craig et al. 2002). These are the first reported natural
product inhibitors of Rce1. Akin to better performing
Rce1 inhibitors, these natural products all carry an iso-
prenoid moiety. Additionally, extracts from the
Indonesian sponge Carteriospongia foliascens contained
two already known and four previously unreported sca-
larane-based sesterterpenoids (Williams et al. 2009). Of
the six compounds, a mixture of two 20,24-bishomosca-
larane ketals, differing in the stereochemistry at C-24,
inhibited activity of HsRce1 (IC
50
¼7.1 lM) despite the
fact that these derivatives do not have an isoprenoid
group (Figure 11).
Small molecule inhibitors
Recent work to identify new inhibitors of Rce1 has
focused on the development of non-peptide based
structures in an effort to identify small, drug-like mole-
cules. Such compounds would be expected to have
improved cell penetration and easier synthetic accessi-
bility compared to peptidomimetic and natural com-
pound-based inhibitors, enabling the use of eukaryotic
cell-based assays of Rce1 activity, which would aid
investigation into the effects of inhibiting Rce1 at the
cellular level.
The first set of small molecule Rce1 inhibitors
reported in the literature were identified as inhibitors of
ScRce1 in a medium throughput screening campaign
using a National Cancer Institute (NCI) library of
approximately 2000 compounds (Manandhar et al.
2007). A final set of nine compounds demonstrated IC
50
values of 6–35 mM against ScRce1. The specificity of the
compounds was evaluated in part by examining their
inhibitory properties toward other proteases. All of the
compounds inhibited ScSte24 (IC
50
¼8–28 lM), but had
Figure 10. Structures of peptidomimetic inhibitors.
Figure 11. Structures of natural product inhibitors of Rce1.
CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY 167
little or no activity on trypsin at 30 lM. For HsRce1
and TbRce1, IC
50
values of 0.4–46 and 0.25–177 lM
were observed, respectively (Mokry et al. 2009). Four of
the compounds were complexes with toxic metals (mer-
cury, palladium, or copper), and one was santowhite
powder, an antioxidant added to rubber and latex. The
additional four hits were NSC609974, NSC73101,
NSC1011, and NSC270718 (Figure 12). In a subsequent
yeast study, NSC1011 and NSC73101 disrupted the
proper membrane localization of a GFP-Ras2p reporter
(Table 1) (Manandhar et al. 2010). In yeast expressing
TbRce1 instead of ScRce1, six of the nine compounds
disrupted GFP-Ras2p membrane localization, including
NSC1011. No species-specific selectivity was observed
for the trypanosomal protein over the human ortholog,
however, which diminishes enthusiasm for selective tar-
geting of TbRce1 in the search for therapeutic
compounds.
NSC609974, NSC73101, and NSC270718 were active
in other screening programs, suggesting that these
might be pan-assay interference compounds (PAINS)
(Baell and Holloway 2010; Thorne et al. 2010; Baell et al.
2013; Baell and Walters 2014; Baell 2016). NSC 609974,
also known as carboxyamidotriazole (CAI) or L651582,
inhibits several calcium-mediated signal transduction
pathways (Kohn and Liotta 1990; Felder et al. 1991;
Kohn et al. 1992). It is an inhibitor of receptor-operated
non-voltage-gated and voltage-gated calcium influx
(Felder et al. 1991; Hupe et al. 1991), calcium-depend-
ent arachidonic acid release (Clark et al. 1991; Felder
et al. 1991), and tyrosine phosphorylation of phospho-
lipase Cc(PLCc) (Gusovsky et al. 1993), and it has been
used to study how calcium influx modulates the expres-
sion of matrix metalloproteinase-2 (MMP2) (Kohn et al.
1994b). NSC609974 and its orotate salt possess anti-pro-
liferative, anti-angiogenic, and anti-metastatic proper-
ties (Kohn et al. 1992; Kohn, Felder, et al. 1994a; Mignen
et al. 2005; Guo et al. 2006; Corrado et al. 2012) and
have been in clinical trials for cancer (Grover et al. 2007;
Taylor et al. 2015; ClinicalTrials.gov 2017). It is tempting
to speculate that the anti-oncogenic activity and per-
haps some of the adverse side effects could be due in
part to the inhibition of Rce1.
NSC73101 was identified in a screen for inhibitors of
poxvirus type I topoisomerase, but it was not the most
promising inhibitor and was not pursued further (Bond
et al. 2006) It was also identified in other screens as an
inhibitor of Vibrio cholera biofilm formation (Peach et al.
2011), an inhibitor of the manganese/magnesium-
dependent phosphatase PP2Ca(Sierecki and Newton
2014), and an irreversible inhibitor of replication protein
A, an essential DNA repair protein (Gavande et al. 2016).
NSC270718 was independently found in multiple
HTS and virtual ligand docking screens to be a moder-
ate inhibitor of angiogenin (K
i
¼75 lM), an inducer of
new blood vessel growth (Jenkins et al. 2002; Kao et al.
2002); the human mitotic kinesin Eg5 (DeBonis et al.
2004); DNA synthesis specific to Kaposi’s sarcoma-asso-
ciated herpes virus (KSHV) (IC
50
¼2.38 lM) (Dorjsuren
et al. 2006); the soluble form of adenylyl cyclase
(Schlicker et al. 2008), and the ATP-dependent MurD lig-
ase (IC
50
¼34 lM in biochemical assays), an enzyme
involved in the biosynthesis of an essential peptidogly-
can component of the bacterial cell wall (Turk et al.
2009). It was also identified in the same screen against
poxvirus type I topoisomerase that NSC73101 was
found (Bond et al. 2006).
Other biological activities for NSC1011 have not
been reported, but its structural analog, NSC66811
(Figure 13) inhibits the interaction between murine
double minute 2 (MDM2) and p53 tumor suppressor
with a K
i
¼120 nM (Lu et al. 2006). The oncogene
MDM2 is the cellular inhibitor of p53, which is an essen-
tial component of cell cycle regulation. In part because
of this relatively higher potency against HsRce1,
NSC1011 was taken forward into an SAR study in an
attempt to improve upon potency and selectivity
toward HsRce1 (Mohammed et al. 2016). A small library
of approximately 56 new compounds was synthesized
around the core 8-hydroxychloroquine scaffold (Figure
13) and tested against HsRce1 using the in vitro IQF pro-
teolysis assay. Many of the derivatives failed to improve
upon potency, although substitutions on the benzoic
Figure 12. Selected examples of Rce1 inhibitors identified in a
medium throughput screen.
168 S. E. HAMPTON ET AL.
acid and phenyl rings were well-tolerated. Changing
the hydroxyquinoline ring to a 1-napthol moiety
showed a modest improvement in IC
50
against HsRce1
from 6.9 mM for NSC1011 to 4.2, 3.9, and 3.8 mM for
compounds 29,31, and 46, respectively (Figure 13).
The hydroxyl group on the quinoline was essential for
biological activity. The most active compounds were
selective for HsRce1 versus HsSte24, and a representa-
tive subset of NSC1011 analogs did not inhibit human
farnesyl transferase. NSC66811 was not active against
HsRce1.
The effectiveness of NSC1011-based compounds to
work in vivo was demonstrated through a Ras mislocali-
zation assay. NSC1011 and compounds 2,5,6, and 17
were effective at mislocalizing Ras to the cytosol in a
human colorectal carcinoma cell line (HCT-116).
Interestingly, the Rce1 inhibitors mislocalized N-Ras to
the greatest extent, followed by H-Ras, then K-Ras4b.
A similar result was found using Rce1 siRNA
knockdowns (Mohammed et al. 2016). Curiously,
NSC1011 and its analogs were more effective than an
FTI at mislocalizing K-Ras4b, which is significant,
because activating K-Ras mutations are more frequently
observed in cancer compared to those of H- and N-Ras
(Prior et al. 2012).
Perspectives
Rce1 has been studied for more than two decades. A
great deal of progress has been made in terms of eluci-
dating the precise involvement of Rce1 in CaaX protein
maturation, yet much remains to be discovered regard-
ing its cleavage specificity and impact of chemical
inhibition, especially in vivo. Efforts to date have
focused on three main areas: genetic inhibition to iden-
tify the impact of down regulation of Rce1, structural
elucidation of a distantly related archeal ortholog, and
development of chemical probes for its inhibition.
Figure 13. Examples of NSC1011 derivatives that remained active against HsRce1. Numbers in parentheses are IC
50
values or the
percent activity of Rce1 after 10 lM treatment as judged using the Rce1 IQF in vitro proteolysis assay (Mohammed et al. 2016).
(see color version of this figure at www.tandfonline.com/ibmg).
CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY 169
Future inhibitor development is certainly currently lim-
ited by the lack of structural data of a eukaryotic
enzyme. This has made it difficult to understand the
mechanistic properties of existing inhibitors and has
delayed structure-based design of inhibitors that may
have better inhibitory properties than those currently
available.
Whereas the current most potent inhibitor of Rce1 is
a farnesylated tetrapeptide (i.e. a likely transition state
substrate mimetic), it is cell impermeable and not useful
to investigate the effects of Rce1 inhibition in vivo.
A crystal structure involving the complex of SA with
Rce1 should be a high priority. If it is a transition state
inhibitor, then the hydroxyl group should displace the
catalytic water molecule in the active site. The activity
of the hydroxy epimer of SA against Rce1 would also be
of interest to give insight into the orientation of the
substrate in the active site. Attempts to overcome the
issue of membrane impermeability of RPI and SA
through development of small molecules are beginning,
but these compounds have so far fallen short of
expected standards for detailed animal studies.
Nevertheless, the small molecule inhibitors identified
thus far with activity in both in vitro and cell-based
assays suggest that potent and selective inhibitors
might yet be developed. New scaffolds may derive from
additional HTS screening and possibly through compu-
tational methods for structure-guided design.
Regardless of how developed, understanding the inhibi-
tory mechanisms of these new compounds will
undoubtedly require new structures of Rce1 orthologs,
especially a eukaryotic one, and preferably that of the
human enzyme.
Once appropriate inhibitory compounds are devel-
oped, it will be interesting to investigate whether Rce1
inhibitors alone or in combination with FTIs will be
more effective than FTIs alone at modulating Ras
activity. It is intriguing to speculate that modulation of
K-Ras4b activity, in particular, might be more effective
when FTIs are applied in combination with Rce1
inhibitors. It is well documented that alternative gera-
nylgeranylation of K-Ras4b occurs in the presence of
FTIs. Concomitant use of FTIs and Rce1 inhibitors could
thus help limit the residual function associated with
geranylgeranylated K-Ras4b. Inhibitors, regardless of
their impact on Ras activity, may also have overlooked
utility in other disease situations. Many CaaX-type iso-
prenylated proteins exist, and they are involved in
many cellular processes. It is not yet clear, however,
which CaaX proteins are cleaved by Rce1 as opposed to
Ste24. Understanding the specificities and mechanisms
of both enzymes is thus intertwined for developing
accurate substrate profiles, which will be needed to
better understand the potential biological impact of
Rce1 inhibition.
Acknowledgements
We thank Drs. Louise Ashall and Emily R. Hildebrandt for
their comments on this manuscript.
Disclosure statement
The authors report no conflicts of interest.
Funding
The work was supported in part by a grant from the National
Institute of General Medical Sciences at the National
Institutes of Health (GM117148) to W. K. S. and research sup-
port from NYU Abu Dhabi to T. M. D.
ORCID
Shahienaz E. Hampton http://orcid.org/0000-0002-6600-
9492
Timothy M. Dore http://orcid.org/0000-0002-3876-5012
Walter K. Schmidt http://orcid.org/0000-0002-3359-3434
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