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Contents lists available at ScienceDirect
Seminars in Cell & Developmental Biology
journal homepage: www.elsevier.com/locate/semcdb
Rhomboid protease inhibitors: Emerging tools and future therapeutics
Kvido Strisovsky
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, Prague 166 10, Czech Republic
a r t i c l e i n f o a b s t r a c t
Article history: Rhomboid-family intramembrane serine proteases are evolutionarily widespread. Their functions in dif-
Received 18 May 2016
ferent organisms are gradually being uncovered and already suggest medical relevance for infectious
Received in revised form 16 August 2016
diseases and cancer. In contrast to these advances, selective inhibitors that could serve as efficient tools for
Accepted 24 August 2016
investigation of physiological functions of rhomboids, validation of their disease relevance or as templates
Available online xxx
for drug development are lacking. In this review I extract what is known about rhomboid protease mech-
anism and specificity, examine the currently used inhibitors, their mechanism of action and limitations,
Keywords:
and conclude by proposing routes for future development of rhomboid protease inhibitors.
Rhomboid protease
Inhibitor © 2016 Elsevier Ltd. All rights reserved. Disease Mechanism
Substrate specificity
Contents
1. Introduction ...... 00
2. Rhomboid proteases as potential future drug targets ...... 00
2.1. Microbial rhomboid proteases ...... 00
2.2. Human rhomboid proteases ...... 00
3. Rhomboid protease mechanism and specificity ...... 00
3.1. Catalytic mechanism ...... 00
3.2. Substrate specificity ...... 00
4. Rhomboid activity assays ...... 00
5. Rhomboid inhibitors ...... 00
5.1. The beginnings ...... 00
5.2. Structure and mechanism based design and future directions ...... 00
6. Concluding remarks and perspectives ...... 00
Acknowledgements...... 00
References ...... 00
1. Introduction
activate the ligands of the epidermal growth factor (EGF) recep-
The first members of the rhomboid-like superfamily were iden-
tor [2]. Rhomboid protease homologs have since been found in
tified in Drosophila [1] as intramembrane serine proteases that
nearly every sequenced genome spanning virtually all life forms,
constituting the most widely occurring family of intramembrane
proteases [3]. More recently, a number of related but proteolyt-
ically inactive members of the superfamily have been identified,
Abbreviations: ABP, activity based probe; ADAM17, a disintegrin and metallopro-
teinase 17; cmk, chloromethylketone; cho, aldehyde; EGF, epidermal growth factor; such as iRhoms or Derlins [reviewed in [4]]. iRhoms are poten-
EGFR, epidermal growth factor receptor; FRET, Foerster resonance energy transfer;
tial novel targets for TNF␣ [5–7] and ADAM17/EGFR [8–10] related
iRhom, inactive rhomboid homologue; PARL, presenilin-associated rhomboid-like;
pathologies, but their mode of action and druggability are unclear
PD, Parkinson’s disease; RHBDD, rhomboid domain-containing protein; RHBDL,
(for more discussion see the article by Lemberg and Adrain in this
rhomboid-like protein; TNF␣, tumor necrosis factor ␣; TACE, TNF␣-converting
enzyme; TMD, transmembrane domain. issue). The main mechanistic and structural model for the rhomboid
E-mail address: [email protected] superfamily have been rhomboid proteases.
http://dx.doi.org/10.1016/j.semcdb.2016.08.021
1084-9521/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: K. Strisovsky, Rhomboid protease inhibitors: Emerging tools and future therapeutics, Semin Cell Dev
Biol (2016), http://dx.doi.org/10.1016/j.semcdb.2016.08.021
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2 K. Strisovsky / Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx
Investigation in as distant fields as parasitology, cancer biol- 2.2. Human rhomboid proteases
ogy or microbiology has revealed exciting functions for rhomboid
proteases in a variety of contexts. Although a large proportion of Rhomboid proteases are cardinal regulators of the EGF receptor
this territory is still unexplored, the few examples below already signaling in Drosophila, but initially it seemed that their function
show that rhomboids are potential therapeutic targets. Investiga- in this pathway has not been conserved, because EGFR ligands
tion of biological roles of rhomboid proteases in multiple organisms in mammals are known to be activated by the ADAM family
would greatly benefit from the availability of selective rhomboid of membrane-bound metalloproteases [reviewed in Ref. [25,26]].
inhibitors, but these are currently not available. Here I review the However, it is clear that the non-protease members of the rhom-
current strategies to inhibit rhomboid proteases and summarize boid family of proteins called iRhoms control EGFR signaling in
what the field has learned from them. I also review the current mammals by activating ADAM17 [8,10,27–29], the main EGFR lig-
knowledge of rhomboid mechanism and specificity, and discuss its and activating enzyme, and there is accumulating evidence that
implications for future strategies of rhomboid inhibitor develop- human rhomboid proteases may participate in fine-tuning of EGFR
ment. Let us first focus on examples and contexts where rhomboid signaling [30–32]. This elevates the interest in understanding the
proteases participate or can participate in disease-relevant pro- role of mammalian rhomboid proteases in greater detail, and in
cesses and where selective rhomboid inhibitors could be employed development of their inhibitors as research tools.
for pharmacological applications. There are four rhomboid proteases located in the secretory
pathway of mammalian cells (‘secretase’ rhomboids RHBDL1-4)
and one in the mitochondria (PARL) [33]. The best studied human
secretase rhomboid is RHBDL2 (located at the plasma membrane
[34]), probably because it is the only RHBDL that readily cleaves
2. Rhomboid proteases as potential future drug targets
model rhomboid substrate Spitz [1] and has thus been amenable
to enzymological and cell biological investigation. RHBDL2 is
2.1. Microbial rhomboid proteases
expressed mainly in epithelia [30], and it has been implicated
in wound healing [35], endothelial angiogenesis [36], EGF recep-
Rhomboid proteases are present in a number of protozoan
tor signaling [30,31], and possibly anoikis resistance [37], but
parasites, such as Plasmodium, Toxoplasma, Eimeria, Cryptosporid-
loss-of-function animal experiments addressing these suggested
ium, Theileria and Babesia [11,12] including serious worldwide
functional hypotheses in vivo are lacking. Disease associations of
pathogens. Their functions have been addressed genetically
RHBDL2 have not been reported apart from a recently found cor-
and biochemically in Plasmodium, Toxoplasma, Entamoeba and
relation between RHBDL2 mRNA levels and histological grade of
Trichomonas so far. In the extracellular protozoan parasite Tri-
breast cancer tumors [38].
chomonas vaginalis, which causes a sexually transmitted infection
The second best studied human secretase rhomboid is RHBDL4
aggravating other disease conditions, rhomboid proteases TvROM1
(also known as RHBDD1), which is located in the endoplasmic retic-
and TvROM3 are active enzymes, and overexpression of the cell-
ulum. It has been implicated in membrane protein quality control
surface localized TvROM1 enhances the association of the parasite
[39], and shown to secrete TGF␣ [32,40] thus promoting the growth
with host cells and their lysis [13]. The extracellular parasite Enta-
of colorectal cancer cells via activation of the EGF receptor [32].
moeba histolytica encodes one rhomboid protease EhROM1 that has
The grade of colorectal cancer biopsies from patients and survival
been implicated in adhesion and phagocytosis [14,15]. The intra-
parameters correlated with RHBDL4 expression, and depletion of
cellular parasite Plasmodium falciparum causing malaria is probably
endogenous RHBDL4 from tumor cells suppressed proliferation
the most serious medical burden of the above named infectious
in vitro and tumor growth in vivo [32], suggesting that inhibitors
agents, affecting millions of people worldwide. The rhomboid pro-
of RHBDL4 could have anti-tumor properties.
teases PfROM1 and PfROM4 of P. falciparum can cleave and shed
The remaining two human secretase rhomboids, RHBDL1 and
several major surface adhesins of the parasite that are implicated
3 are the least characterized ones. They share about 49% sequence
in all stages of its life cycle [16]. Mutations in the transmembrane
identity, and display overlapping but non-identical expression pat-
domain of adhesin EBA-175 inhibiting its cleavage by PfROM4 pre-
terns, suggesting that they have distinct functions. RHBDL1 was
vent the growth of the parasite [17], suggesting that PfROM4 is a
the first human rhomboid protease gene to be identified [41]; it
potential therapeutic target. In Plasmodium berghei, a genetically
is expressed mainly in the brain and kidney [41] and localized to
tractable malaria model, PbROM1 deficiency did not compromise
the Golgi apparatus [34]. RHBDL3 (also known as ventrhoid) is
the infectivity or pathogenicity, but genes encoding rhomboids
expressed in the developing neural ventral tube and in the brain
ROM4, 6, 7 and 8 were refractory to deletion, suggesting that these
[42], and is localized to the Golgi and plasma membrane [34]. It
rhomboid proteases may be essential for the parasite, at least in its
is also expressed in the developing pancreas under control of the
asexual blood stage [18]. Assuming that functions of the P. berghei
neurogenin-3 transcription factor [43], but the significance of this
rhomboids will be conserved in P. falciparum, there is an exciting
observation for pancreas development and function is unclear. The
prospect that inhibitors of several rhomboid proteases could have
expression level of RHBDL3 has been correlated with the chrono-
antimalarial activity.
logical age and it is one of a few candidate markers of aging brain
Beyond protozoan parasites, the rhomboid protease RbdA
[44]. Both RHBDL1 and 3 bear all the sequence hallmarks of active
from Aspergillus fumigatus, an opportunistic pathogenic mold
rhomboid proteases, and RHBDL3 is able to bind an activity-based
encountered in immunocompromised individuals, is required
probe [45], suggesting that it has a functional active site. RHBDL1
for the adaptation of A. fumigatus to hypoxia during infection
and 3 might thus have a markedly different substrate specificity
[19]. The RbdA deficient A. fumigatus is thus more sensitive to
from the model rhomboid proteases (such as RHBDL2), but since
phagocytic killing, elicits weaker immune response and exhibits
no substrates of RHBDL1 and 3 have been identified so far, their
strongly attenuated virulence [19]. Since rhomboid proteases are
molecular functions remain unknown.
widespread, it is likely that other disease-relevant functions in
The mitochondrial rhomboid PARL is the best characterized
microbes will be discovered. In particular, relatively little is known
rhomboid protease in mammals. Mice deficient in PARL have a
about the functions of rhomboid proteases in bacteria [20–23]
pronounced phenotype − muscle wasting and reduced lifespan −
compared to how very widely distributed across prokaryotes rhom-
caused by increased apoptosis [46]. The basis for this effect is that
boids are [3,24].
PARL deficiency results in aberrant mitochondrial cristae, which
Please cite this article in press as: K. Strisovsky, Rhomboid protease inhibitors: Emerging tools and future therapeutics, Semin Cell Dev
Biol (2016), http://dx.doi.org/10.1016/j.semcdb.2016.08.021
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facilitates the release of cytochrome C from mitochondria and sen- (Fig. 1B), while most serine proteases attack the re-face of carbonyl
sitizes the parl −/- cells to intrinsic apoptotic stimuli [46], although carbon [59] apart from few exceptions such as the E. coli leader pep-
further mechanistic details are unclear [47]. In fact, it turns out that tidase [60,61]. These stereochemical differences might be exploited
PARL is intimately involved in controlling mitochondrial health at in the design of rhomboid-specific inhibitors.
several levels. Importantly, it is involved in the regulation of the The structures of the DFP and peptidyl aldehyde inhibitor com-
PINK1-PARK2 pathway that promotes mitophagy by which aber- plexes with GlpG show that the negatively charged oxygen of the
rant mitochondria are degraded. Although the detailed role of PARL covalent tetrahedral intermediates of the reaction (Fig. 1A) is sta-
in this process has been controversial, the most recent research bilised by hydrogen-bonding to the side-chains of amino acids
indicates that PARL-catalysed cleavage of PINK1 leads to its degra- H150 and N154 and the main-chain amide of S201 in a structure
dation in the cytoplasm and suppression of mitophagy [48]. Since termed ‘oxyanion pocket’ [62] (Fig. 1C).
mitochondrial dysfunction emerges as one of the main causes of Rhomboid-catalyzed reaction occurs in the lipid membrane, and
neuronal loss in Parkinson’s disease [49], inhibition (partial loss of the reaction mechanism involves a covalent intermediate acyl-
function) of PARL could stimulate mitophagy and alleviate neuronal enzyme that must be hydrolyzed to complete the reaction cycle.
loss, thereby being beneficial in Parkinson’s disease (PD). Other The catalytic dyad of rhomboid is about 10 Å below the mem-
substrates of PARL have been described and PARL has even been brane surface [54]. The active site is open to bulk solvent, and
proposed to function as a checkpoint of mitochondrial integrity the delivery of water molecules for the deacylation reaction thus
tipping the balance between mitophagy and repair on one hand seems unhindered [57]. Intriguingly however, molecular dynamics
and apoptosis on the other [47]. This is particularly relevant for studies suggest that a cavity near the catalytic serine might act as
human disease, because mitochondrial dysfunction is associated a ‘water retention site’ (Fig. 1D) that facilitates delivery of water
with a number of disease conditions including PD [reviewed in Ref. molecules from bulk solution into the membrane-immersed cat-
[50,51]] and type 2 diabetes [reviewed in Ref. [52]]. For more dis- alytic center, thus potentiating catalytic efficiency of GlpG [63]. This
cussion on PARL refer to the article by DeStrooper in this issue. hypothesis is supported by mutagenesis experiments where muta-
Taken together, the above examples demonstrate that selective tions of amino acids with polar side chains that were suggested to
and potent rhomboid inhibitors and a platform for their develop- constitute a ‘relay‘ mechanism for water molecules (mainly Q189
ment are urgently needed, both for research purposes to validate and S185) decrease catalytic activity of GlpG without impacting on
functional hypotheses and for potential pharmacological use. its thermodynamic stability [63]. It is attractive to speculate that an
accessory mechanism for water delivery into the active site could
be a general feature of intramembrane proteases.
3. Rhomboid protease mechanism and specificity
3.2. Substrate specificity
Before we discuss the possibilities of inhibiting rhomboid pro-
teases, let us first browse through what is known about rhomboid
A separate question important for inhibitor design is that of how
mechanism, because the principles of catalytic mechanism and
rhomboid proteases recognize their substrates. Intramembrane
substrate specificity to a large extent determine the strategies for
protease substrates are transmembrane ␣-helices, and although
inhibitor development.
protease cleavage sites located apparently within secondary struc-
tures such as helices have been described [64], these presumably
3.1. Catalytic mechanism need to be unwound before protease cleavage, because most or
all protease active sites bind substrates or inhibitors in extended
Most globular serine proteases, operating in an aqueous envi- -strand conformations [65,66]. According to this view, intramem-
ronment, use a catalytic triad, typically Asp-Ser-His, to abstract a brane proteases including rhomboids were initially thought not to
proton from the OH group of the catalytic Ser and thus poise it be sequence-specific and to recognize primarily the intrinsic insta-
for nucleophilic attack at a substrate peptide bond [53]. Rhomboid bility of transmembrane helices at the site of cleavage, defined by
proteases hydrolyse peptide bonds in transmembrane substrates the presence of residues with low transmembrane helical propen-
using only a catalytic dyad of Ser and His [54] (Fig. 1A), which brings sity [67]. The intrinsic helical instability was initially thought to be
substantial mechanistic differences. Molecular dynamics and quan- the key feature defining rhomboid substrates [68], but this condi-
tum mechanics studies suggest that the lack of the third, proton tion was insufficient to predict substrates, and did not explain the
abstracting, residue makes the catalytic His254 of the E. coli rhom- position of the cleavage site [69].
boid protease GlpG (hereinafter just GlpG) more acidic (i.e. it has Later it was found that small amino acids were preferred by GlpG
a lower pKa) than the OH group of Ser201 in the unliganded state at the P1 position of the substrate [69], suggesting that rhomboids
of the enzyme [55], which means that Ser201 is protonated in the displayed some level of sequence specificity. Indeed, detailed com-
ground state of the enzyme. Gradual desolvation of the active site parative analysis of cleavage kinetics and site-specificity using the
of GlpG by the incoming ligand (inhibitor or substrate) is thought first natural substrate of a bacterial rhomboid [20] finally revealed
to induce an increase in the pKa of H254, leading to a concerted that rhomboids recognize two elements in their substrates [70]
proton abstraction from OH of Ser201 and nucleophilic attack (Fig. 2 A), one of which is the hydrophobic helical transmembrane
[56]. In contrast, in classical serine proteases containing a catalytic domain (TMD) and the second one is a linear sequence of about 6
triad, such as chymotrypsin, the higher pKa of catalytic His facili- amino acids that binds into the active site and determines the site
tates deprotonation of catalytic Ser and nucleophilic attack can be of cleavage (also called ‘recognition motif’). The recognition motif
energetically and temporally separated into distinct catalytic steps can be part of the TMD of the substrate or be adjacent to it; hence
[56]. This hypothesis could explain the markedly low catalytic effi- the two elements are separable [70]. Numerous rhomboids were
ciency of GlpG compared to classical serine proteases chymotrypsin found to prefer small amino acids in the P1 position, while there
or trypsin, but it is less clear what its implication for rhomboid is more diversity in sequence preferences at other positions. Some
inhibitor design might be. bacterial rhomboids including GlpG, AarA and YqgP prefer large